# Classic Prescription, Kai-Xin-San, Ameliorates Alzheimer’s Disease as an Effective Multitarget Treatment: From Neurotransmitter to Protein Signaling Pathway

Authors: Sirui Guo; Jiahong Wang; Huarong Xu; Weiwei Rong; Cheng Gao; Ziyue Yuan; Fucheng Xie; Kaishun Bi; Zhou Zhang; Qing Li
Journal: Oxidative Medicine and Cellular Longevity
 (2019)

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## Abstract

Alzheimer’s disease (AD) is a widespread neurodegenerative disease caused by complicated disease-causing factors. Unsatisfactorily, curative effects of approved anti-AD drugs were not good enough due to their actions on single-target, which led to desperate requirements for more effective drug therapies involved in multiple pathomechanisms of AD. The anti-AD effect with multiple action targets of Kai-Xin-San (KXS), a classic prescription initially recorded inBei Ji Qian Jin Yao Fang and applied in the treatment of dementia for thousands of years, was deciphered with modern biological methods in our study. Aβ25-35 and D-gal-induced AD rats and Aβ25-35-induced PC12 cells were applied to establish AD models. KXS could significantly improve cognition impairment by decreasing neurotransmitter loss and enhancing the expression of PI3K/Akt. For the first time, KXS was confirmed to improve the expression of PI3K/Akt by neurotransmitter 5-HT. Thereinto, PI3K/Akt could further inhibit Tau hyperphosphorylation as well as the apoptosis induced by oxidative stress and neuroinflammation. Moreover, all above-mentioned effects were verified and blocked by PI3K inhibitor, LY294002, in Aβ25-35-induced PC12 cells, suggesting the precise regulative role of KXS in the PI3K/Akt pathway. The utilization and mechanism elaboration of KXS have been proposed and dissected in the combination of animal, molecular, and protein strategies. Our results demonstrated that KXS could ameliorate AD by regulating neurotransmitter and PI3K/Akt signal pathway as an effective multitarget treatment so that the potential value of this classic prescription could be explored from a novel perspective.


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## Body

## 1. Introduction

Alzheimer’s disease (AD) is the widespread neurodegenerative disease that accounted for 60-70% cases of dementia characterized by amnesia, cognition impairment, and mental behavioral changes [1]. It was reported that there are more than 50 million cases of dementia worldwide in 2018, signifying one new case every 3 seconds [2]. Currently, all FDA-approved drugs were developed for specific single-target, such as cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) and N-methyl-D-aspartate receptor agonist (memantine) [3], and the symptoms of AD could be alleviated to some extent. However, their therapeutic effects were far from the expectation by reason of their actions on single-target, which could not match multiple pathogenetic factors mainly including abnormal β-amyloid and Tau [4, 5], thus providing imperious demands for more effective multitarget drug therapies.Clinical curative effects of traditional Chinese medicine have been continuously verified in thousands of years, and several classic formulas specially aiming at various diseases were gradually developed. As a common formula for the treatment of “amnesia,” the main symptom of AD, KXS was initially recorded in Bei Ji Qian Jin Yao Fang in the Tang dynasty (Year 652), consisting of Polygala tenuifolia Willd (PR), Panax ginseng C.A. Mey (GR), Poria cocos (Schw.) Wolf (PO), and Acorus tatarinowii Schott (AT) [6]. Based on literatures to date, researches of KXS were mainly focused on component identification or mechanism dissection, and most pathological studies were related to neurotransmitter regulation that involved a single pathway, which could not comprehensively elaborate therapeutic effects of KXS and the scientificity of AD treatment [7, 8]. Therefore, there is an urgent need for explaining multidimensional beneficial effects of KXS with modern biological methods.Aβ25-35 and D-gal-induced rats were usually selected as the most pertinent model to simulate characteristic pathological changes of AD [9]. Meanwhile, PC12 cells were commonly used for in vitro research to verify the underlying cellular mechanism against AD, which was important to evaluate biological function. Hence, we explained the pharmacological effects of KXS at animal, molecular, and cellular levels, multidimensionally clarifying the biological mechanism and scientificity for AD treatment, which could provide research reference for studies on the mechanism of Chinese classical prescriptions.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

The purity of the following reference standards was all over 98%. Isoproterenol hydrochloride was purchased from the National Institutes for Food and Drug Control (Beijing, China). Arachidonic acid (AA), leukotriene-B4 (LTB4), thromboxane-B2 (TXB2), 5-hydroxyeicosatetraenoic acid (5-HETE), 8-hydroxyeicosatetraenoic acid (8-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), and 15-hydroxyeicosatetraenoic acid-d8 (15-HETE-d8) were obtained from Cayman. Dopamine (DA), γ-aminobutyric acid (GABA), acetylcholine (ACh), tryptophan (Try), tyrosine (Tyr), 5-hydroxytryptophan (5-HTP), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptamine (5-HT), metanephrine (ME), 3-methoxytyramine (3-MT), Aβ25-35, and D-gal were obtained from Sigma-Aldrich Corporation (USA).


### 2.2. Preparation of Herbal Extracts

The herbal pieces of GR, PR, PO, and AT were purchased from Beijing Tongrentang Co. Ltd., China, and identified by Prof. Ying Jia, Shenyang Pharmaceutical University. The decoction pieces were mixed in a weight ratio of 3 : 2 : 3 : 2, then boiled in 10 volumes of double distilled water for 1 h and extracted twice. The combined extracts were concentrated to 1 mg/mL by a rotary evaporator and stored at -20°C.


### 2.3. Animals

Male SD rats weighing 200-250 g were provided by the Experimental Animal Center of Shenyang Pharmaceutical University. SD rats were maintained in a controlled environment (humidity50±10%, 23±2°C) under a 12 h light/dark cycle and allowed to water and standard diet ad libitum. All experiments were approved by the Guideline of Animal Experimentation of Shenyang Pharmaceutical University. Rats were divided into four groups, namely, the control group, model group, KXS group, and huperzine A (Hup A) group. Briefly, AD rats were injected with D-gal (50 mg/kg/d) intraperitoneally for 6 weeks, and control rats were given the same volume of saline. KXS (10 g/kg/d) and Hup A (30 μg/kg/d) were oral administrated from the third week until the end of the experiment once a day [10]. The stereotaxic injection of 4 μg Aβ25-35 (1 μg/μL) was operated as described previously [11]. At the seventh week, the Morris water maze performance was tested as described previously [12]. Drug administration and Morris water maze test were scheduled according to the experimental timelines (Figure 1).Figure 1
The schematic diagram of the AD rat experimental procedure.


### 2.4. Measurement of Endogenous Substances

The plasma samples were collected after Morris water maze behavioral testing. Aliquots of 200μL plasma samples were spiked with 20 μL of IS (isoproterenol hydrochloride and 15-HETE-d8) and 20 μL of methanol by vortexing for 30 s. The mixtures were extracted with 600 μL acetonitrile (containing 1% formic acid) by vortex-mixing for 3 min. After centrifugation at 12000 rpm, 4°C for 5 min, the supernatants were transferred and evaporated to dryness at 35°C. Then, the residue was reconstituted in 50 μL 0.05% formic acid in methanol-water (15 : 85, v/v) for UFLC-MS/MS analysis.Liquid chromatographic runs were performed on an XR LC-20 AD Prominence™ UFLC system (Shimadzu, Japan). Chromatographic separation was achieved with an Inertsil ODS-EP column (150mm×4.6mm, 5 μm particles), (Shimadzu, Japan) at 30°C. Mobile phase A was 0.05% formic acid in water, and mobile phase B was 0.05% formic acid in methanol. A mobile phase was achieved with gradient elution using a complex gradient, as the following: 85%A→85%A at 0–0.01 min, 85%A→55%A at 0.01–0.5 min, 55%A→0%A at 0.5–5 min, 0%A→0%A at 5–10 min, 0%A→85%A at 10–10.01 min, and 85%A→85%A at 10.01–12 min. Symmetrical and efficient peaks were obtained at a flow rate of 0.5 mL/min with a sample injection volume of 5 μL.Mass spectrometric detection was performed by a 4000 QTrap™ MS/MS system from AB Sciex (Foster City, CA, USA) in the MRM modes. The ion spray voltage was maintained at 5500 V for positive and -4500 V for negative monitoring. The source temperature was set at 500°C with high-purity nitrogen as Gas 1, Gas 2, and curtain gas set at 50, 50, and 20 psi, respectively. The acquisition, instrument control, and analysis of data were dealt with Analyst software (version 1.5.2, AB Sciex, USA).


