# Preparation and Multitarget Anti-AD Activity Study of Chondroitin Sulfate Lithium in AD Mice Induced by Combination of D-Gal/AlCl3

Authors: Debo Gao; Pingli Li; Fei Gao; Yangjun Feng; Xiaolin Li; Delong Li; Yuqin Li; Yuliang Xiao
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

Previous studies have demonstrated that both CS and LiCl possess anti-Alzheimer’s disease (AD) activities. We prepared chondroitin sulfate-Li (CS-Li) and investigated its effect on AD and explored the possible mechanisms bothin vitro and in vivo. We found that CS-Li could inhibit amyloid β (Aβ) aggregation and protect SH-SY5Y cells from Aβ1-42-induced cytotoxicity in vitro. In D-gal and AlCl3-induced AD mouse model, CS-Li improves the spatial learning and memory abilities of AD mice, reverses the nuclear pyknosis and cell edema, and increases the survival rate of neurons in hippocampus of mice. Moreover, CS-Li significantly increased the levels of GSH-Px, Na+/K+-ATPase, and ChAT and decreased the levels of MDA and AchE in AD mice. Western blot results demonstrated that CS-Li could decrease the hyperphosphorylation of tau (Ser396/Ser404) by regulating the expression of p-GSK-3β (Ser9) and PP2A and inhibit the expression of proinflammatory factors through inhibiting NF-κB nuclear translocation by activating the MAPK signaling pathways. In a word, CS-Li can delay AD development through multitarget processes, including Aβ aggregation inhibition, oxidative stress damage, tau hyperphosphorylation, and inflammatory response, thereby improves learning and memory abilities.


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## Body

## 1. Introduction

Alzheimer’s disease (AD) is one of greatest medical care challenges worldwide and is the most prevalent cause of dementia, with the character of progressive neuronal loss and cerebral atrophy [1]. Studies have discovered the main pathogenesis of AD is the accumulation of extracellular amyloid β (Aβ) plaques and intracellular hyperphosphorylated tau protein as neurofibrillary tangles (NFTs) [2]. Aβ production initiates a cascade of pathological changes of brain including inflammation, oxidative damage, and excitotoxicity, while tau protein hyperphosphorylation results in microtubule disassembly, both can lead to neuronal destruction and cognitive deficiencies [3]. Therefore, most clinical trials of disease-modifying treatment (DMT) drugs focus on the primary targets of Aβ and tau protein for AD prevention or therapy, such as solanezumab [4], avagacestat [5], and idalopirdine [6]. However, the results are not always satisfactory. The approved AD drugs by the FDA (donepezil, galantamine, tacrine, rivastigmine, and memantine) acting on acetylcholinesterase (AchE) or N-methyl-D-aspartate (NMDA) receptor can temporarily relieve the clinical symptoms but cannot reverse the disease progression [7]. Recently, a new drug named sodium oligomannate (GV-971) has been conditionally approved in China for the treatment of AD [8]. Sodium oligomannate is a marine algae-derived oral oligosaccharide, and the possible mechanism of action includes gut microbiota dysfunction remodeling, neuroinflammation reduction, and Aβ fibril formation inhibition [9]. The approval of sodium oligomannate suggests that a novel therapeutic strategy, i.e., multitarget effectors mechanism may be an effective solution to treat AD.Chondroitin sulfate (CS) is widely distributed in connective tissues, such as bone, skin, cartilage, spleen, blood vessels, and never tissues, with a variety of biological activities including antioxidation, anti-inflammation, immunoregulation, and antiatherosclerosis [10]. Chondroitin sulfate proteoglycans (CSPGs) are important components of the perineuronal nets (PNN) and play important roles in modulating neurodegeneration, neurodevelopment, and long-term memory [11]. Therefore, the neuroprotective effect of CS has also attracted much attention in recent years. CS exhibits antioxidation and antiapoptosis activities through upregulating nuclear NF-E2-related factor-2 (Nrf2) expression and regulating the mitochondrial pathway to protect SH-SY5Y cells from 6-hydroxydopamine- (6-OHDA-) induced toxicity [12]. Low molecular weight chondroitin sulfate (LMWCS) also possessed neuroprotective effect against Aβ induced toxicity through increasing the level of choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) as well as decreasing the expression of malondialdehyde (MDA) and acetylcholinesterase (AchE) [13, 14]. Our previous study has modified CS with selenium and found selenium-chondroitin sulfate nanoparticles could decrease oxidative stress injury and inhibit Aβ aggregation and tau protein hyperphosphorylation [15, 16]. Therefore, CS and its derivatives have the potential to be developed as anti-AD drugs, especially those with multitarget action mechanisms.It is well recognized that Lithium (Li) has been applied in the treatment of bipolar disorder (BD) for many years in clinical based on its neuroprotective and neurotrophic effects [17], and studies on using Li to treat psychiatric and neurological disorders are still popular, including AD [18–21]. A meta-analysis of randomized placebo-controlled trials indicated Li treatment may be beneficial for patients with mild cognitive impairment (MCI) and AD dementia [19]. The possible mechanism of Li acting on AD may be due to inhibition of glycogen synthase kinase-3 beta (GSK-3β) activity, followed by reducing the hyperphosphorylation of tau and inducing the neuronal death via Aβ overproduction [22]. Besides, Li was also found to increase the level of brain microvascular LRP1 and CSF bulk-flow in APP/PS1 transgenic mice, resulting in the clearance of soluble Aβ protein [23].Our previous studies demonstrated that CS and chondroitin sulfate nanoselenium (CS@Se) exhibit anti-AD activity bothin vitro and in vivo [15, 16]. Since both CS and Li have been reported to have neuroprotective effects, we intended to design a novel multitarget-directed chondroitin sulfate-Li (CS-Li) compound, investigate its therapeutic effect on AD in vitro and in AD mouse model, and explore the possible action mechanisms.


## 2. Materials and Methods

### 2.1. Reagents

CS from shark cartilage was purchased from Jiaxing Hengjie Biopharmaceutical Co., Ltd. (Jiaxing, China). CS (Mw=4.2 kDa) used in this paper was prepared in our laboratory [15]. LiCl was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cation-exchange resin and ultrafiltration membrane with MWCO of 1000 Da and 5000 Da were from Sangon Biotech Co., Ltd. (Shanghai, China). The amyloid-β (1-42) peptide was purchased from GL Biochem Ltd. (Shanghai, China). AchE, ChAT, and Na+/K+-ATPase assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). GSH-Px and MDA assay kits were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Rabbit anti-TNF-α, anti-IL-6, anti-IL-1β, anti-p-p38MAPK, anti-p-Erk1/2, anti-p-NF-κB, anti-p-GSK3β (Ser9), and anti-PP2A antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA). Rabbit anti-p-tau (Ser396/Ser404), anti-tau, and anti-GSK3β antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-IκBa, anti-p-IκBa, and anti-Lamin B were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). The TUNEL assay kit was purchased from Roche (Basel, Switzerland).


### 2.2. Syntheses and Characterization of CS-Li

CS (5 g) was dissolved in deionized water, and residual Na+ in CS was removed with acidified cation-exchange resin. The collected effluent was added in LiCl (2 g) and stirred at 35°C for 6 h. After the reaction, the solution was dialyzed (1000 Da) in water for 48 h to obtain the CS-Li solution, and then 4 times volume of 95% ethanol was added to precipitate. The precipitate was dried and finally got CS-Li compound.The FTIR spectrum and structure of CS-Li was measured by an IRAffinity-1 s spectrometer (Shimadzu, Japan) and nuclear magnetic resonance (NMR) (Bruker Avance 400, Bruker, Germany). The Li content of CS-Li was measured by inductively coupled plasma mass spectrometry (ICP-MS) (ICAP6300, Thermofisher, USA).


### 2.3. Cytotoxicity Study of CS-Li

The cytotoxicity of CS-Li was assessed by its effect on the proliferation of SH-SY5Y cells with MTT method. Briefly, the cells were cultured in 96-well plates at a density of8×103 cells/well with CS-Li (100, 200, 400, and 800 μg/mL) or LiCl (amount of Li was equal to that of CS-Li) for 24 h at 37°C. 10 μL of MTT solution (5 mg/mL) was added to each well for 4 h at 37°C. Then the medium was removed and 150 μL of dimethyl sulfoxide was added. After incubation for 10 min, the optical density (OD) at 570 nm was detected using a microplate reader. The cell viability was calculated according to Formula (1).
(1)Relativecellviability%=OD570sample−OD570blankOD570control−OD570blank×100.


### 2.4. Effect of CS-Li on Aβ1-42 Aggregation and Spatial Conformation

#### 2.4.1. Preparation of the Aβ1-42 Stock Solution

The Aβ1-42 solution was freshly prepared immediately before use: 0.5 mg Aβ1-42 peptide was dissolved in 1.108 mL deionized water, filtrated through 0.22 μm microfiltration membrane, and diluted to a final concentration of 100 μM.


#### 2.4.2. Thioflavine T Fluorescence Spectroscopy Assay

The inhibition of CS-Li on the aggregation process of Aβ1-42 was determined using thioflavine T, which can interact with aggregated fibrils of Aβ peptide [24]. 30 μL of Aβ1-42 stock solution was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL, amount of Li was identical to 400 μg/mL) at 37°C protected from light for 100 h. 30 μL of Aβ solution was taken at different time intervals (0, 10, 24, 48, 72, and 100 h), mixed sufficiently with 400 μL of thioflavine T solution (15 μM in 0.05 M PBS), and incubated in dark for 30 min. The free Aβ peptide was used as the control. The fluorescence intensity was detected by fluorescence spectrophotometer (Cary Eclipse, Varian, USA). The excitation and emission wavelengths of thioflavine T are 440 nm and 480 nm with the slit widths of 20 nm, respectively. Each experiment was repeated three times.


#### 2.4.3. Congo Red-Binding Test

150μL of freshly prepared Aβ1-42 solution (30 μM) was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) at 37°C protected from light for 3 days. Then, Congo red solution was added to the Aβ1-42 solution with equal ratio. After 30 min, the Congo red absorbance (400 nm-650 nm) was detected by UV-vis spectrophotometer.


#### 2.4.4. ANS Fluorescence Assay

30μL of freshly prepared Aβ1-42 solution (30 μM) was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) at 37°C for 3 days, and the Aβ solution was added to 400 μL of ANS solution (50 μM). After 30 min, the fluorescence intensity was detected by fluorescence spectrophotometer. The excitation and emission wavelengths of ANS are 380 nm and 400 nm with the slit widths of 20 nm, respectively. Each experiment was performed in triplet.


#### 2.4.5. Circular Dichroism Measurements

The secondary spatial structure of the Aβ protein was measured by circular dichroism (CD) spectra with a Chirascan CD spectropolarimeter. A freshly prepared Aβ1-42 solution was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) for 3 days at 37°C. The CD spectra of samples were then recorded from 200 nm to 290 nm at a speed of 100 nm per minute with a CD cuvette. The free Aβ peptide treated according to the same steps as the control. Each sample was performed in triplet.


#### 2.4.6. Transmission Electron Microscope

The Aβ solution (100 μM) was incubated with different concentration of CS-Li (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) for 3 days at 37°C. After that, the samples were dropped on the Formvar-coated grid and stained with 2% phosphotungstic acid. The samples were determined using a JEM-1200EXII TEM (Jeol, Japan).


#### 2.4.7. Inhibition of CS-Li on Aβ1-42-Induced Cell Apoptosis

Firstly, Aβ1-42 peptide was prepared according to the literature report [25] with minor modification. Aβ1-42 peptide (0.5 mg) was dissolved in 0.022 mL of dimethyl sulfoxide to make 5 mM Aβ stock. The stock was diluted to 100 μM with phenol red-free DMEM, filtered through 0.22 μm microfiltration membrane, and incubated for 3 days at 37°C.Then, the inhibitory effect of CS-Li on Aβ1-42-induced SH-SY5Y cell apoptosis was studied by MTT method as described in Part 2.3. After the cells were incubated for 24 h, CS-Li (final concentration of 200 μg/mL, 400 μg/mL) or LiCl (final concentration of 14.8 μg/mL) was added to the wells and incubated for 12 h. After that, Aβ1-42 (final concentration of 20 μM) was added and incubated for 24 h. Then, MTT and DMSO were added to each well successively as described above. The OD570 was recorded and the cell viability was calculated with Formula (1).


### 2.5. Effect of CS-Li on Behavioural Tests in AD Mice Induced by D-Gal/AlCl3

#### 2.5.1. Animals

Male C57BL/6 (6-8 weeks old, 18~25 g) were obtained from the Jinan Pengyue Experimental Animal Breeding Co. Ltd. (Jinan, China, license number: SCXK(Lu)-20140007) and maintained at22±2°C temperature and 45-55% humidity under pathogen-free conditions according to the University Animal Care and Use Guidelines. The mice were housed in a 12 h/12 h light/dark cycle, with unrestrained food and water. After 7 days of acclimatization, 75 mice were randomly divided into five groups. The control group mice were orally administrated with normal saline (NS) 0.25 mL every day. The other groups were treated with AlCl3 (200 mg/kg/day, gavage) and D-gal (120 mg/kg/day, injected subcutaneously) for 60 days according to the previous investigation [26]. After 30 days of administration, the study drugs were administrated for the next 30 days. The daily doses of study drugs were 400 mg/kg (CS-Li high dose group, H-CS-Li group), 200 mg/kg (CS-Li low dose group, L-CS-Li group), and 14.8 mg/kg (LiCl group, the amount of Li was equal to that of H-CS-Li group).


#### 2.5.2. Morris Water Maze (MWM)

The MWM is widely applied to study the spatial memory and learning ability of animals [27]. The MWM is a black barrel (110 cm in diameter and 50 cm high) with a camera connecting to a computer to get the swimming pathway of mice. The water in the pool was dyed with nontoxic white dye, and the temperature was maintained at 20±2°C. The pool was divided into four quadrants, and a circular platform (diameter 12 cm) was placed 1 cm below the water surface in the fourth quadrant. The MWM experiment included three continuous phases: initial training (IT), navigation test (NT), and space exploring test (SET). During the IT stage, the mouse was placed into water with the head facing the wall at four different positions, to find the hidden platform within 60 s. If the mouse was able to reach the platform and stayed for 5 s, the test was completed. If the mouse could not find the platform within 60 s, it was guided to the platform and trained to stay on the platform for 10 s, and the time was recorded as 60 s. The mice were trained four sessions every day for 5 days. In the NT phase, the time that each mouse spent in finding and climbing the platform was recorded as the escape latency. The SET was started 24 h after the IT stage. After the platform was removed, the mouse was released in the water at the third quadrant and allowed to swim freely for 60 s. The time spent in the targeted quadrant and the number of platform crossings during the process were recorded.


