CDPS Reduces Cell Apoptosis Induced By Aβ25 － 35 Through Brain-derived Neurotrophic Factor Regulated By Epigenetic Modulation

WU Yan1 ，LIU Dan-dan1 ，MA Zi-xing1 ，MIAO Xin1 ，ZHANG Xiao-fei1 ，LI Gang1，2 ( 1 Inner Mongolia Medical University，Department of Pharmacy，HOHHOT 010110，China; 2 State Key Laboratory of Bioactive Substance and Function of Natural Medicines，Institute of Materia Medica，Chinese Academy of Medical Sciences and Peking Union Medical College，Beijing 100050，China)

［Abstract］ Objective: To investigate the regulatory effect of Cistanche polysaccharides ( CDPS) on the epigenetic changes of BDNF gene induced by Aβ25 － 35 in rat adrenal sinophil tumor cells ( PC12) ，and to analyze the effect of histone acetylation on BDNF gene transcription． Methods: The apoptosis of PC12 cells was induced by Aβ25 － 35 in vitro． Alzheimer's disease ( AD) model was established in vitro． The apoptotic cells were detected by flow cytometry after the positive drug ginsenoside Ｒd ( GSrd) and CDPS were given． The changes of cell morphology were observed by Hoechst33258 staining． The levels of HATs，HDAC2 and BDNF were detected by ELISA method． The expression level of P300 and HDAC2 protein was detected by Western Blotting． The alterations of histone H3 acetylation and HDAC2 in BDNF exon Ⅳ were observed and analyzed by chromatin immunoprecipitation ( CHIP) ．

Chinese herb cistanche-Anti Alzheimer's disease

Results: After exposure of the PC12 cells 48 h by 20 μmol·L － 1 Aβ25 － 35，the model of AD cell apoptosis was established successfully． Compared with model group，each group HATs expression increases (P＜0．05) ，HDAC2 decrease (P＜0．05) ，and BDNF levels increase (P＜0．05) ，and there are dose dependent．ChIP analysis confirmed that after Aβ25－35 induced P12 cells，BDNF exon Ⅳ histone H3 acetylation protein levels decreased significantly，and the reduction of histone acetylation is HDAC2-dependent．48 h after CDPS administration，BDNF exon Ⅳhistone H3 acetylation protein levels increased significantly，and the level of HDAC2 decreased．Conclusion: CDPS had significant protective effects on PC12 cells exposed to Aβ25－35 ．The mechanism may be that the decrease of acetylation of HDAC2-dependent histone H3 induced the suppression of continuous transcription of BDNF gene． 

［Key words］Cistanche polysaccharides; PC12 cells; histone deacetylase 2; histone H3

Cistanche desert cola Y.C.Ma is a dry, scaly, fleshy stem of the Orobaceae plant Cistanche desert cola Y.C.Ma or Cistanche tubulosa (Schrenk) R. Wight. Entering the kidney meridian can replenish kidney yang and benefit essence and blood; Entering the large intestine meridian can moisten the intestines and relieve constipation. Research has shown that most medicinal plant polysaccharides have significant biological activities, such as immune regulation, anti-aging, anti-inflammatory and liver protection, and anti-tumor effects.

 Cistanche tubulosa

Cistanche desert cola Y.C.Ma

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Cognitive decline is the main pathological feature of this disease, but the causes of cognitive decline are currently unclear and there is a lack of effective treatment methods. 

Effects Of Cistanche-Anti Alzheimer's disease

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Numerous studies [4] have found that abnormal histone acetylation is a common epigenetic abnormality in the pathogenesis of AD. In the study of the pathological mechanism of characteristic symptoms of AD, such as cognitive impairment, it was found that histone acetylation modification regulates the expression of memory-related genes [5-7]. The dynamic balance of histone acetylation is jointly regulated by two active enzymes, histone acetylases (HATs) and histone deacetylases (HDACs). HATs mainly carry out reversible acetylation on the lysine residues at the N-terminal tail of the core histone of chromatin nucleosome, neutralize the positive charge between DNA and adjacent histones, promote DNA unwinding, relax the structure of chromatin, and facilitate the binding of transcription factors with DNA functional areas, and finally activate transcription. The currently discovered histone acetylase involved in learning and memory is P300/CBP [8]. In addition, studies have shown that the decrease in HATs activity is involved in neurodegenerative diseases.