### 2.5. Drug Treatment and Cell Viability Detection

Highly differentiated rat pheochromocytoma cells, PC12, were purchased from the Boster Biological Technology Co. Ltd. (Wuhan, China) cultured in H-DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. Cells were incubated at 37°C in moist air containing 5% CO2. PC12 cells were seeded in culture plates (1×105 cells/mL) for 24 h and then treated with series of concentrations of KXS (0, 0.05, 0.1, 0.5, 1, 5, 10, and 20 mg/mL) for 24 h. Subsequently, Aβ25-35 was added to the medium and cocultured with KXS for an additional 24 h. LY294002 which was the inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was employed 1 h prior to KXS treatment. PC12 cells were divided into four groups, namely, the control group, Aβ group, KXS group, and pretreatment of LY294002+KXS group. Meanwhile, PC12 cells were pretreated with LY294002 for 1 h, incubated with 1 μmol/L 5-HT and 20 μmol/L Aβ25-35 for 24 h, and divided into the control group, Aβ group, 5-HT group, and pretreatment of LY294002+5-HT group [13].Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and LDH assay kit. After the KXS treatment, the medium was removed and replaced with 20μL MTT (5 mg/mL, Sigma) in phosphate buffer solution. The plates were incubated for 4 h at 37°C, followed by the addition of 150 μL dimethyl sulfoxide (DMSO). The absorbance was detected at 490 nm wavelength by a microplate reader. The concentrations of LDH assessed as a cytotoxicity assay indicator in cell culture supernatants were evaluated by the LDH assay kit (Nanjing Jiancheng, China) according to the manufacturer’s instructions. The absorbance was detected at 450 nm wavelength. The LDH release activity was calculated as the following:
(1)ODsample−ODcontrolODstandard−ODblank∗concentrationstandard.


### 2.6. Measurement of Intracellular Reactive Oxygen Species (ROS) and Mitochondrial Membrane Potential (MMP)

Intracellular ROS was measured using the ROS assay kit (Nanjing Jiancheng, China). 2,7-Dichlorofluorescin diacetate was a sensitive intracellular reactive oxygen detection probe which was widely used. According to the description of the previous study [14], the fluorescence in fresh hippocampal tissue and PC12 cells was evaluated with the excitation at 500±15nm and the emission at 525±15nm. Rhodamine 123 (Beyotime, China) was a fluorochrome which could penetrate cytomembrane and selectively stain mitochondria in alive cells. Rhodamine 123 was dissolved in DMSO and diluted to the working concentration (2 μmol/L) with cell culture medium. The fluorescence was evaluated with a flow cytometry (BD, USA) at 507 nm excitation and 529 nm emission.


### 2.7. Assessment of Annexin V-FITC/PI Staining and Proinflammatory Factor (IL-1β and TNF-α)

The rate of apoptosis was quantified with an Annexin V-FITC/PI apoptosis detection kit (KeyGen BioTECH, China). The washed cells were resuspended in binding buffer and incubated with annexin V-FITC and propidium iodide. The treated cells were measured with a flow cytometry within 1 h. The annexin V-FITC and PI were tested through FL1 and FL3 and quantified with FlowJo 10 software, respectively. IL-1β and TNF-α ELISA assays (Boster, China) were conducted to determine the level of IL-1β and TNF-α in the hippocampal tissue samples and PC12 cell culture medium obtained from the treated PC12 cells in order to test the anti-inflammatory effect of KXS. Concentrations of IL-1β and TNF-α were measured according to the standard protocol from the manufacturer. The absorbance was evaluated with the microplate reader at 450 nm.


### 2.8. Western Blotting

The protocol of western blotting was proceeded as described previously [15]. In brief, the hippocampal tissue samples and PC12 cells were harvested and lysed with lysis buffer (Beyotime, China) containing 1% phosphatase inhibitors and protease inhibitor. The quantitative proteins (40 μg) were separated on 10% SDS-polyacrylamide gels. The primary antibodies were PI3K p110а (#4249, CST, 1 : 1000), PI3K (ab40776, Abcam, 1 : 1000), Tau (ab32057, Abcam, 1 : 1000), Tau (phospho S396) (ab109390, Abcam, 1 : 5000), Tau (phospho S199) (ab81268, Abcam, 1 : 5000), Bax (ab32503, Abcam, 1 : 1000), bcl-2 (WL01556, Wanleibio, 1 : 800), GSK-3β (#12456, CST, 1 : 1000), phospho-GSK-3β (Ser9) (#5585, CST, 1 : 1000), Akt (#4691, CST, 1 : 1000), Akt (phospho S473) (ab81283, Abcam, 1 : 5000), and cleaved-caspase-3 (25546-1-AP, Proteintech, 1 : 1000). The bands were covered with ECL and photographed with the Chemiluminescent Imaging System (Tanon, China). β-Actin was calculated as a control for protein expression in every membrane.


### 2.9. Immunohistochemistry and Immunofluorescence

In order to detect the phosphorylation of Tau, immunohistochemical analysis was performed on hippocampus sections (4μm thickness) using a monoclonal anti-Tau (phospho S396) antibody (1 : 1000). A Rabbit SP detection kit (ZSBIO) was adopted to visualize immunolabeling according to the manufacturer’s instructions. Images of the immunohistochemical sections were acquired using a CIC microscope (XSP-C204). Histologic sections were rehydrated with gradient alcohol, antigen-repaired with citric acid buffer (pH 6.0), and then quenched with 3% H2O2 for 25 min. After blocking with goat serum, sections were incubated with anti-Tau (phospho S396) antibody at 4°C overnight, followed with streptavidin-horseradish peroxidase (Servicebio, G1211) for 50 min at room temperature. The sections were colored with peroxidase-3,3′-diaminobenzidine (DAB) substrate (Servicebio, G1211) and scanned at 400x.Immunofluorescence for neuronal nuclei (NeuN) expression was performed. Brain tissue sections were rehydrated with gradient alcohol, antigen-repaired with citric acid buffer (pH 6.0), and then blocked with BSA for 30 min. The sections were incubated with NeuN antibody (1 : 200) at 4°C overnight, followed with fluorescence secondary antibodies (Servicebio, 1 : 300) for 50 min at room temperature. NeuN (red fluorescence) was visualized by excitation at 520 nm and detection of emission at 590 nm. DAPI (blue fluorescence) was visualized by excitation at 380 nm and detection of emission at 420 nm. Images of the immunofluorescence slices were acquired with Nikon Eclipse C1. The percentage of the integrated optical density (IOD) occupied by positive expression products above background staining was measured in the hippocampal CA1 region (3 sections per rat) using Image-Pro Plus software.


### 2.10. Statistical Analysis

All statistical data were analyzed using Student’st-test and one-way ANOVA with SPSS 19.0 software. The principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA) were proceeded with the SIMCA-P software package (Umetrics AB, Umea, Sweden). The statistical results were expressed as the mean±SD, and statistical significances were classified as ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.


## 2.1. Chemicals and Reagents

The purity of the following reference standards was all over 98%. Isoproterenol hydrochloride was purchased from the National Institutes for Food and Drug Control (Beijing, China). Arachidonic acid (AA), leukotriene-B4 (LTB4), thromboxane-B2 (TXB2), 5-hydroxyeicosatetraenoic acid (5-HETE), 8-hydroxyeicosatetraenoic acid (8-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), and 15-hydroxyeicosatetraenoic acid-d8 (15-HETE-d8) were obtained from Cayman. Dopamine (DA), γ-aminobutyric acid (GABA), acetylcholine (ACh), tryptophan (Try), tyrosine (Tyr), 5-hydroxytryptophan (5-HTP), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptamine (5-HT), metanephrine (ME), 3-methoxytyramine (3-MT), Aβ25-35, and D-gal were obtained from Sigma-Aldrich Corporation (USA).