#### 2.5.3. Novel Object Recognition (NOR)

The NOR test is applied to estimate the memory ability of the previously familiar objects as well as the visual recognition, learning, and memory ability of the novel items of rodents [28]. The NOR experiment system mainly includes a box (52cm×52cm×52cm) for free activity of mice and a video recording device. In the first stage of the NOR test, two objects (A1, A2) with similar size and same material were put into the box, on the diagonal line with 30 cm apart. The mouse explored the box freely for 5 min, and the time spent on exploring objects A1 (tA1) and A2 (tA2) was recorded. In the second stage, a novel object B is placed in the box to replace the object A2, and the time spent on exploring each object (tA1 and tB) was recorded. The preference index (PI) of each mouse was calculated to evaluate the learning and memory ability according to the following formula:
(2)PI=tBtA1+tB.


### 2.6. Mechanism Study of CS-Li Exhibiting Anti-AD Effect in Mice

#### 2.6.1. Transmission Electron Microscope of Hippocampus Microstructure

After the above behavioral test, the mice were anaesthetized and perfused with 0.1 mol/L precooled phosphate-buffer saline for 5 min and 4% precooled paraformaldehyde until the mice were stiff. Then the hippocampi were extracted and fixed with 3% precooled glutaraldehyde solution for 6 h, and then were cut into 0.5~1 mm3 blocks. The samples were postfixed in 1% osmium tetroxide for 60 min, followed by dehydration in acetone, epoxy resin embedding, and ultrathin section. After stained with lead citrate and uranyl acetate, the ultrathin sections of the cured blocks were visualized by JEM-1200EXII TEM.


#### 2.6.2. Hematoxylin and Eosin (H&E) Staining, Nissl Staining, and TUNEL Staining

The mice were sacrificed and the brains were isolated, then the brains were embedded in paraffin and were cut into 6μm thin slices. The slices were processed for H&E staining, Nissl staining, and TUNEL staining. After that, the sections were imaged and analyzed by microscopy.


#### 2.6.3. Biochemical Parameters Analysis

The hippocampi were isolated as described in Part 2.6.1. Then the hippocampi were homogenized in normal saline containing protease inhibitors. The homogenate was centrifuged at 2500 rpm for 10 min, and the protein concentration in supernatant was measured by BCA method. The level of MDA and the activities of GSH-Px, ChAT, AchE, and Na+K+-ATPase were determined by relevant assay kits.


#### 2.6.4. Western Blot Analysis

Protein contents (35-40μg) were separated by 10% SDS-PAGE and electrotransferred onto PVDF membranes. The membrane was blocked with TBST blocking solution for 1 h, and then incubated with primary antibody overnight at 4°C on a rotating shaker, followed by incubation with HRP conjugated secondary antibody for 2 h. Then the protein bands were detected using enhanced chemiluminescence kit (ECL, Millipore). The protein signal value of each target protein was determined by ImageJ software.


### 2.7. Statistical Analysis

The quantitative data were expressed asmean±SD, and the statistical significance between different groups was analyzed by Student’s t-test one-way analysis of variance (ANOVA). Difference in mean with p<0.05 was considered to demonstrate statistical significance. Statistical analysis was evaluated using SPSS 11.0 software.


## 2.1. Reagents

CS from shark cartilage was purchased from Jiaxing Hengjie Biopharmaceutical Co., Ltd. (Jiaxing, China). CS (Mw=4.2 kDa) used in this paper was prepared in our laboratory [15]. LiCl was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cation-exchange resin and ultrafiltration membrane with MWCO of 1000 Da and 5000 Da were from Sangon Biotech Co., Ltd. (Shanghai, China). The amyloid-β (1-42) peptide was purchased from GL Biochem Ltd. (Shanghai, China). AchE, ChAT, and Na+/K+-ATPase assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). GSH-Px and MDA assay kits were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Rabbit anti-TNF-α, anti-IL-6, anti-IL-1β, anti-p-p38MAPK, anti-p-Erk1/2, anti-p-NF-κB, anti-p-GSK3β (Ser9), and anti-PP2A antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA). Rabbit anti-p-tau (Ser396/Ser404), anti-tau, and anti-GSK3β antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-IκBa, anti-p-IκBa, and anti-Lamin B were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). The TUNEL assay kit was purchased from Roche (Basel, Switzerland).


## 2.2. Syntheses and Characterization of CS-Li

CS (5 g) was dissolved in deionized water, and residual Na+ in CS was removed with acidified cation-exchange resin. The collected effluent was added in LiCl (2 g) and stirred at 35°C for 6 h. After the reaction, the solution was dialyzed (1000 Da) in water for 48 h to obtain the CS-Li solution, and then 4 times volume of 95% ethanol was added to precipitate. The precipitate was dried and finally got CS-Li compound.The FTIR spectrum and structure of CS-Li was measured by an IRAffinity-1 s spectrometer (Shimadzu, Japan) and nuclear magnetic resonance (NMR) (Bruker Avance 400, Bruker, Germany). The Li content of CS-Li was measured by inductively coupled plasma mass spectrometry (ICP-MS) (ICAP6300, Thermofisher, USA).


## 2.3. Cytotoxicity Study of CS-Li

The cytotoxicity of CS-Li was assessed by its effect on the proliferation of SH-SY5Y cells with MTT method. Briefly, the cells were cultured in 96-well plates at a density of8×103 cells/well with CS-Li (100, 200, 400, and 800 μg/mL) or LiCl (amount of Li was equal to that of CS-Li) for 24 h at 37°C. 10 μL of MTT solution (5 mg/mL) was added to each well for 4 h at 37°C. Then the medium was removed and 150 μL of dimethyl sulfoxide was added. After incubation for 10 min, the optical density (OD) at 570 nm was detected using a microplate reader. The cell viability was calculated according to Formula (1).
(1)Relativecellviability%=OD570sample−OD570blankOD570control−OD570blank×100.


## 2.4. Effect of CS-Li on Aβ1-42 Aggregation and Spatial Conformation

### 2.4.1. Preparation of the Aβ1-42 Stock Solution

The Aβ1-42 solution was freshly prepared immediately before use: 0.5 mg Aβ1-42 peptide was dissolved in 1.108 mL deionized water, filtrated through 0.22 μm microfiltration membrane, and diluted to a final concentration of 100 μM.


### 2.4.2. Thioflavine T Fluorescence Spectroscopy Assay

The inhibition of CS-Li on the aggregation process of Aβ1-42 was determined using thioflavine T, which can interact with aggregated fibrils of Aβ peptide [24]. 30 μL of Aβ1-42 stock solution was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL, amount of Li was identical to 400 μg/mL) at 37°C protected from light for 100 h. 30 μL of Aβ solution was taken at different time intervals (0, 10, 24, 48, 72, and 100 h), mixed sufficiently with 400 μL of thioflavine T solution (15 μM in 0.05 M PBS), and incubated in dark for 30 min. The free Aβ peptide was used as the control. The fluorescence intensity was detected by fluorescence spectrophotometer (Cary Eclipse, Varian, USA). The excitation and emission wavelengths of thioflavine T are 440 nm and 480 nm with the slit widths of 20 nm, respectively. Each experiment was repeated three times.


### 2.4.3. Congo Red-Binding Test

150μL of freshly prepared Aβ1-42 solution (30 μM) was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) at 37°C protected from light for 3 days. Then, Congo red solution was added to the Aβ1-42 solution with equal ratio. After 30 min, the Congo red absorbance (400 nm-650 nm) was detected by UV-vis spectrophotometer.


### 2.4.4. ANS Fluorescence Assay

30μL of freshly prepared Aβ1-42 solution (30 μM) was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) at 37°C for 3 days, and the Aβ solution was added to 400 μL of ANS solution (50 μM). After 30 min, the fluorescence intensity was detected by fluorescence spectrophotometer. The excitation and emission wavelengths of ANS are 380 nm and 400 nm with the slit widths of 20 nm, respectively. Each experiment was performed in triplet.


### 2.4.5. Circular Dichroism Measurements

The secondary spatial structure of the Aβ protein was measured by circular dichroism (CD) spectra with a Chirascan CD spectropolarimeter. A freshly prepared Aβ1-42 solution was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) for 3 days at 37°C. The CD spectra of samples were then recorded from 200 nm to 290 nm at a speed of 100 nm per minute with a CD cuvette. The free Aβ peptide treated according to the same steps as the control. Each sample was performed in triplet.


### 2.4.6. Transmission Electron Microscope

The Aβ solution (100 μM) was incubated with different concentration of CS-Li (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) for 3 days at 37°C. After that, the samples were dropped on the Formvar-coated grid and stained with 2% phosphotungstic acid. The samples were determined using a JEM-1200EXII TEM (Jeol, Japan).


### 2.4.7. Inhibition of CS-Li on Aβ1-42-Induced Cell Apoptosis

Firstly, Aβ1-42 peptide was prepared according to the literature report [25] with minor modification. Aβ1-42 peptide (0.5 mg) was dissolved in 0.022 mL of dimethyl sulfoxide to make 5 mM Aβ stock. The stock was diluted to 100 μM with phenol red-free DMEM, filtered through 0.22 μm microfiltration membrane, and incubated for 3 days at 37°C.Then, the inhibitory effect of CS-Li on Aβ1-42-induced SH-SY5Y cell apoptosis was studied by MTT method as described in Part 2.3. After the cells were incubated for 24 h, CS-Li (final concentration of 200 μg/mL, 400 μg/mL) or LiCl (final concentration of 14.8 μg/mL) was added to the wells and incubated for 12 h. After that, Aβ1-42 (final concentration of 20 μM) was added and incubated for 24 h. Then, MTT and DMSO were added to each well successively as described above. The OD570 was recorded and the cell viability was calculated with Formula (1).


## 2.4.1. Preparation of the Aβ1-42 Stock Solution

The Aβ1-42 solution was freshly prepared immediately before use: 0.5 mg Aβ1-42 peptide was dissolved in 1.108 mL deionized water, filtrated through 0.22 μm microfiltration membrane, and diluted to a final concentration of 100 μM.


## 2.4.2. Thioflavine T Fluorescence Spectroscopy Assay

The inhibition of CS-Li on the aggregation process of Aβ1-42 was determined using thioflavine T, which can interact with aggregated fibrils of Aβ peptide [24]. 30 μL of Aβ1-42 stock solution was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL, amount of Li was identical to 400 μg/mL) at 37°C protected from light for 100 h. 30 μL of Aβ solution was taken at different time intervals (0, 10, 24, 48, 72, and 100 h), mixed sufficiently with 400 μL of thioflavine T solution (15 μM in 0.05 M PBS), and incubated in dark for 30 min. The free Aβ peptide was used as the control. The fluorescence intensity was detected by fluorescence spectrophotometer (Cary Eclipse, Varian, USA). The excitation and emission wavelengths of thioflavine T are 440 nm and 480 nm with the slit widths of 20 nm, respectively. Each experiment was repeated three times.


## 2.4.3. Congo Red-Binding Test

150μL of freshly prepared Aβ1-42 solution (30 μM) was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) at 37°C protected from light for 3 days. Then, Congo red solution was added to the Aβ1-42 solution with equal ratio. After 30 min, the Congo red absorbance (400 nm-650 nm) was detected by UV-vis spectrophotometer.


## 2.4.4. ANS Fluorescence Assay

30μL of freshly prepared Aβ1-42 solution (30 μM) was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) at 37°C for 3 days, and the Aβ solution was added to 400 μL of ANS solution (50 μM). After 30 min, the fluorescence intensity was detected by fluorescence spectrophotometer. The excitation and emission wavelengths of ANS are 380 nm and 400 nm with the slit widths of 20 nm, respectively. Each experiment was performed in triplet.


## 2.4.5. Circular Dichroism Measurements

The secondary spatial structure of the Aβ protein was measured by circular dichroism (CD) spectra with a Chirascan CD spectropolarimeter. A freshly prepared Aβ1-42 solution was coincubated with different concentrations of CS-Li solution (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) for 3 days at 37°C. The CD spectra of samples were then recorded from 200 nm to 290 nm at a speed of 100 nm per minute with a CD cuvette. The free Aβ peptide treated according to the same steps as the control. Each sample was performed in triplet.


## 2.4.6. Transmission Electron Microscope

The Aβ solution (100 μM) was incubated with different concentration of CS-Li (final concentration of 200 μg/mL, 400 μg/mL) or LiCl solution (final concentration of 14.8 μg/mL) for 3 days at 37°C. After that, the samples were dropped on the Formvar-coated grid and stained with 2% phosphotungstic acid. The samples were determined using a JEM-1200EXII TEM (Jeol, Japan).


## 2.4.7. Inhibition of CS-Li on Aβ1-42-Induced Cell Apoptosis

Firstly, Aβ1-42 peptide was prepared according to the literature report [25] with minor modification. Aβ1-42 peptide (0.5 mg) was dissolved in 0.022 mL of dimethyl sulfoxide to make 5 mM Aβ stock. The stock was diluted to 100 μM with phenol red-free DMEM, filtered through 0.22 μm microfiltration membrane, and incubated for 3 days at 37°C.Then, the inhibitory effect of CS-Li on Aβ1-42-induced SH-SY5Y cell apoptosis was studied by MTT method as described in Part 2.3. After the cells were incubated for 24 h, CS-Li (final concentration of 200 μg/mL, 400 μg/mL) or LiCl (final concentration of 14.8 μg/mL) was added to the wells and incubated for 12 h. After that, Aβ1-42 (final concentration of 20 μM) was added and incubated for 24 h. Then, MTT and DMSO were added to each well successively as described above. The OD570 was recorded and the cell viability was calculated with Formula (1).


## 2.5. Effect of CS-Li on Behavioural Tests in AD Mice Induced by D-Gal/AlCl3

### 2.5.1. Animals

Male C57BL/6 (6-8 weeks old, 18~25 g) were obtained from the Jinan Pengyue Experimental Animal Breeding Co. Ltd. (Jinan, China, license number: SCXK(Lu)-20140007) and maintained at22±2°C temperature and 45-55% humidity under pathogen-free conditions according to the University Animal Care and Use Guidelines. The mice were housed in a 12 h/12 h light/dark cycle, with unrestrained food and water. After 7 days of acclimatization, 75 mice were randomly divided into five groups. The control group mice were orally administrated with normal saline (NS) 0.25 mL every day. The other groups were treated with AlCl3 (200 mg/kg/day, gavage) and D-gal (120 mg/kg/day, injected subcutaneously) for 60 days according to the previous investigation [26]. After 30 days of administration, the study drugs were administrated for the next 30 days. The daily doses of study drugs were 400 mg/kg (CS-Li high dose group, H-CS-Li group), 200 mg/kg (CS-Li low dose group, L-CS-Li group), and 14.8 mg/kg (LiCl group, the amount of Li was equal to that of H-CS-Li group).