Brain-derived neurotrophin (BDNF), an important neurotrophin of neurons and glia, is considered one of the key proteins in the process of memory formation and is necessary for the survival and normal physiological function of neurons in the mature central and peripheral nervous system [10-11]. Some studies have shown that BDNF is highly expressed in the granular cell layer (GCL) of the dentate gyrus, indicating that it participates in synaptic plasticity and neuronal development through TrkB receptors. In addition, BDNF expression and release can also undergo genetic and epigenetic variations, such as histone modifications [12-13]. P300/CBP is a transcriptional co-activator with HAT activity, which has an important impact on the long-term memory process, depending on the de novo gene expression [14], and the expression of BDNF is regulated by P300/CBP activity [15]. Research has confirmed [16] that not only environmental factors can cause long-term epigenetic changes in the BDNF promoter region, but also drug stimulation, such as imipramine enhancing H3 acetylation of bdnf P4 and P6 through downregulation of HDAC5. However, we are still unclear whether these rapid epigenetic changes are involved in the administration of Cistanche deserticola polysaccharides (CDPS) β 25-35 induces BDNF transcriptional expression in PC12 cells.

This study used GRsd as a positive drug [17], and the results confirmed that in A β The AD cell model induced by 25-35 was administered with three active ingredients of Cistanche deserticola, and the balance between Ac-H3 and HDAC2 was reconstructed to increase BDNF expression, thereby improving and promoting neuronal survival. This study suggests that the expression and release of BDNF are closely related to epigenetic modifications, particularly histone acetylation changes regulated by HDAC and HAT.

Material and Methods

1  Drugs and reagents

DMEM culture medium (Gibco, USA, batch number 12491-015); FBS (Gibco, USA, batch number 10099141); MTT (Sigma, USA, batch number M5655); BDNF Enzyme Linked Immunoassay Kit (Wuhan Xinqidi Biotechnology Co., Ltd., batch number 3374518); HATs enzyme-linked immunosorbent assay kit (Wuhan Xinqidi Biotechnology Co., Ltd., batch number: 6907299); HDAC2 enzyme-linked immunosorbent assay kit (Wuhan Xinqidi Biotechnology Co., Ltd., batch number: 6907236); Ginsenoside (Chengdu Manchester Biotechnology Co., Ltd., batch number A0245); A β 25-35 (Shanghai Jier Biochemical Co., Ltd., batch number: 10-375); HDAC2 polyclonal antibody (Abcam UK, batch number: ab32117); P300 polyclonal antibody (Abcam UK, batch number: ab142163); β- Actin polyclonal antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., batch number: ZM-0001); HRP sheep anti rabbit secondary antibody (Abcam Company, UK, batch number: ab205718); Color pre stained protein molecular weight standard (Sigma Company, USA); ECL color solution (Thermo Fisher Scientific, batch number 10318H08); Nuclear protein extraction kit (Kangwei Century Biotechnology Co., Ltd., batch number: CW0199S); Protein phosphatase inhibitor mixture (Kangwei Century Biotechnology Co., Ltd., batch number: CW2383S); Chromatin immunoprecipitation kit (Merck Millipore, Germany, batch number: 17-371).

2 Main instruments

PL 203 electronic balance (Mettler Toledo Instruments Shanghai Co., Ltd.); TGL 16 ultra-low temperature centrifuge (Changsha Pingfan Instrument Co., Ltd.); TU 1901 Ultraviolet Spectrophotometer (Shanghai Precision Instruments and Meters Co., Ltd.); DYY 6C electrophoresis instrument power supply (Beijing Liuyi Instrument Factory); Polyacrylamide gel electrophoresis (Western Blotting electrophoresis, Beijing Liuyi Instrument Factory); Membrane conversion device (Bio RAD, USA); WH 1 micro vortex mixer (Shanghai Huxi Analytical Instrument Factory); 1/100000 electronic balance (Mettler Toledo, USA); ten μ L，100 μ L，200 μ L and 1000 μ L pipette gun (Rainin Instrument, USA); MDF U3386S low-temperature refrigerator (Sanyo Company, Japan); SB2200 T ultrasonic cleaner (Shanghai Bineng Xin Ultrasonic Co., Ltd.); Model 680 ELISA (Bio RAD, Japan); C6 flow cytometry (BD Ac curi, USA); Odyssey dual color infrared fluorescence imaging system (LI-COR company in the United States); UH 100A Ultrasonic Cell Pulverizer (Tianjin Otsaines Co., Ltd.).