## 2.2. Preparation of Herbal Extracts

The herbal pieces of GR, PR, PO, and AT were purchased from Beijing Tongrentang Co. Ltd., China, and identified by Prof. Ying Jia, Shenyang Pharmaceutical University. The decoction pieces were mixed in a weight ratio of 3 : 2 : 3 : 2, then boiled in 10 volumes of double distilled water for 1 h and extracted twice. The combined extracts were concentrated to 1 mg/mL by a rotary evaporator and stored at -20°C.


## 2.3. Animals

Male SD rats weighing 200-250 g were provided by the Experimental Animal Center of Shenyang Pharmaceutical University. SD rats were maintained in a controlled environment (humidity50±10%, 23±2°C) under a 12 h light/dark cycle and allowed to water and standard diet ad libitum. All experiments were approved by the Guideline of Animal Experimentation of Shenyang Pharmaceutical University. Rats were divided into four groups, namely, the control group, model group, KXS group, and huperzine A (Hup A) group. Briefly, AD rats were injected with D-gal (50 mg/kg/d) intraperitoneally for 6 weeks, and control rats were given the same volume of saline. KXS (10 g/kg/d) and Hup A (30 μg/kg/d) were oral administrated from the third week until the end of the experiment once a day [10]. The stereotaxic injection of 4 μg Aβ25-35 (1 μg/μL) was operated as described previously [11]. At the seventh week, the Morris water maze performance was tested as described previously [12]. Drug administration and Morris water maze test were scheduled according to the experimental timelines (Figure 1).Figure 1
The schematic diagram of the AD rat experimental procedure.


## 2.4. Measurement of Endogenous Substances

The plasma samples were collected after Morris water maze behavioral testing. Aliquots of 200μL plasma samples were spiked with 20 μL of IS (isoproterenol hydrochloride and 15-HETE-d8) and 20 μL of methanol by vortexing for 30 s. The mixtures were extracted with 600 μL acetonitrile (containing 1% formic acid) by vortex-mixing for 3 min. After centrifugation at 12000 rpm, 4°C for 5 min, the supernatants were transferred and evaporated to dryness at 35°C. Then, the residue was reconstituted in 50 μL 0.05% formic acid in methanol-water (15 : 85, v/v) for UFLC-MS/MS analysis.Liquid chromatographic runs were performed on an XR LC-20 AD Prominence™ UFLC system (Shimadzu, Japan). Chromatographic separation was achieved with an Inertsil ODS-EP column (150mm×4.6mm, 5 μm particles), (Shimadzu, Japan) at 30°C. Mobile phase A was 0.05% formic acid in water, and mobile phase B was 0.05% formic acid in methanol. A mobile phase was achieved with gradient elution using a complex gradient, as the following: 85%A→85%A at 0–0.01 min, 85%A→55%A at 0.01–0.5 min, 55%A→0%A at 0.5–5 min, 0%A→0%A at 5–10 min, 0%A→85%A at 10–10.01 min, and 85%A→85%A at 10.01–12 min. Symmetrical and efficient peaks were obtained at a flow rate of 0.5 mL/min with a sample injection volume of 5 μL.Mass spectrometric detection was performed by a 4000 QTrap™ MS/MS system from AB Sciex (Foster City, CA, USA) in the MRM modes. The ion spray voltage was maintained at 5500 V for positive and -4500 V for negative monitoring. The source temperature was set at 500°C with high-purity nitrogen as Gas 1, Gas 2, and curtain gas set at 50, 50, and 20 psi, respectively. The acquisition, instrument control, and analysis of data were dealt with Analyst software (version 1.5.2, AB Sciex, USA).


## 2.5. Drug Treatment and Cell Viability Detection

Highly differentiated rat pheochromocytoma cells, PC12, were purchased from the Boster Biological Technology Co. Ltd. (Wuhan, China) cultured in H-DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. Cells were incubated at 37°C in moist air containing 5% CO2. PC12 cells were seeded in culture plates (1×105 cells/mL) for 24 h and then treated with series of concentrations of KXS (0, 0.05, 0.1, 0.5, 1, 5, 10, and 20 mg/mL) for 24 h. Subsequently, Aβ25-35 was added to the medium and cocultured with KXS for an additional 24 h. LY294002 which was the inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was employed 1 h prior to KXS treatment. PC12 cells were divided into four groups, namely, the control group, Aβ group, KXS group, and pretreatment of LY294002+KXS group. Meanwhile, PC12 cells were pretreated with LY294002 for 1 h, incubated with 1 μmol/L 5-HT and 20 μmol/L Aβ25-35 for 24 h, and divided into the control group, Aβ group, 5-HT group, and pretreatment of LY294002+5-HT group [13].Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and LDH assay kit. After the KXS treatment, the medium was removed and replaced with 20μL MTT (5 mg/mL, Sigma) in phosphate buffer solution. The plates were incubated for 4 h at 37°C, followed by the addition of 150 μL dimethyl sulfoxide (DMSO). The absorbance was detected at 490 nm wavelength by a microplate reader. The concentrations of LDH assessed as a cytotoxicity assay indicator in cell culture supernatants were evaluated by the LDH assay kit (Nanjing Jiancheng, China) according to the manufacturer’s instructions. The absorbance was detected at 450 nm wavelength. The LDH release activity was calculated as the following:
(1)ODsample−ODcontrolODstandard−ODblank∗concentrationstandard.


## 2.6. Measurement of Intracellular Reactive Oxygen Species (ROS) and Mitochondrial Membrane Potential (MMP)

Intracellular ROS was measured using the ROS assay kit (Nanjing Jiancheng, China). 2,7-Dichlorofluorescin diacetate was a sensitive intracellular reactive oxygen detection probe which was widely used. According to the description of the previous study [14], the fluorescence in fresh hippocampal tissue and PC12 cells was evaluated with the excitation at 500±15nm and the emission at 525±15nm. Rhodamine 123 (Beyotime, China) was a fluorochrome which could penetrate cytomembrane and selectively stain mitochondria in alive cells. Rhodamine 123 was dissolved in DMSO and diluted to the working concentration (2 μmol/L) with cell culture medium. The fluorescence was evaluated with a flow cytometry (BD, USA) at 507 nm excitation and 529 nm emission.


## 2.7. Assessment of Annexin V-FITC/PI Staining and Proinflammatory Factor (IL-1β and TNF-α)

The rate of apoptosis was quantified with an Annexin V-FITC/PI apoptosis detection kit (KeyGen BioTECH, China). The washed cells were resuspended in binding buffer and incubated with annexin V-FITC and propidium iodide. The treated cells were measured with a flow cytometry within 1 h. The annexin V-FITC and PI were tested through FL1 and FL3 and quantified with FlowJo 10 software, respectively. IL-1β and TNF-α ELISA assays (Boster, China) were conducted to determine the level of IL-1β and TNF-α in the hippocampal tissue samples and PC12 cell culture medium obtained from the treated PC12 cells in order to test the anti-inflammatory effect of KXS. Concentrations of IL-1β and TNF-α were measured according to the standard protocol from the manufacturer. The absorbance was evaluated with the microplate reader at 450 nm.