### 2.5.2. Morris Water Maze (MWM)

The MWM is widely applied to study the spatial memory and learning ability of animals [27]. The MWM is a black barrel (110 cm in diameter and 50 cm high) with a camera connecting to a computer to get the swimming pathway of mice. The water in the pool was dyed with nontoxic white dye, and the temperature was maintained at 20±2°C. The pool was divided into four quadrants, and a circular platform (diameter 12 cm) was placed 1 cm below the water surface in the fourth quadrant. The MWM experiment included three continuous phases: initial training (IT), navigation test (NT), and space exploring test (SET). During the IT stage, the mouse was placed into water with the head facing the wall at four different positions, to find the hidden platform within 60 s. If the mouse was able to reach the platform and stayed for 5 s, the test was completed. If the mouse could not find the platform within 60 s, it was guided to the platform and trained to stay on the platform for 10 s, and the time was recorded as 60 s. The mice were trained four sessions every day for 5 days. In the NT phase, the time that each mouse spent in finding and climbing the platform was recorded as the escape latency. The SET was started 24 h after the IT stage. After the platform was removed, the mouse was released in the water at the third quadrant and allowed to swim freely for 60 s. The time spent in the targeted quadrant and the number of platform crossings during the process were recorded.


### 2.5.3. Novel Object Recognition (NOR)

The NOR test is applied to estimate the memory ability of the previously familiar objects as well as the visual recognition, learning, and memory ability of the novel items of rodents [28]. The NOR experiment system mainly includes a box (52cm×52cm×52cm) for free activity of mice and a video recording device. In the first stage of the NOR test, two objects (A1, A2) with similar size and same material were put into the box, on the diagonal line with 30 cm apart. The mouse explored the box freely for 5 min, and the time spent on exploring objects A1 (tA1) and A2 (tA2) was recorded. In the second stage, a novel object B is placed in the box to replace the object A2, and the time spent on exploring each object (tA1 and tB) was recorded. The preference index (PI) of each mouse was calculated to evaluate the learning and memory ability according to the following formula:
(2)PI=tBtA1+tB.


## 2.5.1. Animals

Male C57BL/6 (6-8 weeks old, 18~25 g) were obtained from the Jinan Pengyue Experimental Animal Breeding Co. Ltd. (Jinan, China, license number: SCXK(Lu)-20140007) and maintained at22±2°C temperature and 45-55% humidity under pathogen-free conditions according to the University Animal Care and Use Guidelines. The mice were housed in a 12 h/12 h light/dark cycle, with unrestrained food and water. After 7 days of acclimatization, 75 mice were randomly divided into five groups. The control group mice were orally administrated with normal saline (NS) 0.25 mL every day. The other groups were treated with AlCl3 (200 mg/kg/day, gavage) and D-gal (120 mg/kg/day, injected subcutaneously) for 60 days according to the previous investigation [26]. After 30 days of administration, the study drugs were administrated for the next 30 days. The daily doses of study drugs were 400 mg/kg (CS-Li high dose group, H-CS-Li group), 200 mg/kg (CS-Li low dose group, L-CS-Li group), and 14.8 mg/kg (LiCl group, the amount of Li was equal to that of H-CS-Li group).


## 2.5.2. Morris Water Maze (MWM)

The MWM is widely applied to study the spatial memory and learning ability of animals [27]. The MWM is a black barrel (110 cm in diameter and 50 cm high) with a camera connecting to a computer to get the swimming pathway of mice. The water in the pool was dyed with nontoxic white dye, and the temperature was maintained at 20±2°C. The pool was divided into four quadrants, and a circular platform (diameter 12 cm) was placed 1 cm below the water surface in the fourth quadrant. The MWM experiment included three continuous phases: initial training (IT), navigation test (NT), and space exploring test (SET). During the IT stage, the mouse was placed into water with the head facing the wall at four different positions, to find the hidden platform within 60 s. If the mouse was able to reach the platform and stayed for 5 s, the test was completed. If the mouse could not find the platform within 60 s, it was guided to the platform and trained to stay on the platform for 10 s, and the time was recorded as 60 s. The mice were trained four sessions every day for 5 days. In the NT phase, the time that each mouse spent in finding and climbing the platform was recorded as the escape latency. The SET was started 24 h after the IT stage. After the platform was removed, the mouse was released in the water at the third quadrant and allowed to swim freely for 60 s. The time spent in the targeted quadrant and the number of platform crossings during the process were recorded.


## 2.5.3. Novel Object Recognition (NOR)

The NOR test is applied to estimate the memory ability of the previously familiar objects as well as the visual recognition, learning, and memory ability of the novel items of rodents [28]. The NOR experiment system mainly includes a box (52cm×52cm×52cm) for free activity of mice and a video recording device. In the first stage of the NOR test, two objects (A1, A2) with similar size and same material were put into the box, on the diagonal line with 30 cm apart. The mouse explored the box freely for 5 min, and the time spent on exploring objects A1 (tA1) and A2 (tA2) was recorded. In the second stage, a novel object B is placed in the box to replace the object A2, and the time spent on exploring each object (tA1 and tB) was recorded. The preference index (PI) of each mouse was calculated to evaluate the learning and memory ability according to the following formula:
(2)PI=tBtA1+tB.


## 2.6. Mechanism Study of CS-Li Exhibiting Anti-AD Effect in Mice

### 2.6.1. Transmission Electron Microscope of Hippocampus Microstructure

After the above behavioral test, the mice were anaesthetized and perfused with 0.1 mol/L precooled phosphate-buffer saline for 5 min and 4% precooled paraformaldehyde until the mice were stiff. Then the hippocampi were extracted and fixed with 3% precooled glutaraldehyde solution for 6 h, and then were cut into 0.5~1 mm3 blocks. The samples were postfixed in 1% osmium tetroxide for 60 min, followed by dehydration in acetone, epoxy resin embedding, and ultrathin section. After stained with lead citrate and uranyl acetate, the ultrathin sections of the cured blocks were visualized by JEM-1200EXII TEM.


### 2.6.2. Hematoxylin and Eosin (H&E) Staining, Nissl Staining, and TUNEL Staining

The mice were sacrificed and the brains were isolated, then the brains were embedded in paraffin and were cut into 6μm thin slices. The slices were processed for H&E staining, Nissl staining, and TUNEL staining. After that, the sections were imaged and analyzed by microscopy.


### 2.6.3. Biochemical Parameters Analysis

The hippocampi were isolated as described in Part 2.6.1. Then the hippocampi were homogenized in normal saline containing protease inhibitors. The homogenate was centrifuged at 2500 rpm for 10 min, and the protein concentration in supernatant was measured by BCA method. The level of MDA and the activities of GSH-Px, ChAT, AchE, and Na+K+-ATPase were determined by relevant assay kits.


### 2.6.4. Western Blot Analysis

Protein contents (35-40μg) were separated by 10% SDS-PAGE and electrotransferred onto PVDF membranes. The membrane was blocked with TBST blocking solution for 1 h, and then incubated with primary antibody overnight at 4°C on a rotating shaker, followed by incubation with HRP conjugated secondary antibody for 2 h. Then the protein bands were detected using enhanced chemiluminescence kit (ECL, Millipore). The protein signal value of each target protein was determined by ImageJ software.


## 2.6.1. Transmission Electron Microscope of Hippocampus Microstructure

After the above behavioral test, the mice were anaesthetized and perfused with 0.1 mol/L precooled phosphate-buffer saline for 5 min and 4% precooled paraformaldehyde until the mice were stiff. Then the hippocampi were extracted and fixed with 3% precooled glutaraldehyde solution for 6 h, and then were cut into 0.5~1 mm3 blocks. The samples were postfixed in 1% osmium tetroxide for 60 min, followed by dehydration in acetone, epoxy resin embedding, and ultrathin section. After stained with lead citrate and uranyl acetate, the ultrathin sections of the cured blocks were visualized by JEM-1200EXII TEM.


## 2.6.2. Hematoxylin and Eosin (H&E) Staining, Nissl Staining, and TUNEL Staining

The mice were sacrificed and the brains were isolated, then the brains were embedded in paraffin and were cut into 6μm thin slices. The slices were processed for H&E staining, Nissl staining, and TUNEL staining. After that, the sections were imaged and analyzed by microscopy.


## 2.6.3. Biochemical Parameters Analysis

The hippocampi were isolated as described in Part 2.6.1. Then the hippocampi were homogenized in normal saline containing protease inhibitors. The homogenate was centrifuged at 2500 rpm for 10 min, and the protein concentration in supernatant was measured by BCA method. The level of MDA and the activities of GSH-Px, ChAT, AchE, and Na+K+-ATPase were determined by relevant assay kits.


## 2.6.4. Western Blot Analysis

Protein contents (35-40μg) were separated by 10% SDS-PAGE and electrotransferred onto PVDF membranes. The membrane was blocked with TBST blocking solution for 1 h, and then incubated with primary antibody overnight at 4°C on a rotating shaker, followed by incubation with HRP conjugated secondary antibody for 2 h. Then the protein bands were detected using enhanced chemiluminescence kit (ECL, Millipore). The protein signal value of each target protein was determined by ImageJ software.


## 2.7. Statistical Analysis

The quantitative data were expressed asmean±SD, and the statistical significance between different groups was analyzed by Student’s t-test one-way analysis of variance (ANOVA). Difference in mean with p<0.05 was considered to demonstrate statistical significance. Statistical analysis was evaluated using SPSS 11.0 software.


## 3. Results and Discussion

### 3.1. Preparation and Characterization of CS-Li

CS-Li was successfully prepared and characterized by FTIR,1HNMR, and ICP-MS analysis. The results of ICP-MS analysis indicated the lithium content in CS-Li was about 0.61%. 1HNMR results showed no significant difference between CS and CS-Li (Figure 1(a)), since the major structure of CS was consistent during the reaction, and only partial H+ was substituted by Li+. The FTIR results are shown in Figure 1(b). It can be observed that the wide band at 3200-3600 cm−1 was the stretching vibrations of the –OH and –NH groups, and the peak at 2930 cm−1 was the antisymmetric stretching vibrations of methyl and methylene groups. The peaks at 1647 cm−1 and 1568 cm−1 were strengthened, which might contribute to the formation of carboxylates. This result indicated that Li has been successfully bound to the carboxyl group of the CS.Figure 1
Characterization of CS and CS-Li. (a)1H NMR spectra of CS and CS-Li and (b) FTIR spectra of CS and CS-Li. (c) Cytotoxicity of CS-Li in SH-SY5Y cells: 1: control; 2: CS-Li 100 μg/mL and LiCl (amount of Li was equal to 100 μg/mL of CS-Li); 3: CS-Li 200 μg/mL and LiCl (amount of Li was equal to 200 μg/mL of CS-Li); 4: CS-Li 400 μg/mL and LiCl (amount of Li was equal to 400 μg/mL of CS-Li); 5: CS-Li 800 μg/mL and LiCl (amount of Li was equal to 800 μg/mL of CS-Li); ∗∗∗p<0.001 vs. the CS-Li group.
(a)(b)(c)The safety of CS was evaluated by SH-SY5Y cells with MTT method. The concentration of Li in CS-Li and LiCl were 0.61% and 16.37%, respectively. As shown in Figure1(c), CS-Li exhibited extremely low toxicity to the SH-SY5Y cells, even at high concentration (400 μg/mL), whereas LiCl showed relatively high toxicity at the same amount of Li (p<0.001). The result indicated CS-Li was much safer than LiCl with equivalent Li content.


### 3.2. Effect of CS-Li on Aβ1-42 Aggregation and Spatial Conformation

#### 3.2.1. Thioflavine T Fluorescence Spectroscopy Assay

Thioflavine T can bind specifically toβ-sheet in amyloid, resulting in increased fluorescence intensity. The inhibitory activity of CS-Li on Aβ1-42 aggregation could be reflected by measuring thioflavine T fluorescence anisotropy. The results are shown in Figure 2(a), the growth kinetic of Aβ1-42 was a semi-S curve when it was incubated alone, which was consisted with previous study [29]. When Aβ1-42 was incubated with CS-Li (200, 400 μg/mL), the thioflavine T fluorescence intensity decreased to 93.3% and 62.1%, respectively. LiCl (14.8 μg/mL) exhibited a weaker effect on suppressing the Aβ1-42 aggregation than that of CS-Li (400 μg/mL) with the same Li content. The above results showed CS-Li could suppress the Aβ1-42 aggregation.Figure 2
Effect of CS-Li on Aβ1-42 aggregation and spatial conformation. (a) ThT fluorescence kinetics of Aβ1-42 aggregation in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for different time (0, 10, 24, 48, 72, and 100 h). (b) Congo red-binding absorption spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (c) ANS fluorescence spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (d) CD spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (e) TEM images of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days, scalebars=500 nm. (f) Effect of CS-Li on the cell cytotoxicity of Aβ1-42 fibrils. The viability was estimated by MTT assay. ##p<0.01 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the Aβ1-42 group.
(a)(b)(c)(d)(e)(f)


#### 3.2.2. Congo Red-Binding Test

The purpose of the Congo red-binding test was to evaluate the suppressing effect of CS-Li on the Aβ1-42 aggregation. As shown in Figure 2(b), in the range of 400-600 nm spectrum, the absorbance of Congo red increased significantly after binding with Aβ1-42, and the absorption peak was red-shifted. After incubation with CS-Li and LiCl, Aβ1-42 binding with Congo red displayed a marked dose-dependent decrease in the absorbance. Combined with thioflavine T assay result, CS-Li exhibited a higher suppressing effect on the aggregation and fiber formation of Aβ1-42 aggregation and fiber formation than that of LiCl, and the suppressing effect was proportional to the CS-Li concentration.


#### 3.2.3. ANS-Binding Assay

ANS fluorescence microscopy can bind to the hydrophobic region of Aβ1-42 to produce specific fluorescence. The fluorescence intensity of ANS was often used to investigate the influence of drugs on the hydrophobicity of Aβ1-42. The results are shown in Figure 2(c), after Aβ1-42 was treated with CS-Li (200, 400 μg/mL), the ANS fluorescence intensity decreased from (579.1±8.3) to (488.1±2.2) and (464.0±0.9), respectively. While LiCl at 14.8 μg/mL showed no significant influence on the ANS fluorescence intensity, these results indicated the hydrophobic region of Aβ1-42 was reduced after incubation with CS-Li. CS-Li had the ability to improve the water solubility of Aβ1-42 and inhibit Aβ1-42 fiber aggregation.


#### 3.2.4. Circular Dichroism Assay

Circular dichroism (CD) is a fast technique to analyze the conformation of nucleic acids and proteins [30] and was used to evaluate the influence of CS-Li on the Aβ1-42 secondary structure. The results are shown in Figure 2(d), the structure of freshly prepared Aβ1-42 displayed a typical random coil form [15]. After incubation for 72 h, the CD spectrum of Aβ1-42 exhibited obvious structure transition from random coil to β-sheet-rich oligomeric form, and the maximum negative value of the spectrum appeared at 213 nm. After treated with 200 μg/mL of CS-Li, Aβ1-42 showed a coli form similar to that of the incubated Aβ1-42, but the intensity of peak at 213 nm was obvious reduced. However, after Aβ1-42 was treated with 400 μg/mL of CS-Li, Aβ1-42 exhibited almost no β-sheet and showed a random coli form similar to that of Aβ1-42 without incubation. The soluble Aβ oligomer has been reported to be the main form of Aβ protein to induce neurotoxicity. These results suggested that CS-Li at 400 μg/mL could prevent the transition Aβ1-42 from random coils to β-sheet crystals, indicating CS-Li was able to suppress the formation of Aβ oligomer and attenuate Aβ-related neurotoxicity.


#### 3.2.5. Ultrastructural Analysis of Aβ1-42 Fibril Morphology

To further find out whether CS-Li could inhibit Aβ1-42 aggregation, TEM was applied to study the effect of CS-Li on Aβ1-42 fibril ultrastructure. As shown in Figure 2(e), Aβ1-42 formed long and bifurcated fibrils after incubated for 72 h alone, which was one of the most basic structure of amyloids [31]. When incubated with CS-Li at 200 μg/mL for 72 h, the fibers of Aβ1-42 were obviously shortened, but the visible amorphous oligomers could still be observed. When CS-Li increased to 400 μg/mL, the long fibers and visible amorphous oligomers of Aβ1-42 decreased significantly, and the aggregates were fragment and almost no fiber structure was observed. However, the Aβ1-42 maintained fibrillar structure without obvious shortening after incubation with LiCl.


#### 3.2.6. Effect of CS-Li on Aβ1-42-Induced Damage of SH-SY5Y

Aβ deposition-induced neuronal damage is believed as a main reason in the pathogenesis of AD [32]. After being treated with Aβ1-42 (20 μM) for 24 h, the cells viability reduced to 73.52±2.26% compared with the control group (Figure 2(f)), indicating the cytotoxicity of Aβ1-42 on cells. After pretreated Aβ1-42 with CS-Li (200, 400 μg/mL) for 12 h, the cell viability increased to 89.96±6.20% and 97.27±5.31% (p<0.05 and p<0.01, respectively). Therefore, CS-Li could inhibit Aβ1-42-induced damage of SH-SY5Y cells, indicating the potential cytoprotective activity of CS-Li on Aβ-induced neuronal cell injury.


### 3.3. Effect of CS-Li on the Learning and Memory Dysfunction in AD Mice

To investigate whether CS-Li was able to improve the dysfunction in learning and memory of AD mice, MWM and NOR tests were designed and performed to study the spatial memory and learning ability. The results of positioning and navigation experiments are shown in Figure3(a). Compared to the control group, the escape latency of the model group was prolonged significantly from the 4th day D-gal/AlCl3 treatment (p<0.01), indicating the spatial memory deficit in model AD mice. With the extension of MWM training time, the escape latency showed no obviously change in model group, whereas the escape latencies were decreased significantly in control, H-CS-Li, L-CS-Li, and LiCl groups. On the fourth and fifth day, the escape latencies were reduced obviously in H-CS-Li (p<0.01), L-CS-Li (p<0.05), and LiCl (p<0.05), respectively.Figure 3
Effects of CS-Li on learning and memory abilities in AD mice induced by D-gal/AlCl3. (a) Escape latency determination in the positioning and navigation experiment. (b) Number of platform crossings in the spatial exploration experiment. (c) Time spent in target quadrant during the spatial exploration experiment. (d) Representative trajectory in the spatial exploration experiment. (e) Preference index in the novel object recognition experiment. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.
(a)(b)(c)(d)(e)After five days of navigation test, the platform was dismantled to give way to the space exploring test. As displayed in Figure3(b) and Figure 3(c), compared with the control group, the time of latency to find the targeted quadrant was increased and the number of platform crossings during the process was reduced significantly in model group (p<0.001, p<0.01). Upon treatment with CS-Li and LiCl, the time spent to find the targeted quadrant was decreased in the H-CS-Li group (p<0.001), L-CS-Li group (p<0.001), and LiCl group (p<0.01) compared with the model group. Meanwhile, the number of platform crossings was also raised in the H-CS-Li group (p<0.001), L-CS-Li group (p<0.001), and LiCl group (n.s.) compared to the model group. The swimming distance in target quadrant was apparently shorter in the model group than that in the control group, as presented in Figure 3(d). After 30 days of treatment with CS-Li, the distance was prolonged markedly in the H-CS-Li and L-CS-Li groups than that in the model group. These results indicated CS-Li has the potential to improve the spatial memory deficiency induced by combination of D-gal/AlCl3.NOR test is a valuable measure of cognition to assess nonspatial working ability of rodents [33]. As displayed in Figure 3(e), compared to the control group, the times of new object touching behavior was reduced, indicating the cognitive ability was decreased significantly (p<0.05) in the model group. After CS-Li and LiCl treatment, the time of new object touching behavior was increased obviously in the H-CS-Li groups compare with the model group (p<0.01), whereas LiCl showed no significant improvement on the recognition ability of the mice.All these results demonstrated both CS-Li and LiCl could improve the learning and memory abilities of mouse at the tested concentrations, and H-CS-Li showed better effect than that of LiCl with the same Li content.


### 3.4. Effect of CS-Li on Hippocampal Neurons in AD Mice Induced by D-Gal/AlCl3

#### 3.4.1. H&E Staining

The effect of CS-Li on the neuronal injury in AD mice was probed by H&E staining test to observe the morphological change of neurons in hippocampus, which reflected the damage of hippocampal tissue. Figure4 shows the H&E staining of CA1-CA4 and DG regions in the hippocampus of mice. It was found that the neurocytes in CA1 region were well-arranged and structurally intact in the control group, and the nucleoli were obvious, without dense staining and pyknosis (red arrows). On the other hand, in the model group, the cell nucleus in CA1 region were obviously pyknotic (blue arrows), and some cells were edematous (green arrows). The cell width in CA1 region was significantly reduced with the disordered arrangement and neuron loss. Meanwhile, the cells in the model group were pyknotic, stained deeply, and exhibited disorderly arrangement, and some neurons were found to have undergone apoptosis in CA2, CA3, and CA4 regions. There is also an obvious wide edema zone in the DG region in the model group. These results indicated D-gal/AlCl3 had damaged the hippocampal region of the mice. After treatment with CS-Li, the neurons in CA1-CA4 regions of hippocampus were arranged tightly and orderly, the morphology was improved, the cell edema and pyknosis were reduced, and the number of neurons was increased. The edema zone of DG region was also significantly reduced compared with the model group. The hippocampal damage of AD mice was also reversed in LiCl group, but the reversal effect was not as obvious as that of H-CS-Li and L-CS-Li groups. These results suggested CS-Li could reverse and alleviate the damage of hippocampal neurons in AD mice induced by D-gal/AlCl3.Figure 4
Histopathological staining showing hippocampal morphology in AD mice (×100, ×400): normal neurocytes (red arrows), pyknosis (blue arrows), and cell edema (green arrows). CS-Li reduced the cell edema and pyknosis and increased the number of neurons in the hippocampus of AD mice.Scalebar=100 μm and 25 μm.


#### 3.4.2. Nissl Staining

The Nissl body in neuron is the main site to synthesize proteins and plays significant roles in neuron excitation and conduction [34]. Nissl staining was used to evaluate the damage of Nissl body in hippocampus. As shown in Figure 5, the neurons of mice in the control group were morphologically normal with distinct hierarchical structure and regular arrangement, and the Nissl bodies were abundant in CA1-CA4 and DG regions. While in model group, the neurons in CA1 and DG regions were pyknotic and shaped into diamond and triangle. Compared with the control group, the number of Nissl bodies (purple arrows) was reduced largely in model group. After CS-Li treatment, the hippocampal neurons were arranged neatly and the Nissl body reappeared in CA1-CA4 regions. The results demonstrated that CS-Li could maintain neuronal cell morphology and improve neuronal protein synthesis function in AD mice, and the effect was more significant than that of LiCl.Figure 5
Nissl staining showing hippocampal morphology in AD mice (×100, ×400): Nissl bodies (purple arrows) and nucleoli (red arrows). CS-Li increased the number of Nissl body in the hippocampus of AD mice.Scalebar=100 μm and 25 μm.


#### 3.4.3. TUNEL Staining

TUNEL staining was performed to examine the effect of CS-Li on suppressing the apoptosis of hippocampal neurons in AD mice. The apoptotic cell nuclei with positive expression appeared brownish. The results are shown in Figure6, TUNEL-positive cells appeared in the hippocampus of mice in the control group, and the apoptosis rates in the C1-C4 and DG regions were about 56.9±3.8%, 55.6±6.0%, 69.3±7.1%, 52.9±3.6%, and 49.7±2.2%, while the neurons in the hippocampus of mice were arranged regularly, and the nuclei were clear (red arrows) with no apoptosis in the CA1-CA4 and DG regions. While in the model group, TUNEL-positive cells increased largely in hippocampus, the apoptosis rate in the C1-C4 and DG regions were about 73.6±7.3%, 71.8±4.1%, 71.7±5.4%, 65.9±5.7%, and 67.7±6.0% (p<0.05, p<0.01, p>0.05, p<0.05, and p<0.01), the nucleoli disappeared, the cell gap was widened, and some nerve cells were degenerated by apoptosis. After CS-Li treatment, the apoptosis rate of neurons significantly decreased in CA1-CA4 regions (p<0.05, p<0.05, p<0.05, p<0.05, and p<0.05), and the edema in DG region was also ameliorated (green arrows). The neuronal apoptosis of AD mice was also reversed in LiCl group, but the reversal effect was not as obvious as that of H-CS-Li group. The TUNEL staining results demonstrated that CS-Li could reduce apoptosis of nerve cells in AD mice.Figure 6
TUNEL staining showing hippocampal morphology in AD mice (×100, ×400): nucleus (red arrows), normal neurocytes (purple arrows), cell edema (green arrows), and pyknosis (blue arrows).Scalebar=100 μm and 25 μm. The number of positive cells in the CA1, CA2, CA3, CA4, and DG regions (a, b, c, d, e, and f) of hippocampal cells was counted under a light microscope and quantified as percentage of total neuron cells among different groups (n=3 per group). CS-Li reduced the apoptosis of nerve cells in the hippocampus of AD mice. #p<0.05 and ##p<0.01 vs. the control group; ∗p<0.05 vs. the model group.
(a)(b)(c)(d)(e)(f)


#### 3.4.4. Effect of CS-Li on the Ultrastructure in the Hippocampi of Mice

To study the ultrastructural changes in hippocampi of AD mice, TEM was applied to examine the ultrastructure of hippocampal neurons, the number of synapses, and the microstructure of mitochondria. The results are shown in Figure7(a), the synapse exhibited damaged structure, decreased density, shortening, and flattening. After treatment with H-CS-Li, the synaptic density of hippocampal neurons was increased (blue arrows), and the damage of synaptic structure was improved significantly. The mitochondrial microstructure of hippocampal neurons in mice in Figure 7(b) shows that the mitochondria of hippocampal neurons displayed obvious cristae degeneration (yellow arrows) and membrane rupture (green arrows). Compared with the model group, the mitochondrial structure of hippocampal neurons was relatively intact (red arrows), and the membrane rupture caused by mitochondrial swelling was improved in H-CS-Li group. The TEM results suggested that CS-Li can reduce the damage of hippocampal mitochondria, improve the abnormal changes of mitochondrial ultrastructure, protect hippocampal neurons, and increase the survival of neurons in AD mice.Figure 7
TEM images of neuron mitochondria and synapses in the hippocampi of mice: synaptic density of hippocampal neurons (blue arrows), mitochondria (red arrows), cristae degeneration (yellow arrows), and membrane disruption (green arrows). CS-Li increased the synaptic density of hippocampal neurons and improved the damage of hippocampal mitochondria in AD mice.Scalebar=200 nm.
(a)(b)


### 3.5. Mechanism Study of CS-Li Exhibiting Anti-AD Effect in Mice

#### 3.5.1. Effect of CS-Li on Oxidative Stress in the Hippocampi of Mice

Oxidative stress is the imbalance between the generation of reactive oxygen and nitrogen species. Studies have demonstrated that oxidative stress is a major factor in AD pathogenesis [35]. In this study, we measured the levels of GSH-Px and end product of oxidation MDA, and the results are shown in Figures 8(a) and 8(b). Compared to the control group, the activity of GSH-Px was decreased, while the level of MDA was increased obviously in hippocampus of AD mice in the model group (p<0.05 and p<0.05, respectively), indicating D-gal/AlCl3 could induce the peroxidation of lipid in hippocampus of AD mice. After CS-Li and LiCl treatment, the activity of GSH-Px was increased appreciably in the H-CS-Li, L-CS-Li, and LiCl groups than that in the model group (p<0.001, p<0.01, and p<0.001, respectively), and the level of MDA was decreased in H-CS-Li, L-CS-Li, and LiCl markedly compared with the model group (p<0.01, p<0.01, and p<0.01). These results showed that both CS-Li and LiCl at the tested concentration could improve the activity of GSH-Px and inhibit the level of MDA, and further protect the brain from oxidative damage generated by free radicals.Figure 8
Effects of CS-Li on oxidative stress in the hippocampi of mice. CS-Li decreased the levels of MDA (a) and AchE (e) and increased the activities of GSH-Px (b), Na+/K+-ATPase (c), and ChAT (d) in the hippocampi of mice.#p<0.05 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.
(a)(b)(c)(d)(e)Na+/K+-ATPase plays important roles in ATP hydrolysis, Na+/K+ exchange, and transmembrane fluxes of Ca2+ and excitatory neurotransmitter. Oxidative metabolism is very active in brain and a large amount of chemical energy as ATP is consumed [36]. Studies have indicated that Na+/K+-ATPase activity is significantly lower in AD brains than that in age-matched control brains, and inhibition of Na+/K+-ATPase activity impairs learning and memory abilities [37]. Figure 8(c) shows the activity of Na+/K+-ATPase in the model group decreased largely (p<0.05), indicating D-gal/AlCl3 could induce disorders of energy metabolism in mouse hippocampi. After drug administration, the activities of Na+/K+-ATPase were remarkably increased in the H-CS-Li, L-CS-Li, and LiCl groups (p<0.001, p<0.001, and p<0.001) compared with the model group. The results suggest that CS-Li and LiCl could increase Na+/K+-ATPase activity and energy metabolism in hippocampal cells in mice brain and reduce edema and cell death at CNS level.