## 2.8. Western Blotting

The protocol of western blotting was proceeded as described previously [15]. In brief, the hippocampal tissue samples and PC12 cells were harvested and lysed with lysis buffer (Beyotime, China) containing 1% phosphatase inhibitors and protease inhibitor. The quantitative proteins (40 μg) were separated on 10% SDS-polyacrylamide gels. The primary antibodies were PI3K p110а (#4249, CST, 1 : 1000), PI3K (ab40776, Abcam, 1 : 1000), Tau (ab32057, Abcam, 1 : 1000), Tau (phospho S396) (ab109390, Abcam, 1 : 5000), Tau (phospho S199) (ab81268, Abcam, 1 : 5000), Bax (ab32503, Abcam, 1 : 1000), bcl-2 (WL01556, Wanleibio, 1 : 800), GSK-3β (#12456, CST, 1 : 1000), phospho-GSK-3β (Ser9) (#5585, CST, 1 : 1000), Akt (#4691, CST, 1 : 1000), Akt (phospho S473) (ab81283, Abcam, 1 : 5000), and cleaved-caspase-3 (25546-1-AP, Proteintech, 1 : 1000). The bands were covered with ECL and photographed with the Chemiluminescent Imaging System (Tanon, China). β-Actin was calculated as a control for protein expression in every membrane.


## 2.9. Immunohistochemistry and Immunofluorescence

In order to detect the phosphorylation of Tau, immunohistochemical analysis was performed on hippocampus sections (4μm thickness) using a monoclonal anti-Tau (phospho S396) antibody (1 : 1000). A Rabbit SP detection kit (ZSBIO) was adopted to visualize immunolabeling according to the manufacturer’s instructions. Images of the immunohistochemical sections were acquired using a CIC microscope (XSP-C204). Histologic sections were rehydrated with gradient alcohol, antigen-repaired with citric acid buffer (pH 6.0), and then quenched with 3% H2O2 for 25 min. After blocking with goat serum, sections were incubated with anti-Tau (phospho S396) antibody at 4°C overnight, followed with streptavidin-horseradish peroxidase (Servicebio, G1211) for 50 min at room temperature. The sections were colored with peroxidase-3,3′-diaminobenzidine (DAB) substrate (Servicebio, G1211) and scanned at 400x.Immunofluorescence for neuronal nuclei (NeuN) expression was performed. Brain tissue sections were rehydrated with gradient alcohol, antigen-repaired with citric acid buffer (pH 6.0), and then blocked with BSA for 30 min. The sections were incubated with NeuN antibody (1 : 200) at 4°C overnight, followed with fluorescence secondary antibodies (Servicebio, 1 : 300) for 50 min at room temperature. NeuN (red fluorescence) was visualized by excitation at 520 nm and detection of emission at 590 nm. DAPI (blue fluorescence) was visualized by excitation at 380 nm and detection of emission at 420 nm. Images of the immunofluorescence slices were acquired with Nikon Eclipse C1. The percentage of the integrated optical density (IOD) occupied by positive expression products above background staining was measured in the hippocampal CA1 region (3 sections per rat) using Image-Pro Plus software.


## 2.10. Statistical Analysis

All statistical data were analyzed using Student’st-test and one-way ANOVA with SPSS 19.0 software. The principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA) were proceeded with the SIMCA-P software package (Umetrics AB, Umea, Sweden). The statistical results were expressed as the mean±SD, and statistical significances were classified as ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.


## 3. Results

### 3.1. KXS Improved Learning and Memory Impaired in AD Rats Induced by D-Gal and Aβ25-35

The AD animal model induced by D-gal and Aβ25-35 was applied in order to simulate human pathological processes. The quality control of KXS was achieved by HPLC fingerprint (Figure S1). The water maze test was adopted to detect the effect of KXS on learning and memory in AD rats. Hup A (AChEIs), an alkaloid isolated from the Chinese herbal medicine Huperzia serrata, was licensed as a nutraceutical in the United States and widely used for AD treatment in China [16]. From the second day of training trials, the rats in the model group showed longer escape latency and swimming distance, meaning obvious learning and memory impairment compared to the control (Con) group (Figures 2(a) and 2(b)). On the fifth day of the Morris water maze, the spatial probe test was tested. The rats treated with Hup A or KXS showed significantly less escape latency and swimming distance to find the removal platform in the target quadrant (Figures 2(c)–2(e)). In addition, Hup A and KXS group rats showed more platform crossings than the model group (Figure 2(f)). Neuronal nuclei (NeuN), a neuronal special nuclear marker, was detected by immunohistochemistry staining to evaluate the neuroprotection of KXS on neuronal density in hippocampal CA1 regions [17]. The fluorescence intensity of NeuN in the model group was significantly reduced, which was a hallmark pathology of neuronal damage in AD and indicated a successful AD model. KXS treatment observably increased fluorescence intensity of NeuN compared with the model group (Figures 2(g) and 2(h)). The results of the Morris water maze and immunohistochemistry staining of NeuN suggested that KXS could be potentially used for the prevention and/or treatment of AD.Figure 2
KXS treatment ameliorated memory and learning impairment. The mean escape latency (a) and swimming distances (b) were obtained from different test days, and the KXS group spent less time and distances to reach the fourth quadrant platform. Representative rat search paths from different groups (c) were shown on the fifth day. The rats treated with Hup A or KXS showed significantly less escape latency (d), swimming distance (e), and platform crossings (f) to find the removal platform in the fourth quadrant. Impact of KXS on modulating hippocampal NeuN levels was assessed by immunohistochemistry. The fluorescence intensity was quantified using Image-Pro Plus software (g and h). The treatment of KXS showed improvement in spatial memory. Values were themean±SD (n=6). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the model group; $$P<0.01vs. the Hup A group. Scalebar=20μm.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


### 3.2. KXS Treatment Increased the Concentration of Central Neurotransmitters in AD Rats

In order to study the influence of KXS on central neurotransmitter loss, a typical pathologic manifestation, the UFLC-MS/MS method was used to detect the concentrations of related endogenous neurotransmitters. The method was validated according to the European Medicines Agency guidance for bioanalytical method validation (TablesS1-S2) [18]. Compared with the model group, concentrations of four types of neurotransmitters, namely, ACh, GABA, dopamine (Tyr, dopamine, ME, and 3-MT), and 5-HT (5-HTP, 5-HT, and 5-HIAA), were increased in rat plasmas after drug treatment (Table 1). Therefore, KXS could protect an impaired central nervous system by ameliorating neurotransmitter loss. Neurotransmitter data were handled with PCA. In the light of unsupervised PCA score plots, plasma samples in KXS and Hup A groups were located between the AD model group and the control group. Nevertheless, there was no significant difference between the KXS group and the Hup A group (Figure 3(a)). The PCA result also proved the efficacy of KXS on regulating the levels of central neurotransmitters. After PLS-DA processing, 5-HIAA, 5-HT, ME, ACh, 5-HTP, and DA were successively screened from detected neurotransmitters based on the values of variable importance in the projection (VIP>1) (Figures 3(b) and 3(c)). These all demonstrated that the 5-HT pathway played an important role in inhibiting effect of KXS on neurotransmitter loss.Table 1
The concentrations of central neurotransmitters were determined by UFLC-MS/MS.
Analytes
Control (ng/mL)
Model (ng/mL)
KXS (ng/mL)
Hup A (ng/mL)
ACh
166.7±21.8
99.0±10.0∗∗∗
133.4±10.6###
125.7±7.6##
Tyr
7170±897
4918±716∗∗∗
5886±608#
6407±637##
DA
6.785±1.678
2.673±0.773∗∗∗
5.601±1.383##
5.218±0.965##
ME
10.76±1.45
4.507±0.6441∗∗∗
8.96±1.10###
9.00±0.72###
3-MT
1.821±0.434
0.6645±0.3005∗∗∗
1.505±0.405##
1.425±0.272##
Try
1.637×104±0.332×104
1.580×104±0.435×104
1.848×104±0.465×104
1.763×104±0.219×104
5-HTP
15.56±3.96
4.144±0.985∗∗∗
10.10±3.38###
11.12±1.38###
5-HT
959±30
509.4±110.7∗∗∗
787.9±27.5###
719.1±46.0###
5-HIAA
32.28±2.80
5.864±0.847∗∗∗
29.00±1.43###$$$
24.52±2.33###
GABA
53.17±7.05
37.02±10.26∗
49.05±9.19#
48.07±7.34#
Data were expressed as themean±SD (n=6). ∗P<0.05 and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the model group; and $$$P<0.001vs. the Hup A group.Figure 3
KXS treatment regulated central neurotransmitters in AD rats. The score plot of neurotransmitters in plasma was showed in unsupervised PCA (a) and PLS-DA (b). Control group, the model group, the KXS group, and the Hup A group generated distinct metabolic phenotypes. The VIP of neurotransmitters was represented according to the PLS-DA (c).
(a)
(b)
(c)