#### 3.5.2. Effect of CS-Li on the Activity of Cholinesterase in the Hippocampi of Mice

The cholinergic system plays crucial roles in neurological and cognitive function. Acetyl choline (ACh) is a neurotransmitter and the increase of ACh level in brain can improve the symptoms of AD. The dynamic equilibrium of ACh is regulated by ChAT and AchE, which is responsible for ACh synthesis and degradation, respectively [38]. Figure 8(d) shows the activity of ChAT was significantly lower in the model group than that in the control group (p<0.05), suggesting D-gal/AlCl3 could inhibit the synthesis of ACh, decrease the cholinergic system function, and further influence the learning and memory abilities of mice. After CS-Li and LiCl treatment, the activities of ACh in the H-CS-Li, L-CS-Li, and LiCl groups were generally improved compared with the model group (p<0.001, p<0.001, and p<0.01).AchE inhibitors have been developed as the first line therapy for AD, such as donepezil, galantamine, and rivastigmine [39]. As shown in Figure 8(e), the activity of AchE in the model mice was higher than that of the control group (p<0.05). The results indicated D-gal and AlCl3 could increase the activity of AchE in mice brain, finally resulting in the reduction in ACh level. The activity of AchE in the hippocampus of H-CS-Li, L-CS-Li, and LiCl groups was decreased significantly compared with the model group (p<0.001, p<0.001, and p<0.001). The above results indicated that both CS-Li and LiCl at the test concentrations could influence the activity of ChAT and AchE in the brain of AD mice, further affecting the balance of ACh and improve the learning and memory abilities in AD mice.


#### 3.5.3. Effect of CS-Li on Tau Hyperphosphorylation in the Hippocampi of Mice

Tau plays important roles in promoting assembly and stability of microtubule [40]. In the pathogenesis of AD, hyperphosphorylation of tau deprives it from the physiological function of microtubule binding and further destroys the neuronal cytoskeleton, causing aggregation to form fibrillary tangles, and eventually leading to neuronal degeneration and damage [41]. Therefore, the phosphorylation of tau has been recognized as the international gold standard to evaluate AD progression [42]. The phosphorylation sites Ser396 and Ser404 are reported to be involved in tau aggregation [43], and Western blot results indicated that the expression of tau (Ser396) and tau(Ser404) in model group was generally higher than that of the control group (p<0.05 and p<0.001) (Figure 9). After CS-Li treatment, the expression levels of tau (Ser396) and tau (Ser404) were reduced obviously in the H-CS-Li group (p<0.05 and p<0.01). The results which disclosed CS-Li at 400 μg/mL could inhibit the hyperphosphorylation of tau at Ser396 and Ser404 sites, while L-CS-Li and LiCl showed little effect on the hyperphosphorylation of tau at both sites.Figure 9
Effects of CS-Li on tau phosphorylation in the hippocampi of mice. Western blotting of GSK-3β, p-GSK-3β (Ser9), p-tau (Ser396), p-tau (Ser404), and tau, GAPDH was used as the internal control; Western blotting of PP2A, β-actin was used as the internal control. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.The regulation of tau phosphorylation depends on several protein kinases and phosphatases, among which GSK-3β and PP2A act a pivotal part in tau hyperphosphorylation [44]. Both higher level of GSK-3β and reduced activity of PP2A in tangles bearing neurons have been found in AD patient brains [45]. Hyperphosphorylation of tau and cognitive impairment can be alleviated by inhibiting GSK-3β activity and increasing PP2A activity [46, 47]. To gain insight into the mechanism of CS-Li in regulating tau hyperphosphorylation, Western blot assay was performed to examine the effect of CS-Li on the expression of GSK-3β and PP2A in AD mice. The results shown in Figure 9 suggest that the expression of p-GSK-3β (Ser9) was increased in the model group compared with the control group (p<0.001), whereas the expression of GSK-3β showed no obvious difference between the two groups, indicating phosphorylation of GSK-3β at Ser9 site had been induced by D-gal/AlCl3. The expression of PP2A in the model group was nearly the same as that of the control group. After treatment with CS-Li and LiCl, the GSK-3β phosphorylation was reduced significantly in the L-CS-Li, H-CS-Li, and LiCl groups compared with the model group (p<0.001, p<0.001, and p<0.01, respectively). In addition, the expression of PP2A in H-CS-Li group was increased significantly compared with the model group (p<0.05), showing better results than the L-CS-Li and LiCl groups. The above results demonstrated CS-Li could reduce the tau protein hyperphosphorylation at Ser396 and Ser404 sites by regulating the phosphorylation of GSK-3β at Ser9 site and the activity of PP2A.


#### 3.5.4. Effect of CS-Li on Inflammatory Responses in the Hippocampi of Mice

Neuroinflammation is associated with neurodegenerative diseases, including AD. The proinflammatory cytokine levels in serum are elevated both in human and experimental models [48]. Proinflammatory cytokines can activate the microglia to express proinflammatory factors such as IL-6, IL-1β, TNF-α, nitric oxide, and proteases, to exert detrimental effects in neuropathy. To explore the effect of CS-Li in regulating neuroinflammatory response, Western blot assay was preformed to study the effect of CS-Li on the expression of TNF-α, IL-6, and IL-1β in hippocampus of AD mice. As shown in Figure 10, the levels of IL-6, IL-1β, and TNF-α were generally higher in the model group than those of the control group (p<0.05, p<0.01, and p<0.05, respectively). Compared with the model group, the expressions of IL-6, IL-1β, and TNF-α were all decreased obviously in the H-CS-Li, L-CS-Li, and CS-Li groups (p<0.05 or p<0.01). These results suggested that both CS-Li and LiCl can inhibit the expressions of proinflammatory cytokines in the hippocampi of AD mice, indicating CS-Li and LiCl exert neuroprotective effect by suppressing hippocampal inflammatory responses.Figure 10
Effects of CS-Li on inflammatory responses in the hippocampi of mice. Western blotting of IL-6, IL-1β, and TNF-α, β-actin was used as the internal control; Western blotting of p-Erk1/2 and p-38, GAPDH was used as the internal control; Western blotting of p-NF-κB, IκBα, and p-IκBα, Lamin B was used as the internal control. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.The activation of inflammatory response can be mediated by multiple signaling pathways including MAPK and NF-κB [49]. To figure out whether CS-Li could regulate the expression of proinflammatory cytokines through MAPK/NF-κB signaling pathway in AD mice, Western blot was used to analyze the expressions of p-NF-κB p65, p-Erk, p-p38, IκBa, and p-IκBa in hippocampal of AD mice. As displayed in Figure 10, the expression of p-Erk1/2 in H-CS-Li group was decreased significantly compared with the model group (p<0.05); the expression of p-p38 was reduced obviously in H-CS-Li, L-CS-Li, and LiCl groups (p<0.01, p<0.01, and p<0.01). The expression of p-NF-κB p65 and p-IκBa in H-CS-Li groups were decreased compared with the model group (p<0.05 and p<0.01), and the expression of IκBa in H-CS-Li group was increased obviously compared with the model group (p<0.05). The results indicated that CS-Li could activate MAPK pathway through p38 and Erk1/2 signaling, and then suppress the degradation of IκBa, further regulating the nuclear translocation of NF-κB, eventually inhibiting the expressions of IL-6, IL-1β, and TNF-α.


## 3.1. Preparation and Characterization of CS-Li

CS-Li was successfully prepared and characterized by FTIR,1HNMR, and ICP-MS analysis. The results of ICP-MS analysis indicated the lithium content in CS-Li was about 0.61%. 1HNMR results showed no significant difference between CS and CS-Li (Figure 1(a)), since the major structure of CS was consistent during the reaction, and only partial H+ was substituted by Li+. The FTIR results are shown in Figure 1(b). It can be observed that the wide band at 3200-3600 cm−1 was the stretching vibrations of the –OH and –NH groups, and the peak at 2930 cm−1 was the antisymmetric stretching vibrations of methyl and methylene groups. The peaks at 1647 cm−1 and 1568 cm−1 were strengthened, which might contribute to the formation of carboxylates. This result indicated that Li has been successfully bound to the carboxyl group of the CS.Figure 1
Characterization of CS and CS-Li. (a)1H NMR spectra of CS and CS-Li and (b) FTIR spectra of CS and CS-Li. (c) Cytotoxicity of CS-Li in SH-SY5Y cells: 1: control; 2: CS-Li 100 μg/mL and LiCl (amount of Li was equal to 100 μg/mL of CS-Li); 3: CS-Li 200 μg/mL and LiCl (amount of Li was equal to 200 μg/mL of CS-Li); 4: CS-Li 400 μg/mL and LiCl (amount of Li was equal to 400 μg/mL of CS-Li); 5: CS-Li 800 μg/mL and LiCl (amount of Li was equal to 800 μg/mL of CS-Li); ∗∗∗p<0.001 vs. the CS-Li group.
(a)(b)(c)The safety of CS was evaluated by SH-SY5Y cells with MTT method. The concentration of Li in CS-Li and LiCl were 0.61% and 16.37%, respectively. As shown in Figure1(c), CS-Li exhibited extremely low toxicity to the SH-SY5Y cells, even at high concentration (400 μg/mL), whereas LiCl showed relatively high toxicity at the same amount of Li (p<0.001). The result indicated CS-Li was much safer than LiCl with equivalent Li content.


## 3.2. Effect of CS-Li on Aβ1-42 Aggregation and Spatial Conformation

### 3.2.1. Thioflavine T Fluorescence Spectroscopy Assay

Thioflavine T can bind specifically toβ-sheet in amyloid, resulting in increased fluorescence intensity. The inhibitory activity of CS-Li on Aβ1-42 aggregation could be reflected by measuring thioflavine T fluorescence anisotropy. The results are shown in Figure 2(a), the growth kinetic of Aβ1-42 was a semi-S curve when it was incubated alone, which was consisted with previous study [29]. When Aβ1-42 was incubated with CS-Li (200, 400 μg/mL), the thioflavine T fluorescence intensity decreased to 93.3% and 62.1%, respectively. LiCl (14.8 μg/mL) exhibited a weaker effect on suppressing the Aβ1-42 aggregation than that of CS-Li (400 μg/mL) with the same Li content. The above results showed CS-Li could suppress the Aβ1-42 aggregation.Figure 2
Effect of CS-Li on Aβ1-42 aggregation and spatial conformation. (a) ThT fluorescence kinetics of Aβ1-42 aggregation in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for different time (0, 10, 24, 48, 72, and 100 h). (b) Congo red-binding absorption spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (c) ANS fluorescence spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (d) CD spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (e) TEM images of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days, scalebars=500 nm. (f) Effect of CS-Li on the cell cytotoxicity of Aβ1-42 fibrils. The viability was estimated by MTT assay. ##p<0.01 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the Aβ1-42 group.
(a)(b)(c)(d)(e)(f)


### 3.2.2. Congo Red-Binding Test

The purpose of the Congo red-binding test was to evaluate the suppressing effect of CS-Li on the Aβ1-42 aggregation. As shown in Figure 2(b), in the range of 400-600 nm spectrum, the absorbance of Congo red increased significantly after binding with Aβ1-42, and the absorption peak was red-shifted. After incubation with CS-Li and LiCl, Aβ1-42 binding with Congo red displayed a marked dose-dependent decrease in the absorbance. Combined with thioflavine T assay result, CS-Li exhibited a higher suppressing effect on the aggregation and fiber formation of Aβ1-42 aggregation and fiber formation than that of LiCl, and the suppressing effect was proportional to the CS-Li concentration.


### 3.2.3. ANS-Binding Assay

ANS fluorescence microscopy can bind to the hydrophobic region of Aβ1-42 to produce specific fluorescence. The fluorescence intensity of ANS was often used to investigate the influence of drugs on the hydrophobicity of Aβ1-42. The results are shown in Figure 2(c), after Aβ1-42 was treated with CS-Li (200, 400 μg/mL), the ANS fluorescence intensity decreased from (579.1±8.3) to (488.1±2.2) and (464.0±0.9), respectively. While LiCl at 14.8 μg/mL showed no significant influence on the ANS fluorescence intensity, these results indicated the hydrophobic region of Aβ1-42 was reduced after incubation with CS-Li. CS-Li had the ability to improve the water solubility of Aβ1-42 and inhibit Aβ1-42 fiber aggregation.


### 3.2.4. Circular Dichroism Assay

Circular dichroism (CD) is a fast technique to analyze the conformation of nucleic acids and proteins [30] and was used to evaluate the influence of CS-Li on the Aβ1-42 secondary structure. The results are shown in Figure 2(d), the structure of freshly prepared Aβ1-42 displayed a typical random coil form [15]. After incubation for 72 h, the CD spectrum of Aβ1-42 exhibited obvious structure transition from random coil to β-sheet-rich oligomeric form, and the maximum negative value of the spectrum appeared at 213 nm. After treated with 200 μg/mL of CS-Li, Aβ1-42 showed a coli form similar to that of the incubated Aβ1-42, but the intensity of peak at 213 nm was obvious reduced. However, after Aβ1-42 was treated with 400 μg/mL of CS-Li, Aβ1-42 exhibited almost no β-sheet and showed a random coli form similar to that of Aβ1-42 without incubation. The soluble Aβ oligomer has been reported to be the main form of Aβ protein to induce neurotoxicity. These results suggested that CS-Li at 400 μg/mL could prevent the transition Aβ1-42 from random coils to β-sheet crystals, indicating CS-Li was able to suppress the formation of Aβ oligomer and attenuate Aβ-related neurotoxicity.


### 3.2.5. Ultrastructural Analysis of Aβ1-42 Fibril Morphology

To further find out whether CS-Li could inhibit Aβ1-42 aggregation, TEM was applied to study the effect of CS-Li on Aβ1-42 fibril ultrastructure. As shown in Figure 2(e), Aβ1-42 formed long and bifurcated fibrils after incubated for 72 h alone, which was one of the most basic structure of amyloids [31]. When incubated with CS-Li at 200 μg/mL for 72 h, the fibers of Aβ1-42 were obviously shortened, but the visible amorphous oligomers could still be observed. When CS-Li increased to 400 μg/mL, the long fibers and visible amorphous oligomers of Aβ1-42 decreased significantly, and the aggregates were fragment and almost no fiber structure was observed. However, the Aβ1-42 maintained fibrillar structure without obvious shortening after incubation with LiCl.


### 3.2.6. Effect of CS-Li on Aβ1-42-Induced Damage of SH-SY5Y

Aβ deposition-induced neuronal damage is believed as a main reason in the pathogenesis of AD [32]. After being treated with Aβ1-42 (20 μM) for 24 h, the cells viability reduced to 73.52±2.26% compared with the control group (Figure 2(f)), indicating the cytotoxicity of Aβ1-42 on cells. After pretreated Aβ1-42 with CS-Li (200, 400 μg/mL) for 12 h, the cell viability increased to 89.96±6.20% and 97.27±5.31% (p<0.05 and p<0.01, respectively). Therefore, CS-Li could inhibit Aβ1-42-induced damage of SH-SY5Y cells, indicating the potential cytoprotective activity of CS-Li on Aβ-induced neuronal cell injury.


## 3.2.1. Thioflavine T Fluorescence Spectroscopy Assay

Thioflavine T can bind specifically toβ-sheet in amyloid, resulting in increased fluorescence intensity. The inhibitory activity of CS-Li on Aβ1-42 aggregation could be reflected by measuring thioflavine T fluorescence anisotropy. The results are shown in Figure 2(a), the growth kinetic of Aβ1-42 was a semi-S curve when it was incubated alone, which was consisted with previous study [29]. When Aβ1-42 was incubated with CS-Li (200, 400 μg/mL), the thioflavine T fluorescence intensity decreased to 93.3% and 62.1%, respectively. LiCl (14.8 μg/mL) exhibited a weaker effect on suppressing the Aβ1-42 aggregation than that of CS-Li (400 μg/mL) with the same Li content. The above results showed CS-Li could suppress the Aβ1-42 aggregation.Figure 2
Effect of CS-Li on Aβ1-42 aggregation and spatial conformation. (a) ThT fluorescence kinetics of Aβ1-42 aggregation in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for different time (0, 10, 24, 48, 72, and 100 h). (b) Congo red-binding absorption spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (c) ANS fluorescence spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (d) CD spectra of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days. (e) TEM images of Aβ1-42 in absence and presence of CS-Li (200 μg/mL, 400 μg/mL) at 37°C for 3 days, scalebars=500 nm. (f) Effect of CS-Li on the cell cytotoxicity of Aβ1-42 fibrils. The viability was estimated by MTT assay. ##p<0.01 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the Aβ1-42 group.
(a)(b)(c)(d)(e)(f)


## 3.2.2. Congo Red-Binding Test

The purpose of the Congo red-binding test was to evaluate the suppressing effect of CS-Li on the Aβ1-42 aggregation. As shown in Figure 2(b), in the range of 400-600 nm spectrum, the absorbance of Congo red increased significantly after binding with Aβ1-42, and the absorption peak was red-shifted. After incubation with CS-Li and LiCl, Aβ1-42 binding with Congo red displayed a marked dose-dependent decrease in the absorbance. Combined with thioflavine T assay result, CS-Li exhibited a higher suppressing effect on the aggregation and fiber formation of Aβ1-42 aggregation and fiber formation than that of LiCl, and the suppressing effect was proportional to the CS-Li concentration.


## 3.2.3. ANS-Binding Assay

ANS fluorescence microscopy can bind to the hydrophobic region of Aβ1-42 to produce specific fluorescence. The fluorescence intensity of ANS was often used to investigate the influence of drugs on the hydrophobicity of Aβ1-42. The results are shown in Figure 2(c), after Aβ1-42 was treated with CS-Li (200, 400 μg/mL), the ANS fluorescence intensity decreased from (579.1±8.3) to (488.1±2.2) and (464.0±0.9), respectively. While LiCl at 14.8 μg/mL showed no significant influence on the ANS fluorescence intensity, these results indicated the hydrophobic region of Aβ1-42 was reduced after incubation with CS-Li. CS-Li had the ability to improve the water solubility of Aβ1-42 and inhibit Aβ1-42 fiber aggregation.


## 3.2.4. Circular Dichroism Assay

Circular dichroism (CD) is a fast technique to analyze the conformation of nucleic acids and proteins [30] and was used to evaluate the influence of CS-Li on the Aβ1-42 secondary structure. The results are shown in Figure 2(d), the structure of freshly prepared Aβ1-42 displayed a typical random coil form [15]. After incubation for 72 h, the CD spectrum of Aβ1-42 exhibited obvious structure transition from random coil to β-sheet-rich oligomeric form, and the maximum negative value of the spectrum appeared at 213 nm. After treated with 200 μg/mL of CS-Li, Aβ1-42 showed a coli form similar to that of the incubated Aβ1-42, but the intensity of peak at 213 nm was obvious reduced. However, after Aβ1-42 was treated with 400 μg/mL of CS-Li, Aβ1-42 exhibited almost no β-sheet and showed a random coli form similar to that of Aβ1-42 without incubation. The soluble Aβ oligomer has been reported to be the main form of Aβ protein to induce neurotoxicity. These results suggested that CS-Li at 400 μg/mL could prevent the transition Aβ1-42 from random coils to β-sheet crystals, indicating CS-Li was able to suppress the formation of Aβ oligomer and attenuate Aβ-related neurotoxicity.


## 3.2.5. Ultrastructural Analysis of Aβ1-42 Fibril Morphology

To further find out whether CS-Li could inhibit Aβ1-42 aggregation, TEM was applied to study the effect of CS-Li on Aβ1-42 fibril ultrastructure. As shown in Figure 2(e), Aβ1-42 formed long and bifurcated fibrils after incubated for 72 h alone, which was one of the most basic structure of amyloids [31]. When incubated with CS-Li at 200 μg/mL for 72 h, the fibers of Aβ1-42 were obviously shortened, but the visible amorphous oligomers could still be observed. When CS-Li increased to 400 μg/mL, the long fibers and visible amorphous oligomers of Aβ1-42 decreased significantly, and the aggregates were fragment and almost no fiber structure was observed. However, the Aβ1-42 maintained fibrillar structure without obvious shortening after incubation with LiCl.


## 3.2.6. Effect of CS-Li on Aβ1-42-Induced Damage of SH-SY5Y

Aβ deposition-induced neuronal damage is believed as a main reason in the pathogenesis of AD [32]. After being treated with Aβ1-42 (20 μM) for 24 h, the cells viability reduced to 73.52±2.26% compared with the control group (Figure 2(f)), indicating the cytotoxicity of Aβ1-42 on cells. After pretreated Aβ1-42 with CS-Li (200, 400 μg/mL) for 12 h, the cell viability increased to 89.96±6.20% and 97.27±5.31% (p<0.05 and p<0.01, respectively). Therefore, CS-Li could inhibit Aβ1-42-induced damage of SH-SY5Y cells, indicating the potential cytoprotective activity of CS-Li on Aβ-induced neuronal cell injury.


## 3.3. Effect of CS-Li on the Learning and Memory Dysfunction in AD Mice

To investigate whether CS-Li was able to improve the dysfunction in learning and memory of AD mice, MWM and NOR tests were designed and performed to study the spatial memory and learning ability. The results of positioning and navigation experiments are shown in Figure3(a). Compared to the control group, the escape latency of the model group was prolonged significantly from the 4th day D-gal/AlCl3 treatment (p<0.01), indicating the spatial memory deficit in model AD mice. With the extension of MWM training time, the escape latency showed no obviously change in model group, whereas the escape latencies were decreased significantly in control, H-CS-Li, L-CS-Li, and LiCl groups. On the fourth and fifth day, the escape latencies were reduced obviously in H-CS-Li (p<0.01), L-CS-Li (p<0.05), and LiCl (p<0.05), respectively.Figure 3
Effects of CS-Li on learning and memory abilities in AD mice induced by D-gal/AlCl3. (a) Escape latency determination in the positioning and navigation experiment. (b) Number of platform crossings in the spatial exploration experiment. (c) Time spent in target quadrant during the spatial exploration experiment. (d) Representative trajectory in the spatial exploration experiment. (e) Preference index in the novel object recognition experiment. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.
(a)(b)(c)(d)(e)After five days of navigation test, the platform was dismantled to give way to the space exploring test. As displayed in Figure3(b) and Figure 3(c), compared with the control group, the time of latency to find the targeted quadrant was increased and the number of platform crossings during the process was reduced significantly in model group (p<0.001, p<0.01). Upon treatment with CS-Li and LiCl, the time spent to find the targeted quadrant was decreased in the H-CS-Li group (p<0.001), L-CS-Li group (p<0.001), and LiCl group (p<0.01) compared with the model group. Meanwhile, the number of platform crossings was also raised in the H-CS-Li group (p<0.001), L-CS-Li group (p<0.001), and LiCl group (n.s.) compared to the model group. The swimming distance in target quadrant was apparently shorter in the model group than that in the control group, as presented in Figure 3(d). After 30 days of treatment with CS-Li, the distance was prolonged markedly in the H-CS-Li and L-CS-Li groups than that in the model group. These results indicated CS-Li has the potential to improve the spatial memory deficiency induced by combination of D-gal/AlCl3.NOR test is a valuable measure of cognition to assess nonspatial working ability of rodents [33]. As displayed in Figure 3(e), compared to the control group, the times of new object touching behavior was reduced, indicating the cognitive ability was decreased significantly (p<0.05) in the model group. After CS-Li and LiCl treatment, the time of new object touching behavior was increased obviously in the H-CS-Li groups compare with the model group (p<0.01), whereas LiCl showed no significant improvement on the recognition ability of the mice.All these results demonstrated both CS-Li and LiCl could improve the learning and memory abilities of mouse at the tested concentrations, and H-CS-Li showed better effect than that of LiCl with the same Li content.


## 3.4. Effect of CS-Li on Hippocampal Neurons in AD Mice Induced by D-Gal/AlCl3

### 3.4.1. H&E Staining

The effect of CS-Li on the neuronal injury in AD mice was probed by H&E staining test to observe the morphological change of neurons in hippocampus, which reflected the damage of hippocampal tissue. Figure4 shows the H&E staining of CA1-CA4 and DG regions in the hippocampus of mice. It was found that the neurocytes in CA1 region were well-arranged and structurally intact in the control group, and the nucleoli were obvious, without dense staining and pyknosis (red arrows). On the other hand, in the model group, the cell nucleus in CA1 region were obviously pyknotic (blue arrows), and some cells were edematous (green arrows). The cell width in CA1 region was significantly reduced with the disordered arrangement and neuron loss. Meanwhile, the cells in the model group were pyknotic, stained deeply, and exhibited disorderly arrangement, and some neurons were found to have undergone apoptosis in CA2, CA3, and CA4 regions. There is also an obvious wide edema zone in the DG region in the model group. These results indicated D-gal/AlCl3 had damaged the hippocampal region of the mice. After treatment with CS-Li, the neurons in CA1-CA4 regions of hippocampus were arranged tightly and orderly, the morphology was improved, the cell edema and pyknosis were reduced, and the number of neurons was increased. The edema zone of DG region was also significantly reduced compared with the model group. The hippocampal damage of AD mice was also reversed in LiCl group, but the reversal effect was not as obvious as that of H-CS-Li and L-CS-Li groups. These results suggested CS-Li could reverse and alleviate the damage of hippocampal neurons in AD mice induced by D-gal/AlCl3.Figure 4
Histopathological staining showing hippocampal morphology in AD mice (×100, ×400): normal neurocytes (red arrows), pyknosis (blue arrows), and cell edema (green arrows). CS-Li reduced the cell edema and pyknosis and increased the number of neurons in the hippocampus of AD mice.Scalebar=100 μm and 25 μm.


### 3.4.2. Nissl Staining

The Nissl body in neuron is the main site to synthesize proteins and plays significant roles in neuron excitation and conduction [34]. Nissl staining was used to evaluate the damage of Nissl body in hippocampus. As shown in Figure 5, the neurons of mice in the control group were morphologically normal with distinct hierarchical structure and regular arrangement, and the Nissl bodies were abundant in CA1-CA4 and DG regions. While in model group, the neurons in CA1 and DG regions were pyknotic and shaped into diamond and triangle. Compared with the control group, the number of Nissl bodies (purple arrows) was reduced largely in model group. After CS-Li treatment, the hippocampal neurons were arranged neatly and the Nissl body reappeared in CA1-CA4 regions. The results demonstrated that CS-Li could maintain neuronal cell morphology and improve neuronal protein synthesis function in AD mice, and the effect was more significant than that of LiCl.Figure 5
Nissl staining showing hippocampal morphology in AD mice (×100, ×400): Nissl bodies (purple arrows) and nucleoli (red arrows). CS-Li increased the number of Nissl body in the hippocampus of AD mice.Scalebar=100 μm and 25 μm.


### 3.4.3. TUNEL Staining

TUNEL staining was performed to examine the effect of CS-Li on suppressing the apoptosis of hippocampal neurons in AD mice. The apoptotic cell nuclei with positive expression appeared brownish. The results are shown in Figure6, TUNEL-positive cells appeared in the hippocampus of mice in the control group, and the apoptosis rates in the C1-C4 and DG regions were about 56.9±3.8%, 55.6±6.0%, 69.3±7.1%, 52.9±3.6%, and 49.7±2.2%, while the neurons in the hippocampus of mice were arranged regularly, and the nuclei were clear (red arrows) with no apoptosis in the CA1-CA4 and DG regions. While in the model group, TUNEL-positive cells increased largely in hippocampus, the apoptosis rate in the C1-C4 and DG regions were about 73.6±7.3%, 71.8±4.1%, 71.7±5.4%, 65.9±5.7%, and 67.7±6.0% (p<0.05, p<0.01, p>0.05, p<0.05, and p<0.01), the nucleoli disappeared, the cell gap was widened, and some nerve cells were degenerated by apoptosis. After CS-Li treatment, the apoptosis rate of neurons significantly decreased in CA1-CA4 regions (p<0.05, p<0.05, p<0.05, p<0.05, and p<0.05), and the edema in DG region was also ameliorated (green arrows). The neuronal apoptosis of AD mice was also reversed in LiCl group, but the reversal effect was not as obvious as that of H-CS-Li group. The TUNEL staining results demonstrated that CS-Li could reduce apoptosis of nerve cells in AD mice.Figure 6
TUNEL staining showing hippocampal morphology in AD mice (×100, ×400): nucleus (red arrows), normal neurocytes (purple arrows), cell edema (green arrows), and pyknosis (blue arrows).Scalebar=100 μm and 25 μm. The number of positive cells in the CA1, CA2, CA3, CA4, and DG regions (a, b, c, d, e, and f) of hippocampal cells was counted under a light microscope and quantified as percentage of total neuron cells among different groups (n=3 per group). CS-Li reduced the apoptosis of nerve cells in the hippocampus of AD mice. #p<0.05 and ##p<0.01 vs. the control group; ∗p<0.05 vs. the model group.
(a)(b)(c)(d)(e)(f)