### 3.3. KXS Played a Role in Neuroprotective Effect against AD via the PI3K/Akt Signaling Pathway

Previous studies have shown that neurotransmitter 5-HT could regulate PI3K/Akt and glycogen synthase kinase-3 (GSK-3) signaling pathways [19]. Hence, we examined the effect of KXS on the PI3K/Akt/GSK-3β protein signaling pathway by immunoblotting. Our data showed that AD model rats had dramatically impaired PI3K/Akt/GSK-3β signaling caused by Aβ25-35 and D-gal, including reduced p-PI3K, p-Akt, and p-GSK-3β (Ser9), while the rats treated with KXS or Hup A resulted in sufficient restoration of PI3K/Akt GSK-3β signaling (Figures 4(a) and 4(b)). NFTs, another typical pathological change in AD, are mainly caused by the Tau hyperphosphorylation activated by GSK-3β [20]. To address this, we evaluated the p-Tau level with immunoblot and immunohistochemistry. The results of western blot (Figures 4(a) and 4(b)) and immunohistochemistry (Figures 4(c) and 4(d)) showed that KXS could prominently reduce the expression of p-Tau (S199 and S396). In brief, the above results demonstrated that the neuroprotection of KXS was achieved with modulating Tau hyperphosphorylation via the PI3K/Akt/GSK-3β signaling pathway.Figure 4
KXS inhibited hyperphosphorylation of Tau by activating the PI3K/Akt/GSK-3β signaling pathway. KXS upregulated the PI3K/Akt signaling pathway, restored the phosphorylation activity of GSK-3β (Ser9), and decreased the degree of Tau phosphorylation caused by Aβ25-35 and D-gal. Representative western images (a) and quantification (b) of protein related to the Tau signaling pathway in the rat hippocampus were represented. The area fraction of the hippocampus occupied by p-Tau (S396) was calculated using Image-Pro Plus software (c and d). Values were the mean±SD (n=6). ∗∗∗P<0.001 vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001 vs. the model group; $P<0.05 and $$P<0.01 vs. the Hup A group. Scalebar=25μm.
(a)
(b)
(c)
(d)Inhibition of inflammatory factor and ROS-induced neuronal apoptosis via PI3K/Akt and GSK-3β pathways has been approved as an available therapeutic strategy for AD [21]. Various studies have indicated that there was a vicious circle between oxidative stress and neuroinflammation which were representative pathomechanisms of AD [22]. With the treatment of KXS, the ROS level measured with dichlorofluorescein (DCF) fluorescence and concentrations of inflammatory factors such as IL-1β and TNF-α were significantly reduced in the rat hippocampus (Figures 5(a)–5(c)). Moreover, the arachidonic acid pathway, leading to the increase of proinflammatory mediators, was evaluated by the UFLC-MS/MS to examine the anti-inflammatory effects of KXS [23]. We found that KXS could significantly decrease the concentration of AA as well as its metabolites like 5-HETE, LTB4, and TXB2 (Table 2). In the light of unsupervised PCA score plots, plasma samples of four groups were completely separated, and KXS was more effective than Hup A for a comprehensive evaluation on the regulation of AAs (Figure 5(f)). Meanwhile, increased apoptosis in the rat hippocampus, measured with the expression of Bax/bcl-2 and cleaved-caspase-3, were downregulated by KXS treatment (Figures 5(d) and 5(e)). Based on the above results, KXS could reduce the neuroinflammation and ROS-induced neuronal apoptosis via PI3K/Akt/GSK-3β pathways.Figure 5
KXS inhibited oxidative stress, neuroinflammation, and apoptosis in AD rat hippocampus. The levels of ROS (a), IL-1β (b), and TNF-α (c) in the rat hippocampus were measured using commercial kits. The representative western images (d) and quantification (e) of Bax/bcl-2 and cleaved-caspase-3 in the rat hippocampus were showed. The score plot of arachidonic acid and its metabolites in plasma was showed in unsupervised PCA (f). Values were the mean±SD (n=6). ∗∗∗P<0.001vs. the control group; ###P<0.001vs. the model group; and $P<0.05 and $$$P<0.001vs. the Hup A group.
(a)
(b)
(c)
(d)
(e)
(f)Table 2
The concentrations of arachidonic acid and its metabolites in rat plasma determined by UFLC-MS/MS.
Analytes
Control (ng/mL)
Model (ng/mL)
KXS (ng/mL)
Hup A (ng/mL)
5-HETE
0.807±0.276
1.748±0.248∗∗∗
0.934±0.192###$$
1.344±0.0695##
8-HETE
39.19±6.00
55.78±10.24∗∗∗
34.68±7.88###
38.21±4.67###
12-HETE
35.19±8.97
69.06±8.73∗∗∗
45.14±7.57###
52.82±3.67###
15-HETE
28.33±2.92
47.90±6.83∗∗∗
31.33±2.04###$$$
48.22±5.87
AA
1541±228
3571±242∗∗∗
1684±136###$$$
3076±391##
LTB4
20.10±6.68
34.92±6.78∗∗∗
27.63±4.37#
31.67±4.58
TXB2
474.1±83.7
975.2±106.7∗∗∗
676.9±64.4###$
850±172
Values were themean±SD (n=6). ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the model group; and $P<0.05, $$P<0.01, and $$$P<0.001vs. the Hup A group.


### 3.4. KXS Treatment Decreased the Activity of PC12 Cells and the Expression of Tau Signaling PathwayIn Vitro

To investigate whether KXS treatment could protect PC12 cells against Aβ25-35-induced damage, we measured the cell viability with MTT and LDH release assays [24]. According to MTT results, alive PC12 cells at 24 h coculture with Aβ25-35 at 10, 20, and 40 μmol/L were 76.96±2.85%, 57.23±3.67%, and 16.26±1.54%, respectively (Figure 6(a)). 20 μmol/L Aβ25-35 was chosen in the later experiments which would result in 50-60% cell death. In the presence of an increased dose of KXS (0.05 to 20 mg/mL), we found that KXS at 1 mg/mL could inhibit cell death induced by Aβ25-35; however, with the higher concentration of KXS (20 mg/mL), the cell viability was significantly reduced (Figure 6(b)). Meanwhile, KXS could also effectively meliorate Aβ25-35-caused LDH release in PC12 cells (Figure 6(c)). LY294002 (20 μmol/L), a classic PI3K inhibitor, was applied to further verify the effect of KXS on the neuronal activity via the PI3K signaling pathway (Figure 6(d)). Then, the effect of 5-HT on the expression of p-Akt was explored. After the treatment of 5-HT, the expression of p-Akt was significantly increased, while the regulation of 5-HT was inhibited by LY294002. It was verified that KXS could improve the expression of PI3K/Akt by neurotransmitter 5-HT (Figure 6(e)). What is more, the treatment with KXS upregulated the PI3K/Akt signaling pathway, restored the phosphorylation activity of GSK-3β (Ser9) affected by Aβ25-35, and promoted the downregulation of phosphorylated Tau (S199 and S396) (Figures 6(f) and 6(g)). The use of inhibitor affected the efficacy of KXS, further demonstrating that KXS exerted its efficacy through the PI3K signaling pathway, which was in consistent with the results of KXS in vivo.Figure 6
KXS treatment regulated the activity of neurons and the expression of the Tau signaling pathwayin vitro. The treatment of PC12 cells with Aβ25-35 (10, 20, and 40 μmol/L) alone for 24 h (a) or pretreatment with different concentrations of KXS for 24 h (b) was represented by the MTT assay. The concentration of LDH was assessed as the cytotoxicity assay indicator to assess the effect of different concentrations of KXS (c). The inhibiting effects of LY294002 (5, 10, 20, and 40 μmol/L) were investigated through pretreatment for 1 h (d). The expression of p-Akt was significantly increased by 5-HT (e). KXS had neuroprotection by modulating the hyperphosphorylation of Tau through the PI3K/Akt signaling pathway. Representative western images (f) and quantification (g) of proteins related to the Tau signaling pathway in PC12 cells were represented. Values were the mean±SD (n=6). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the Aβ group; &P<0.05, &&P<0.01, and &&&P<0.001vs. the LY+KXS group; and $$P<0.01vs. the LY+5-HT group.
(a)
(b)
(c)
(d)
(e)
(f)
(g)