### 3.4.4. Effect of CS-Li on the Ultrastructure in the Hippocampi of Mice

To study the ultrastructural changes in hippocampi of AD mice, TEM was applied to examine the ultrastructure of hippocampal neurons, the number of synapses, and the microstructure of mitochondria. The results are shown in Figure7(a), the synapse exhibited damaged structure, decreased density, shortening, and flattening. After treatment with H-CS-Li, the synaptic density of hippocampal neurons was increased (blue arrows), and the damage of synaptic structure was improved significantly. The mitochondrial microstructure of hippocampal neurons in mice in Figure 7(b) shows that the mitochondria of hippocampal neurons displayed obvious cristae degeneration (yellow arrows) and membrane rupture (green arrows). Compared with the model group, the mitochondrial structure of hippocampal neurons was relatively intact (red arrows), and the membrane rupture caused by mitochondrial swelling was improved in H-CS-Li group. The TEM results suggested that CS-Li can reduce the damage of hippocampal mitochondria, improve the abnormal changes of mitochondrial ultrastructure, protect hippocampal neurons, and increase the survival of neurons in AD mice.Figure 7
TEM images of neuron mitochondria and synapses in the hippocampi of mice: synaptic density of hippocampal neurons (blue arrows), mitochondria (red arrows), cristae degeneration (yellow arrows), and membrane disruption (green arrows). CS-Li increased the synaptic density of hippocampal neurons and improved the damage of hippocampal mitochondria in AD mice.Scalebar=200 nm.
(a)(b)


## 3.4.1. H&E Staining

The effect of CS-Li on the neuronal injury in AD mice was probed by H&E staining test to observe the morphological change of neurons in hippocampus, which reflected the damage of hippocampal tissue. Figure4 shows the H&E staining of CA1-CA4 and DG regions in the hippocampus of mice. It was found that the neurocytes in CA1 region were well-arranged and structurally intact in the control group, and the nucleoli were obvious, without dense staining and pyknosis (red arrows). On the other hand, in the model group, the cell nucleus in CA1 region were obviously pyknotic (blue arrows), and some cells were edematous (green arrows). The cell width in CA1 region was significantly reduced with the disordered arrangement and neuron loss. Meanwhile, the cells in the model group were pyknotic, stained deeply, and exhibited disorderly arrangement, and some neurons were found to have undergone apoptosis in CA2, CA3, and CA4 regions. There is also an obvious wide edema zone in the DG region in the model group. These results indicated D-gal/AlCl3 had damaged the hippocampal region of the mice. After treatment with CS-Li, the neurons in CA1-CA4 regions of hippocampus were arranged tightly and orderly, the morphology was improved, the cell edema and pyknosis were reduced, and the number of neurons was increased. The edema zone of DG region was also significantly reduced compared with the model group. The hippocampal damage of AD mice was also reversed in LiCl group, but the reversal effect was not as obvious as that of H-CS-Li and L-CS-Li groups. These results suggested CS-Li could reverse and alleviate the damage of hippocampal neurons in AD mice induced by D-gal/AlCl3.Figure 4
Histopathological staining showing hippocampal morphology in AD mice (×100, ×400): normal neurocytes (red arrows), pyknosis (blue arrows), and cell edema (green arrows). CS-Li reduced the cell edema and pyknosis and increased the number of neurons in the hippocampus of AD mice.Scalebar=100 μm and 25 μm.


## 3.4.2. Nissl Staining

The Nissl body in neuron is the main site to synthesize proteins and plays significant roles in neuron excitation and conduction [34]. Nissl staining was used to evaluate the damage of Nissl body in hippocampus. As shown in Figure 5, the neurons of mice in the control group were morphologically normal with distinct hierarchical structure and regular arrangement, and the Nissl bodies were abundant in CA1-CA4 and DG regions. While in model group, the neurons in CA1 and DG regions were pyknotic and shaped into diamond and triangle. Compared with the control group, the number of Nissl bodies (purple arrows) was reduced largely in model group. After CS-Li treatment, the hippocampal neurons were arranged neatly and the Nissl body reappeared in CA1-CA4 regions. The results demonstrated that CS-Li could maintain neuronal cell morphology and improve neuronal protein synthesis function in AD mice, and the effect was more significant than that of LiCl.Figure 5
Nissl staining showing hippocampal morphology in AD mice (×100, ×400): Nissl bodies (purple arrows) and nucleoli (red arrows). CS-Li increased the number of Nissl body in the hippocampus of AD mice.Scalebar=100 μm and 25 μm.


## 3.4.3. TUNEL Staining

TUNEL staining was performed to examine the effect of CS-Li on suppressing the apoptosis of hippocampal neurons in AD mice. The apoptotic cell nuclei with positive expression appeared brownish. The results are shown in Figure6, TUNEL-positive cells appeared in the hippocampus of mice in the control group, and the apoptosis rates in the C1-C4 and DG regions were about 56.9±3.8%, 55.6±6.0%, 69.3±7.1%, 52.9±3.6%, and 49.7±2.2%, while the neurons in the hippocampus of mice were arranged regularly, and the nuclei were clear (red arrows) with no apoptosis in the CA1-CA4 and DG regions. While in the model group, TUNEL-positive cells increased largely in hippocampus, the apoptosis rate in the C1-C4 and DG regions were about 73.6±7.3%, 71.8±4.1%, 71.7±5.4%, 65.9±5.7%, and 67.7±6.0% (p<0.05, p<0.01, p>0.05, p<0.05, and p<0.01), the nucleoli disappeared, the cell gap was widened, and some nerve cells were degenerated by apoptosis. After CS-Li treatment, the apoptosis rate of neurons significantly decreased in CA1-CA4 regions (p<0.05, p<0.05, p<0.05, p<0.05, and p<0.05), and the edema in DG region was also ameliorated (green arrows). The neuronal apoptosis of AD mice was also reversed in LiCl group, but the reversal effect was not as obvious as that of H-CS-Li group. The TUNEL staining results demonstrated that CS-Li could reduce apoptosis of nerve cells in AD mice.Figure 6
TUNEL staining showing hippocampal morphology in AD mice (×100, ×400): nucleus (red arrows), normal neurocytes (purple arrows), cell edema (green arrows), and pyknosis (blue arrows).Scalebar=100 μm and 25 μm. The number of positive cells in the CA1, CA2, CA3, CA4, and DG regions (a, b, c, d, e, and f) of hippocampal cells was counted under a light microscope and quantified as percentage of total neuron cells among different groups (n=3 per group). CS-Li reduced the apoptosis of nerve cells in the hippocampus of AD mice. #p<0.05 and ##p<0.01 vs. the control group; ∗p<0.05 vs. the model group.
(a)(b)(c)(d)(e)(f)


## 3.4.4. Effect of CS-Li on the Ultrastructure in the Hippocampi of Mice

To study the ultrastructural changes in hippocampi of AD mice, TEM was applied to examine the ultrastructure of hippocampal neurons, the number of synapses, and the microstructure of mitochondria. The results are shown in Figure7(a), the synapse exhibited damaged structure, decreased density, shortening, and flattening. After treatment with H-CS-Li, the synaptic density of hippocampal neurons was increased (blue arrows), and the damage of synaptic structure was improved significantly. The mitochondrial microstructure of hippocampal neurons in mice in Figure 7(b) shows that the mitochondria of hippocampal neurons displayed obvious cristae degeneration (yellow arrows) and membrane rupture (green arrows). Compared with the model group, the mitochondrial structure of hippocampal neurons was relatively intact (red arrows), and the membrane rupture caused by mitochondrial swelling was improved in H-CS-Li group. The TEM results suggested that CS-Li can reduce the damage of hippocampal mitochondria, improve the abnormal changes of mitochondrial ultrastructure, protect hippocampal neurons, and increase the survival of neurons in AD mice.Figure 7
TEM images of neuron mitochondria and synapses in the hippocampi of mice: synaptic density of hippocampal neurons (blue arrows), mitochondria (red arrows), cristae degeneration (yellow arrows), and membrane disruption (green arrows). CS-Li increased the synaptic density of hippocampal neurons and improved the damage of hippocampal mitochondria in AD mice.Scalebar=200 nm.
(a)(b)


## 3.5. Mechanism Study of CS-Li Exhibiting Anti-AD Effect in Mice

### 3.5.1. Effect of CS-Li on Oxidative Stress in the Hippocampi of Mice

Oxidative stress is the imbalance between the generation of reactive oxygen and nitrogen species. Studies have demonstrated that oxidative stress is a major factor in AD pathogenesis [35]. In this study, we measured the levels of GSH-Px and end product of oxidation MDA, and the results are shown in Figures 8(a) and 8(b). Compared to the control group, the activity of GSH-Px was decreased, while the level of MDA was increased obviously in hippocampus of AD mice in the model group (p<0.05 and p<0.05, respectively), indicating D-gal/AlCl3 could induce the peroxidation of lipid in hippocampus of AD mice. After CS-Li and LiCl treatment, the activity of GSH-Px was increased appreciably in the H-CS-Li, L-CS-Li, and LiCl groups than that in the model group (p<0.001, p<0.01, and p<0.001, respectively), and the level of MDA was decreased in H-CS-Li, L-CS-Li, and LiCl markedly compared with the model group (p<0.01, p<0.01, and p<0.01). These results showed that both CS-Li and LiCl at the tested concentration could improve the activity of GSH-Px and inhibit the level of MDA, and further protect the brain from oxidative damage generated by free radicals.Figure 8
Effects of CS-Li on oxidative stress in the hippocampi of mice. CS-Li decreased the levels of MDA (a) and AchE (e) and increased the activities of GSH-Px (b), Na+/K+-ATPase (c), and ChAT (d) in the hippocampi of mice.#p<0.05 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.
(a)(b)(c)(d)(e)Na+/K+-ATPase plays important roles in ATP hydrolysis, Na+/K+ exchange, and transmembrane fluxes of Ca2+ and excitatory neurotransmitter. Oxidative metabolism is very active in brain and a large amount of chemical energy as ATP is consumed [36]. Studies have indicated that Na+/K+-ATPase activity is significantly lower in AD brains than that in age-matched control brains, and inhibition of Na+/K+-ATPase activity impairs learning and memory abilities [37]. Figure 8(c) shows the activity of Na+/K+-ATPase in the model group decreased largely (p<0.05), indicating D-gal/AlCl3 could induce disorders of energy metabolism in mouse hippocampi. After drug administration, the activities of Na+/K+-ATPase were remarkably increased in the H-CS-Li, L-CS-Li, and LiCl groups (p<0.001, p<0.001, and p<0.001) compared with the model group. The results suggest that CS-Li and LiCl could increase Na+/K+-ATPase activity and energy metabolism in hippocampal cells in mice brain and reduce edema and cell death at CNS level.


### 3.5.2. Effect of CS-Li on the Activity of Cholinesterase in the Hippocampi of Mice

The cholinergic system plays crucial roles in neurological and cognitive function. Acetyl choline (ACh) is a neurotransmitter and the increase of ACh level in brain can improve the symptoms of AD. The dynamic equilibrium of ACh is regulated by ChAT and AchE, which is responsible for ACh synthesis and degradation, respectively [38]. Figure 8(d) shows the activity of ChAT was significantly lower in the model group than that in the control group (p<0.05), suggesting D-gal/AlCl3 could inhibit the synthesis of ACh, decrease the cholinergic system function, and further influence the learning and memory abilities of mice. After CS-Li and LiCl treatment, the activities of ACh in the H-CS-Li, L-CS-Li, and LiCl groups were generally improved compared with the model group (p<0.001, p<0.001, and p<0.01).AchE inhibitors have been developed as the first line therapy for AD, such as donepezil, galantamine, and rivastigmine [39]. As shown in Figure 8(e), the activity of AchE in the model mice was higher than that of the control group (p<0.05). The results indicated D-gal and AlCl3 could increase the activity of AchE in mice brain, finally resulting in the reduction in ACh level. The activity of AchE in the hippocampus of H-CS-Li, L-CS-Li, and LiCl groups was decreased significantly compared with the model group (p<0.001, p<0.001, and p<0.001). The above results indicated that both CS-Li and LiCl at the test concentrations could influence the activity of ChAT and AchE in the brain of AD mice, further affecting the balance of ACh and improve the learning and memory abilities in AD mice.


### 3.5.3. Effect of CS-Li on Tau Hyperphosphorylation in the Hippocampi of Mice

Tau plays important roles in promoting assembly and stability of microtubule [40]. In the pathogenesis of AD, hyperphosphorylation of tau deprives it from the physiological function of microtubule binding and further destroys the neuronal cytoskeleton, causing aggregation to form fibrillary tangles, and eventually leading to neuronal degeneration and damage [41]. Therefore, the phosphorylation of tau has been recognized as the international gold standard to evaluate AD progression [42]. The phosphorylation sites Ser396 and Ser404 are reported to be involved in tau aggregation [43], and Western blot results indicated that the expression of tau (Ser396) and tau(Ser404) in model group was generally higher than that of the control group (p<0.05 and p<0.001) (Figure 9). After CS-Li treatment, the expression levels of tau (Ser396) and tau (Ser404) were reduced obviously in the H-CS-Li group (p<0.05 and p<0.01). The results which disclosed CS-Li at 400 μg/mL could inhibit the hyperphosphorylation of tau at Ser396 and Ser404 sites, while L-CS-Li and LiCl showed little effect on the hyperphosphorylation of tau at both sites.Figure 9
Effects of CS-Li on tau phosphorylation in the hippocampi of mice. Western blotting of GSK-3β, p-GSK-3β (Ser9), p-tau (Ser396), p-tau (Ser404), and tau, GAPDH was used as the internal control; Western blotting of PP2A, β-actin was used as the internal control. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.The regulation of tau phosphorylation depends on several protein kinases and phosphatases, among which GSK-3β and PP2A act a pivotal part in tau hyperphosphorylation [44]. Both higher level of GSK-3β and reduced activity of PP2A in tangles bearing neurons have been found in AD patient brains [45]. Hyperphosphorylation of tau and cognitive impairment can be alleviated by inhibiting GSK-3β activity and increasing PP2A activity [46, 47]. To gain insight into the mechanism of CS-Li in regulating tau hyperphosphorylation, Western blot assay was performed to examine the effect of CS-Li on the expression of GSK-3β and PP2A in AD mice. The results shown in Figure 9 suggest that the expression of p-GSK-3β (Ser9) was increased in the model group compared with the control group (p<0.001), whereas the expression of GSK-3β showed no obvious difference between the two groups, indicating phosphorylation of GSK-3β at Ser9 site had been induced by D-gal/AlCl3. The expression of PP2A in the model group was nearly the same as that of the control group. After treatment with CS-Li and LiCl, the GSK-3β phosphorylation was reduced significantly in the L-CS-Li, H-CS-Li, and LiCl groups compared with the model group (p<0.001, p<0.001, and p<0.01, respectively). In addition, the expression of PP2A in H-CS-Li group was increased significantly compared with the model group (p<0.05), showing better results than the L-CS-Li and LiCl groups. The above results demonstrated CS-Li could reduce the tau protein hyperphosphorylation at Ser396 and Ser404 sites by regulating the phosphorylation of GSK-3β at Ser9 site and the activity of PP2A.


### 3.5.4. Effect of CS-Li on Inflammatory Responses in the Hippocampi of Mice

Neuroinflammation is associated with neurodegenerative diseases, including AD. The proinflammatory cytokine levels in serum are elevated both in human and experimental models [48]. Proinflammatory cytokines can activate the microglia to express proinflammatory factors such as IL-6, IL-1β, TNF-α, nitric oxide, and proteases, to exert detrimental effects in neuropathy. To explore the effect of CS-Li in regulating neuroinflammatory response, Western blot assay was preformed to study the effect of CS-Li on the expression of TNF-α, IL-6, and IL-1β in hippocampus of AD mice. As shown in Figure 10, the levels of IL-6, IL-1β, and TNF-α were generally higher in the model group than those of the control group (p<0.05, p<0.01, and p<0.05, respectively). Compared with the model group, the expressions of IL-6, IL-1β, and TNF-α were all decreased obviously in the H-CS-Li, L-CS-Li, and CS-Li groups (p<0.05 or p<0.01). These results suggested that both CS-Li and LiCl can inhibit the expressions of proinflammatory cytokines in the hippocampi of AD mice, indicating CS-Li and LiCl exert neuroprotective effect by suppressing hippocampal inflammatory responses.Figure 10
Effects of CS-Li on inflammatory responses in the hippocampi of mice. Western blotting of IL-6, IL-1β, and TNF-α, β-actin was used as the internal control; Western blotting of p-Erk1/2 and p-38, GAPDH was used as the internal control; Western blotting of p-NF-κB, IκBα, and p-IκBα, Lamin B was used as the internal control. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.The activation of inflammatory response can be mediated by multiple signaling pathways including MAPK and NF-κB [49]. To figure out whether CS-Li could regulate the expression of proinflammatory cytokines through MAPK/NF-κB signaling pathway in AD mice, Western blot was used to analyze the expressions of p-NF-κB p65, p-Erk, p-p38, IκBa, and p-IκBa in hippocampal of AD mice. As displayed in Figure 10, the expression of p-Erk1/2 in H-CS-Li group was decreased significantly compared with the model group (p<0.05); the expression of p-p38 was reduced obviously in H-CS-Li, L-CS-Li, and LiCl groups (p<0.01, p<0.01, and p<0.01). The expression of p-NF-κB p65 and p-IκBa in H-CS-Li groups were decreased compared with the model group (p<0.05 and p<0.01), and the expression of IκBa in H-CS-Li group was increased obviously compared with the model group (p<0.05). The results indicated that CS-Li could activate MAPK pathway through p38 and Erk1/2 signaling, and then suppress the degradation of IκBa, further regulating the nuclear translocation of NF-κB, eventually inhibiting the expressions of IL-6, IL-1β, and TNF-α.


## 3.5.1. Effect of CS-Li on Oxidative Stress in the Hippocampi of Mice

Oxidative stress is the imbalance between the generation of reactive oxygen and nitrogen species. Studies have demonstrated that oxidative stress is a major factor in AD pathogenesis [35]. In this study, we measured the levels of GSH-Px and end product of oxidation MDA, and the results are shown in Figures 8(a) and 8(b). Compared to the control group, the activity of GSH-Px was decreased, while the level of MDA was increased obviously in hippocampus of AD mice in the model group (p<0.05 and p<0.05, respectively), indicating D-gal/AlCl3 could induce the peroxidation of lipid in hippocampus of AD mice. After CS-Li and LiCl treatment, the activity of GSH-Px was increased appreciably in the H-CS-Li, L-CS-Li, and LiCl groups than that in the model group (p<0.001, p<0.01, and p<0.001, respectively), and the level of MDA was decreased in H-CS-Li, L-CS-Li, and LiCl markedly compared with the model group (p<0.01, p<0.01, and p<0.01). These results showed that both CS-Li and LiCl at the tested concentration could improve the activity of GSH-Px and inhibit the level of MDA, and further protect the brain from oxidative damage generated by free radicals.Figure 8
Effects of CS-Li on oxidative stress in the hippocampi of mice. CS-Li decreased the levels of MDA (a) and AchE (e) and increased the activities of GSH-Px (b), Na+/K+-ATPase (c), and ChAT (d) in the hippocampi of mice.#p<0.05 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.
(a)(b)(c)(d)(e)Na+/K+-ATPase plays important roles in ATP hydrolysis, Na+/K+ exchange, and transmembrane fluxes of Ca2+ and excitatory neurotransmitter. Oxidative metabolism is very active in brain and a large amount of chemical energy as ATP is consumed [36]. Studies have indicated that Na+/K+-ATPase activity is significantly lower in AD brains than that in age-matched control brains, and inhibition of Na+/K+-ATPase activity impairs learning and memory abilities [37]. Figure 8(c) shows the activity of Na+/K+-ATPase in the model group decreased largely (p<0.05), indicating D-gal/AlCl3 could induce disorders of energy metabolism in mouse hippocampi. After drug administration, the activities of Na+/K+-ATPase were remarkably increased in the H-CS-Li, L-CS-Li, and LiCl groups (p<0.001, p<0.001, and p<0.001) compared with the model group. The results suggest that CS-Li and LiCl could increase Na+/K+-ATPase activity and energy metabolism in hippocampal cells in mice brain and reduce edema and cell death at CNS level.


## 3.5.2. Effect of CS-Li on the Activity of Cholinesterase in the Hippocampi of Mice

The cholinergic system plays crucial roles in neurological and cognitive function. Acetyl choline (ACh) is a neurotransmitter and the increase of ACh level in brain can improve the symptoms of AD. The dynamic equilibrium of ACh is regulated by ChAT and AchE, which is responsible for ACh synthesis and degradation, respectively [38]. Figure 8(d) shows the activity of ChAT was significantly lower in the model group than that in the control group (p<0.05), suggesting D-gal/AlCl3 could inhibit the synthesis of ACh, decrease the cholinergic system function, and further influence the learning and memory abilities of mice. After CS-Li and LiCl treatment, the activities of ACh in the H-CS-Li, L-CS-Li, and LiCl groups were generally improved compared with the model group (p<0.001, p<0.001, and p<0.01).AchE inhibitors have been developed as the first line therapy for AD, such as donepezil, galantamine, and rivastigmine [39]. As shown in Figure 8(e), the activity of AchE in the model mice was higher than that of the control group (p<0.05). The results indicated D-gal and AlCl3 could increase the activity of AchE in mice brain, finally resulting in the reduction in ACh level. The activity of AchE in the hippocampus of H-CS-Li, L-CS-Li, and LiCl groups was decreased significantly compared with the model group (p<0.001, p<0.001, and p<0.001). The above results indicated that both CS-Li and LiCl at the test concentrations could influence the activity of ChAT and AchE in the brain of AD mice, further affecting the balance of ACh and improve the learning and memory abilities in AD mice.


## 3.5.3. Effect of CS-Li on Tau Hyperphosphorylation in the Hippocampi of Mice

Tau plays important roles in promoting assembly and stability of microtubule [40]. In the pathogenesis of AD, hyperphosphorylation of tau deprives it from the physiological function of microtubule binding and further destroys the neuronal cytoskeleton, causing aggregation to form fibrillary tangles, and eventually leading to neuronal degeneration and damage [41]. Therefore, the phosphorylation of tau has been recognized as the international gold standard to evaluate AD progression [42]. The phosphorylation sites Ser396 and Ser404 are reported to be involved in tau aggregation [43], and Western blot results indicated that the expression of tau (Ser396) and tau(Ser404) in model group was generally higher than that of the control group (p<0.05 and p<0.001) (Figure 9). After CS-Li treatment, the expression levels of tau (Ser396) and tau (Ser404) were reduced obviously in the H-CS-Li group (p<0.05 and p<0.01). The results which disclosed CS-Li at 400 μg/mL could inhibit the hyperphosphorylation of tau at Ser396 and Ser404 sites, while L-CS-Li and LiCl showed little effect on the hyperphosphorylation of tau at both sites.Figure 9
Effects of CS-Li on tau phosphorylation in the hippocampi of mice. Western blotting of GSK-3β, p-GSK-3β (Ser9), p-tau (Ser396), p-tau (Ser404), and tau, GAPDH was used as the internal control; Western blotting of PP2A, β-actin was used as the internal control. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.The regulation of tau phosphorylation depends on several protein kinases and phosphatases, among which GSK-3β and PP2A act a pivotal part in tau hyperphosphorylation [44]. Both higher level of GSK-3β and reduced activity of PP2A in tangles bearing neurons have been found in AD patient brains [45]. Hyperphosphorylation of tau and cognitive impairment can be alleviated by inhibiting GSK-3β activity and increasing PP2A activity [46, 47]. To gain insight into the mechanism of CS-Li in regulating tau hyperphosphorylation, Western blot assay was performed to examine the effect of CS-Li on the expression of GSK-3β and PP2A in AD mice. The results shown in Figure 9 suggest that the expression of p-GSK-3β (Ser9) was increased in the model group compared with the control group (p<0.001), whereas the expression of GSK-3β showed no obvious difference between the two groups, indicating phosphorylation of GSK-3β at Ser9 site had been induced by D-gal/AlCl3. The expression of PP2A in the model group was nearly the same as that of the control group. After treatment with CS-Li and LiCl, the GSK-3β phosphorylation was reduced significantly in the L-CS-Li, H-CS-Li, and LiCl groups compared with the model group (p<0.001, p<0.001, and p<0.01, respectively). In addition, the expression of PP2A in H-CS-Li group was increased significantly compared with the model group (p<0.05), showing better results than the L-CS-Li and LiCl groups. The above results demonstrated CS-Li could reduce the tau protein hyperphosphorylation at Ser396 and Ser404 sites by regulating the phosphorylation of GSK-3β at Ser9 site and the activity of PP2A.


## 3.5.4. Effect of CS-Li on Inflammatory Responses in the Hippocampi of Mice

Neuroinflammation is associated with neurodegenerative diseases, including AD. The proinflammatory cytokine levels in serum are elevated both in human and experimental models [48]. Proinflammatory cytokines can activate the microglia to express proinflammatory factors such as IL-6, IL-1β, TNF-α, nitric oxide, and proteases, to exert detrimental effects in neuropathy. To explore the effect of CS-Li in regulating neuroinflammatory response, Western blot assay was preformed to study the effect of CS-Li on the expression of TNF-α, IL-6, and IL-1β in hippocampus of AD mice. As shown in Figure 10, the levels of IL-6, IL-1β, and TNF-α were generally higher in the model group than those of the control group (p<0.05, p<0.01, and p<0.05, respectively). Compared with the model group, the expressions of IL-6, IL-1β, and TNF-α were all decreased obviously in the H-CS-Li, L-CS-Li, and CS-Li groups (p<0.05 or p<0.01). These results suggested that both CS-Li and LiCl can inhibit the expressions of proinflammatory cytokines in the hippocampi of AD mice, indicating CS-Li and LiCl exert neuroprotective effect by suppressing hippocampal inflammatory responses.Figure 10
Effects of CS-Li on inflammatory responses in the hippocampi of mice. Western blotting of IL-6, IL-1β, and TNF-α, β-actin was used as the internal control; Western blotting of p-Erk1/2 and p-38, GAPDH was used as the internal control; Western blotting of p-NF-κB, IκBα, and p-IκBα, Lamin B was used as the internal control. #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group; ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. the model group.The activation of inflammatory response can be mediated by multiple signaling pathways including MAPK and NF-κB [49]. To figure out whether CS-Li could regulate the expression of proinflammatory cytokines through MAPK/NF-κB signaling pathway in AD mice, Western blot was used to analyze the expressions of p-NF-κB p65, p-Erk, p-p38, IκBa, and p-IκBa in hippocampal of AD mice. As displayed in Figure 10, the expression of p-Erk1/2 in H-CS-Li group was decreased significantly compared with the model group (p<0.05); the expression of p-p38 was reduced obviously in H-CS-Li, L-CS-Li, and LiCl groups (p<0.01, p<0.01, and p<0.01). The expression of p-NF-κB p65 and p-IκBa in H-CS-Li groups were decreased compared with the model group (p<0.05 and p<0.01), and the expression of IκBa in H-CS-Li group was increased obviously compared with the model group (p<0.05). The results indicated that CS-Li could activate MAPK pathway through p38 and Erk1/2 signaling, and then suppress the degradation of IκBa, further regulating the nuclear translocation of NF-κB, eventually inhibiting the expressions of IL-6, IL-1β, and TNF-α.


## 4. Conclusion

In this study, CS-Li was prepared and its anti-AD activity was investigated bothin vitro and in vivo. Compared to LiCl, CS-Li displays much less toxicity and higher safety to SH-SY5Y cells at the same Li content. CS-Li could inhibit the secondary spatial structure formation, improve the water solubility, suppress the aggregation, and reduce the toxicity of Aβ1-42, which would effectively inhibit Aβ1-42 induced neuron apoptosis. The in vivo study demonstrated the protective effect of CS-Li on the D-gal/AlCl3-induced AD mice model. The results demonstrated that CS-Li can improve the spatial learning and memory deficit, reverse the nuclear pyknosis and cell edema, improve the abnormal changes of ultrastructure in the hippocampi of mice, and increase the survival of neurons in the hippocampal CA1-CA4 and DG regions in mice. CS-Li could increase the activities of GSH-Px, ChAT, and Na+/K+-ATPase and reduce the level of MDA and AchE to improve the energy metabolism of brain cells and imbalance the level of ACh, resulting in brain tissue protection from oxidative damage and cholinergic neuronal injury. Western blot results revealed that CS-Li inhibited the hyperphosphorylation of tau protein (Ser396/Ser404) by controlling the expression of p-GSK-3β (Ser9) and PP2A and reducing the expressions of proinflammatory factors by regulating the nuclear translocation of NF-κB/p65 through MAPK (Erk/p38) signaling pathways. In conclusion, the above results show that CS-Li possesses the ability to inhibit the aggregation of Aβ, reduce the damage of oxidative stress, regulate tau hyperphosphorylation, decrease inflammation response to decelerate AD progression, and improve learning and memory abilities of AD mice by simultaneously acting on multitargets. More studies are currently performed in our laboratory to verify the anti-AD effect of CS-Li to explore its potential as a more effective and safer anti-AD polysaccharide agent.



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*Source: 9466166-2022-11-12.xml*