### 3.5. KXS Modulated Oxidation and Neuroinflammation-Induced ApoptosisIn Vitro

There were abundant evidences suggesting that excess ROS production and neuroinflammation were involved in Aβ-induced neurotoxicity and harmful to mitochondrial function [25]. Coculture of KXS at 1 mg/mL could dramatically reverse Aβ25-35-induced ROS accumulation (Figure 7(a)) and reduced the MMP level (Figure 7(b)). Interestingly, pretreating the cells with LY294002 resulted in the blockage of ROS reduction and MMP increase, suggesting that PI3K signaling played a role in oxidative stress. We also found that the treatment of Aβ25-35 alone significantly increased the levels of IL-1β and TNF-α compared to the control (Figures 7(c) and 7(d)). However, the increase was significantly reduced in the presence of KXS at 1 mg/mL. The neuroinflammation and ROS production resulting in mitochondrial dysfunction could also activate the following caspase cascade up to apoptosis [26]. KXS treatment could significantly reduce the expressions of Bax/bcl-2 and cleaved-caspase-3 (Figures 7(e) and 7(f)) and the percentage of apoptotic cells (Figures 7(g) and 7(h)) compared to the control, which was evidenced by immunoblotting and flow cytometry. Furthermore, the pretreatment of LY294002 influenced the effect of KXS on inhibiting oxidation and neuroinflammation-induced apoptosis, indicating the PI3K signaling pathway played a vital role in neuroprotection of KXS.Figure 7
KXS treatment inhibited oxidative stress and neuroinflammation-induced apoptosisin vitro. KXS treatment decreases the levels of ROS (a) and MMP (b), the concentration of IL-1β and TNF-α (c and d), and the expressions of Bax/bcl-2 and cleaved-caspase-3 (e and f), and the cell apoptosis rate (g and h) enhanced by Aβ25-35 in PC12 cells. Values were the mean±SD (n=6). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the Aβ group; and &P<0.05, &&P<0.01, and &&&P<0.001vs. the LY+KXS group.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.1. KXS Improved Learning and Memory Impaired in AD Rats Induced by D-Gal and Aβ25-35

The AD animal model induced by D-gal and Aβ25-35 was applied in order to simulate human pathological processes. The quality control of KXS was achieved by HPLC fingerprint (Figure S1). The water maze test was adopted to detect the effect of KXS on learning and memory in AD rats. Hup A (AChEIs), an alkaloid isolated from the Chinese herbal medicine Huperzia serrata, was licensed as a nutraceutical in the United States and widely used for AD treatment in China [16]. From the second day of training trials, the rats in the model group showed longer escape latency and swimming distance, meaning obvious learning and memory impairment compared to the control (Con) group (Figures 2(a) and 2(b)). On the fifth day of the Morris water maze, the spatial probe test was tested. The rats treated with Hup A or KXS showed significantly less escape latency and swimming distance to find the removal platform in the target quadrant (Figures 2(c)–2(e)). In addition, Hup A and KXS group rats showed more platform crossings than the model group (Figure 2(f)). Neuronal nuclei (NeuN), a neuronal special nuclear marker, was detected by immunohistochemistry staining to evaluate the neuroprotection of KXS on neuronal density in hippocampal CA1 regions [17]. The fluorescence intensity of NeuN in the model group was significantly reduced, which was a hallmark pathology of neuronal damage in AD and indicated a successful AD model. KXS treatment observably increased fluorescence intensity of NeuN compared with the model group (Figures 2(g) and 2(h)). The results of the Morris water maze and immunohistochemistry staining of NeuN suggested that KXS could be potentially used for the prevention and/or treatment of AD.Figure 2
KXS treatment ameliorated memory and learning impairment. The mean escape latency (a) and swimming distances (b) were obtained from different test days, and the KXS group spent less time and distances to reach the fourth quadrant platform. Representative rat search paths from different groups (c) were shown on the fifth day. The rats treated with Hup A or KXS showed significantly less escape latency (d), swimming distance (e), and platform crossings (f) to find the removal platform in the fourth quadrant. Impact of KXS on modulating hippocampal NeuN levels was assessed by immunohistochemistry. The fluorescence intensity was quantified using Image-Pro Plus software (g and h). The treatment of KXS showed improvement in spatial memory. Values were themean±SD (n=6). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the model group; $$P<0.01vs. the Hup A group. Scalebar=20μm.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.2. KXS Treatment Increased the Concentration of Central Neurotransmitters in AD Rats

In order to study the influence of KXS on central neurotransmitter loss, a typical pathologic manifestation, the UFLC-MS/MS method was used to detect the concentrations of related endogenous neurotransmitters. The method was validated according to the European Medicines Agency guidance for bioanalytical method validation (TablesS1-S2) [18]. Compared with the model group, concentrations of four types of neurotransmitters, namely, ACh, GABA, dopamine (Tyr, dopamine, ME, and 3-MT), and 5-HT (5-HTP, 5-HT, and 5-HIAA), were increased in rat plasmas after drug treatment (Table 1). Therefore, KXS could protect an impaired central nervous system by ameliorating neurotransmitter loss. Neurotransmitter data were handled with PCA. In the light of unsupervised PCA score plots, plasma samples in KXS and Hup A groups were located between the AD model group and the control group. Nevertheless, there was no significant difference between the KXS group and the Hup A group (Figure 3(a)). The PCA result also proved the efficacy of KXS on regulating the levels of central neurotransmitters. After PLS-DA processing, 5-HIAA, 5-HT, ME, ACh, 5-HTP, and DA were successively screened from detected neurotransmitters based on the values of variable importance in the projection (VIP>1) (Figures 3(b) and 3(c)). These all demonstrated that the 5-HT pathway played an important role in inhibiting effect of KXS on neurotransmitter loss.Table 1
The concentrations of central neurotransmitters were determined by UFLC-MS/MS.
Analytes
Control (ng/mL)
Model (ng/mL)
KXS (ng/mL)
Hup A (ng/mL)
ACh
166.7±21.8
99.0±10.0∗∗∗
133.4±10.6###
125.7±7.6##
Tyr
7170±897
4918±716∗∗∗
5886±608#
6407±637##
DA
6.785±1.678
2.673±0.773∗∗∗
5.601±1.383##
5.218±0.965##
ME
10.76±1.45
4.507±0.6441∗∗∗
8.96±1.10###
9.00±0.72###
3-MT
1.821±0.434
0.6645±0.3005∗∗∗
1.505±0.405##
1.425±0.272##
Try
1.637×104±0.332×104
1.580×104±0.435×104
1.848×104±0.465×104
1.763×104±0.219×104
5-HTP
15.56±3.96
4.144±0.985∗∗∗
10.10±3.38###
11.12±1.38###
5-HT
959±30
509.4±110.7∗∗∗
787.9±27.5###
719.1±46.0###
5-HIAA
32.28±2.80
5.864±0.847∗∗∗
29.00±1.43###$$$
24.52±2.33###
GABA
53.17±7.05
37.02±10.26∗
49.05±9.19#
48.07±7.34#
Data were expressed as themean±SD (n=6). ∗P<0.05 and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the model group; and $$$P<0.001vs. the Hup A group.Figure 3
KXS treatment regulated central neurotransmitters in AD rats. The score plot of neurotransmitters in plasma was showed in unsupervised PCA (a) and PLS-DA (b). Control group, the model group, the KXS group, and the Hup A group generated distinct metabolic phenotypes. The VIP of neurotransmitters was represented according to the PLS-DA (c).
(a)
(b)
(c)


## 3.3. KXS Played a Role in Neuroprotective Effect against AD via the PI3K/Akt Signaling Pathway

Previous studies have shown that neurotransmitter 5-HT could regulate PI3K/Akt and glycogen synthase kinase-3 (GSK-3) signaling pathways [19]. Hence, we examined the effect of KXS on the PI3K/Akt/GSK-3β protein signaling pathway by immunoblotting. Our data showed that AD model rats had dramatically impaired PI3K/Akt/GSK-3β signaling caused by Aβ25-35 and D-gal, including reduced p-PI3K, p-Akt, and p-GSK-3β (Ser9), while the rats treated with KXS or Hup A resulted in sufficient restoration of PI3K/Akt GSK-3β signaling (Figures 4(a) and 4(b)). NFTs, another typical pathological change in AD, are mainly caused by the Tau hyperphosphorylation activated by GSK-3β [20]. To address this, we evaluated the p-Tau level with immunoblot and immunohistochemistry. The results of western blot (Figures 4(a) and 4(b)) and immunohistochemistry (Figures 4(c) and 4(d)) showed that KXS could prominently reduce the expression of p-Tau (S199 and S396). In brief, the above results demonstrated that the neuroprotection of KXS was achieved with modulating Tau hyperphosphorylation via the PI3K/Akt/GSK-3β signaling pathway.Figure 4
KXS inhibited hyperphosphorylation of Tau by activating the PI3K/Akt/GSK-3β signaling pathway. KXS upregulated the PI3K/Akt signaling pathway, restored the phosphorylation activity of GSK-3β (Ser9), and decreased the degree of Tau phosphorylation caused by Aβ25-35 and D-gal. Representative western images (a) and quantification (b) of protein related to the Tau signaling pathway in the rat hippocampus were represented. The area fraction of the hippocampus occupied by p-Tau (S396) was calculated using Image-Pro Plus software (c and d). Values were the mean±SD (n=6). ∗∗∗P<0.001 vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001 vs. the model group; $P<0.05 and $$P<0.01 vs. the Hup A group. Scalebar=25μm.
(a)
(b)
(c)
(d)Inhibition of inflammatory factor and ROS-induced neuronal apoptosis via PI3K/Akt and GSK-3β pathways has been approved as an available therapeutic strategy for AD [21]. Various studies have indicated that there was a vicious circle between oxidative stress and neuroinflammation which were representative pathomechanisms of AD [22]. With the treatment of KXS, the ROS level measured with dichlorofluorescein (DCF) fluorescence and concentrations of inflammatory factors such as IL-1β and TNF-α were significantly reduced in the rat hippocampus (Figures 5(a)–5(c)). Moreover, the arachidonic acid pathway, leading to the increase of proinflammatory mediators, was evaluated by the UFLC-MS/MS to examine the anti-inflammatory effects of KXS [23]. We found that KXS could significantly decrease the concentration of AA as well as its metabolites like 5-HETE, LTB4, and TXB2 (Table 2). In the light of unsupervised PCA score plots, plasma samples of four groups were completely separated, and KXS was more effective than Hup A for a comprehensive evaluation on the regulation of AAs (Figure 5(f)). Meanwhile, increased apoptosis in the rat hippocampus, measured with the expression of Bax/bcl-2 and cleaved-caspase-3, were downregulated by KXS treatment (Figures 5(d) and 5(e)). Based on the above results, KXS could reduce the neuroinflammation and ROS-induced neuronal apoptosis via PI3K/Akt/GSK-3β pathways.Figure 5
KXS inhibited oxidative stress, neuroinflammation, and apoptosis in AD rat hippocampus. The levels of ROS (a), IL-1β (b), and TNF-α (c) in the rat hippocampus were measured using commercial kits. The representative western images (d) and quantification (e) of Bax/bcl-2 and cleaved-caspase-3 in the rat hippocampus were showed. The score plot of arachidonic acid and its metabolites in plasma was showed in unsupervised PCA (f). Values were the mean±SD (n=6). ∗∗∗P<0.001vs. the control group; ###P<0.001vs. the model group; and $P<0.05 and $$$P<0.001vs. the Hup A group.
(a)
(b)
(c)
(d)
(e)
(f)Table 2
The concentrations of arachidonic acid and its metabolites in rat plasma determined by UFLC-MS/MS.
Analytes
Control (ng/mL)
Model (ng/mL)
KXS (ng/mL)
Hup A (ng/mL)
5-HETE
0.807±0.276
1.748±0.248∗∗∗
0.934±0.192###$$
1.344±0.0695##
8-HETE
39.19±6.00
55.78±10.24∗∗∗
34.68±7.88###
38.21±4.67###
12-HETE
35.19±8.97
69.06±8.73∗∗∗
45.14±7.57###
52.82±3.67###
15-HETE
28.33±2.92
47.90±6.83∗∗∗
31.33±2.04###$$$
48.22±5.87
AA
1541±228
3571±242∗∗∗
1684±136###$$$
3076±391##
LTB4
20.10±6.68
34.92±6.78∗∗∗
27.63±4.37#
31.67±4.58
TXB2
474.1±83.7
975.2±106.7∗∗∗
676.9±64.4###$
850±172
Values were themean±SD (n=6). ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the model group; and $P<0.05, $$P<0.01, and $$$P<0.001vs. the Hup A group.


## 3.4. KXS Treatment Decreased the Activity of PC12 Cells and the Expression of Tau Signaling PathwayIn Vitro

To investigate whether KXS treatment could protect PC12 cells against Aβ25-35-induced damage, we measured the cell viability with MTT and LDH release assays [24]. According to MTT results, alive PC12 cells at 24 h coculture with Aβ25-35 at 10, 20, and 40 μmol/L were 76.96±2.85%, 57.23±3.67%, and 16.26±1.54%, respectively (Figure 6(a)). 20 μmol/L Aβ25-35 was chosen in the later experiments which would result in 50-60% cell death. In the presence of an increased dose of KXS (0.05 to 20 mg/mL), we found that KXS at 1 mg/mL could inhibit cell death induced by Aβ25-35; however, with the higher concentration of KXS (20 mg/mL), the cell viability was significantly reduced (Figure 6(b)). Meanwhile, KXS could also effectively meliorate Aβ25-35-caused LDH release in PC12 cells (Figure 6(c)). LY294002 (20 μmol/L), a classic PI3K inhibitor, was applied to further verify the effect of KXS on the neuronal activity via the PI3K signaling pathway (Figure 6(d)). Then, the effect of 5-HT on the expression of p-Akt was explored. After the treatment of 5-HT, the expression of p-Akt was significantly increased, while the regulation of 5-HT was inhibited by LY294002. It was verified that KXS could improve the expression of PI3K/Akt by neurotransmitter 5-HT (Figure 6(e)). What is more, the treatment with KXS upregulated the PI3K/Akt signaling pathway, restored the phosphorylation activity of GSK-3β (Ser9) affected by Aβ25-35, and promoted the downregulation of phosphorylated Tau (S199 and S396) (Figures 6(f) and 6(g)). The use of inhibitor affected the efficacy of KXS, further demonstrating that KXS exerted its efficacy through the PI3K signaling pathway, which was in consistent with the results of KXS in vivo.Figure 6
KXS treatment regulated the activity of neurons and the expression of the Tau signaling pathwayin vitro. The treatment of PC12 cells with Aβ25-35 (10, 20, and 40 μmol/L) alone for 24 h (a) or pretreatment with different concentrations of KXS for 24 h (b) was represented by the MTT assay. The concentration of LDH was assessed as the cytotoxicity assay indicator to assess the effect of different concentrations of KXS (c). The inhibiting effects of LY294002 (5, 10, 20, and 40 μmol/L) were investigated through pretreatment for 1 h (d). The expression of p-Akt was significantly increased by 5-HT (e). KXS had neuroprotection by modulating the hyperphosphorylation of Tau through the PI3K/Akt signaling pathway. Representative western images (f) and quantification (g) of proteins related to the Tau signaling pathway in PC12 cells were represented. Values were the mean±SD (n=6). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the Aβ group; &P<0.05, &&P<0.01, and &&&P<0.001vs. the LY+KXS group; and $$P<0.01vs. the LY+5-HT group.
(a)
(b)
(c)
(d)
(e)
(f)
(g)


## 3.5. KXS Modulated Oxidation and Neuroinflammation-Induced ApoptosisIn Vitro

There were abundant evidences suggesting that excess ROS production and neuroinflammation were involved in Aβ-induced neurotoxicity and harmful to mitochondrial function [25]. Coculture of KXS at 1 mg/mL could dramatically reverse Aβ25-35-induced ROS accumulation (Figure 7(a)) and reduced the MMP level (Figure 7(b)). Interestingly, pretreating the cells with LY294002 resulted in the blockage of ROS reduction and MMP increase, suggesting that PI3K signaling played a role in oxidative stress. We also found that the treatment of Aβ25-35 alone significantly increased the levels of IL-1β and TNF-α compared to the control (Figures 7(c) and 7(d)). However, the increase was significantly reduced in the presence of KXS at 1 mg/mL. The neuroinflammation and ROS production resulting in mitochondrial dysfunction could also activate the following caspase cascade up to apoptosis [26]. KXS treatment could significantly reduce the expressions of Bax/bcl-2 and cleaved-caspase-3 (Figures 7(e) and 7(f)) and the percentage of apoptotic cells (Figures 7(g) and 7(h)) compared to the control, which was evidenced by immunoblotting and flow cytometry. Furthermore, the pretreatment of LY294002 influenced the effect of KXS on inhibiting oxidation and neuroinflammation-induced apoptosis, indicating the PI3K signaling pathway played a vital role in neuroprotection of KXS.Figure 7
KXS treatment inhibited oxidative stress and neuroinflammation-induced apoptosisin vitro. KXS treatment decreases the levels of ROS (a) and MMP (b), the concentration of IL-1β and TNF-α (c and d), and the expressions of Bax/bcl-2 and cleaved-caspase-3 (e and f), and the cell apoptosis rate (g and h) enhanced by Aβ25-35 in PC12 cells. Values were the mean±SD (n=6). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001vs. the control group; #P<0.05, ##P<0.01, and ###P<0.001vs. the Aβ group; and &P<0.05, &&P<0.01, and &&&P<0.001vs. the LY+KXS group.
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## 4. Discussion

Nowadays, FDA-approved drugs for the treatment of AD were single-target, which did not have satisfactory therapeutic effects aiming at various pathologic changes including abnormalβ-amyloid and Tau [27, 28]. For these reasons, a perfect therapeutic strategy against AD should be comprehensive pharmacological improvements with multiple targets [29]. In the last years, the pharmaceutical development has turned to search for Chinese herbs with multiple targets and pathways rather than just pursuit medicines that specifically target a single disease-causing factor [30]. The classical prescription, KXS, was used for dementia treatment for thousands of years by means of restoring the body dynamic balances [31]. The development of KXS was restrained due to the lack of a definite mechanism. Thus, KXS was investigated by previously optimizing the pharmacokinetic study on AD rats [10], and the underlying mechanisms of KXS against AD were researched in vivo and in vitro.In China, Hup A, a natural alkaloid, was widely applied to improve cognition impairments of dementia in the clinical application via multiple pathways [32]. Therefore, Hup A was used as the positive control in all animal studies and proved the improvements on cognition impairment, neurotransmitter imbalance, oxidative stress, etc. The behavioral experiments indicated the improving effect of KXS on learning and memory abilities. To explore the underlying mechanism, the effects of KXS on the classic pathologic pathways of AD were further studied. Based on our finding, a model for the neuroprotective effect of KXS against AD pathogenesis was proposed (Figure 8). Firstly, the mechanism research was carried out with quantitative determination of neurotransmitters which could be potential biomarkers and provide the evidences for early diagnosis and treatment of AD. The pathogenesis of AD was closely related to the changes of neurotransmitter homeostasis [33]. Aβ could impair patient’s memory and executive function by reducing the concentration of neurotransmitters in the cerebral cortex and hippocampus such as dopamine and 5-HT [34]. The detected neurotransmitters mainly include GABA, ACh, dopamine pathway, and 5-HT pathway in target metabolomics, and it was confirmed that KXS played a crucial role in treating AD by increasing those neurotransmitters (Table 1). In PCA result, there was no significant separation between the KXS group and the Hup A group which also proved that KXS and Hup A have the same effect on improving neurotransmitters. According to PLS-DA result, the 5-HT pathway made great contribution to the sample grouping. Previously, it was reported that 5-HT receptors could modulate the release of ACh [35]. These all demonstrated that 5-HT played an important role in inhibiting effect of KXS on neurotransmitter loss. Moreover, the PI3K/Akt/GSK-3 signal pathway was affected by the central neurotransmitter signaling pathways such as 5-HT [19]. We also confirmed that it was reasonable to ameliorate the expression of PI3K/Akt by improving the concentration of 5-HT.Figure 8
KXS ameliorated behavioral and pathological deterioration via the improvement of neurotransmitters and PI3K/Akt signaling pathways on the AD model.GSK-3, a serine/threonine protein kinase, which was found closely related to Aβ and Tau, belonged to the proline-directed kinase and had two subtypes: GSK-3α and GSK-3β [36]. The GSK-3α was involved in regulating the production of Aβ peptide, while the latter was involved in the phosphorylation of Tau and the formation of NFTs [37]. Deposition of Aβ could further cause synaptic changes, abnormal phosphorylation of Tau proteins, transmitter loss, and memory dysfunction [38, 39]. The abnormal hyperphosphorylated Tau was present in the neurofibrillary tangles, disrupted microtubule system, and constituted a threat to the stability of neurons. Therefore, it was involved in the pathogenesis of AD as the main pathological feature of AD [40]. It was demonstrated that KXS could upregulate PI3K/Akt, along with the inhibition of GSK-3β activation as well as the progressive reduction of the phosphorylation level of Tau protein (S199 and S396) in the hippocampus, and these protein signaling pathways were mediated by central neurotransmitters.There were often metabolic disorders and increased oxidative stress in the brain of AD patients [41]. Deposition of Aβ could produce a large number of free radicals and further cause apoptosis [42]. Available evidences showed that overaccumulation of ROS would cause mitochondrial membrane damage [43]. Our experiments found that KXS could significantly improve mitochondrial function by reducing ROS levels and increasing mitochondrial membrane potential. Several studies suggest that a vicious circle between oxidative stress and neuroinflammation was a significant pathogenic feature in AD where the circle might aggravate the Aβ-induced pathology and the hyperphosphorylation of Tau [44]. In vitro studies have demonstrated that these proinflammatory cytokines such as IL-1β and TNF-α were neurotoxic and could induce neuronal death [45]. AA is an essential fatty acid that plays an important role in signal transduction of neuronal cells and glial cells, which can be converted into prostaglandin, thromboxane, leukotriene, etc. In AD, the increased ROS and proinflammatory factors induced by Aβ can cause excessive activation of phospholipase A2 (PLA2) and release of excessive AA, which ultimately leads to nerve cell apoptosis [46]. We found that KXS had significantly reduced inflammatory factors (IL-1β and TNF-α) and AA as well as its metabolites in plasma, indicating that KXS has the anti-inflammatory effect. Thus, we thought that the antiapoptosis effect of KXS was achieved by inhibiting mitochondrial dysfunction and inflammation.In summary, our studies indicated that the treatment of KXS significantly improved cognition impairment by decreasing neurotransmitter loss and further causing the intensified expression of PI3K/Akt, which could further inhibit the hyperphosphorylation of Tau as well as the apoptosis induced by oxidative stress and neuroinflammation. We dissected the KXS as a new therapy for the treatment of AD at the animal, molecular, and cellular levels; it may be considered as a valuable modality to explore the value of Chinese classical prescription.



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*Source: 9096409-2019-07-01.xml*