3 Preparation and Identification of 3 CDPS

The raw medicinal material of Cistanche deserticola ma is soaked in hot ethanol for 3 hours, filtered with gauze, and the filtrate is discarded. Boil the medicinal residue with water three times, each time for 1-2 hours, filter, and merge the filtrate (showing a brownish-red color). After evaporation and concentration, centrifuge at 5000 r · min-1 for 10 minutes to precipitate. The supernatant was precipitated with 2-3 times the volume of 95% ethanol and left to stand at 4 ℃ for 24 hours. The next day, centrifuge at 6000 r · min-1 (4 ℃) for 20 minutes to collect the precipitate. CDPS was obtained from the precipitate after water solubilization, deproteinization, dialysis, and freeze-drying. The content of polysaccharides is more than 90% by UV spectrophotometry, and the rest is a few monosaccharides and oligosaccharides.

Chinese herb cistanche

4 cells

PC12 cell line (Beijing Xiehe Cell Resource Center).

5 Establishment of a 5-cell model

Take PC12 cells with logarithmic growth phase, and measure them at a rate of 3% per milliliter × Inoculate 104 pieces of density into a 96-well culture plate, with 0.1 mL per well. After 48 hours of cultivation, use A β 25-35 and CDPS (50 and 150 μ After 48 hours of co-action with g · mL-1, cells were collected for the experiment.

6 Detection of apoptosis by flow cytometry

Use deionized water to mix 10 × Binding Buffer diluted to 1 × Binding Buffer; Centrifuge at 2000 r · min-1 (room temperature) for 5-10 minutes to collect cells; Cell washing: pre-cooled at 4 ℃ with 1 × Resuspended PBS cells, centrifuged at 2000 r · min-1 for 5-10 minutes, and washed cells; Add 300 μ 1 of L × Binding Buffer suspension cells; Annexin VFITC Flag: Add 5 μ Mix L's Annexin V-FITC well and incubate at room temperature for 15 minutes in a dark place; Add 5 more minutes before starting the machine μ PI staining of L. Within 1 hour, perform flow cytometry detection and observation.

7 Enzyme-linked immunosorbent assay for the determination of HATs, HDAC2, and BDNF

After drug treatment, the PC12 cell culture medium was collected, centrifuged at 2500 r · min-1 for 10 minutes, and the supernatant was taken for testing. Create a standard curve, follow the operation steps of the reagent kit, add the specified volume of detection reagent, and incubate in dark for 30 minutes. Measure the absorbance (A) value at 450 nm on an enzyme-linked immunosorbent assay (ELISA) and calculate the content of HATs, HDAC2, and BDNF. Each experimental group was set up with 3 compound wells, and the experiment was repeated 3 times.

8 Western Blotting Electrophoresis

RIPA buffer was used to lyse PC12 cells, extract nuclear proteins, and quantify protein content using the BCA method. Add RIAP buffer and adjust the protein levels of each group to be consistent. SDS-PAGE electrophoresis was performed until bromophenol blue reached the bottom of the separation gel and was transferred to the NC membrane (100 V, 2 hours). The protein marker was pre-stained to determine the standard molecular weight position of the protein. Seal with 50 g · L-1 skim milk powder for 1 hour. First antibody P300/CBP (1:5000), HADC2 (1:5000), and β- The actin (1:200) polyclonal antibody was diluted proportionally with a blocking solution, incubated overnight at 4 ℃, and then decolorized 3 times with TBS for 10 minutes each time. The horseradish peroxidase-labeled goat anti-rabbit polyclonal antibody (1:5000) was incubated at room temperature for 1 hour, and DAB was added for color development. The band grayscale was analyzed using Bandscan analysis software for semi-quantitative comparative analysis, and self-grayscale values were used to correct the band grayscale of the target gene and the housekeeping factor β- The grayscale ratio of actin represents the protein expression level.

9 chromatin immune coprecipitation (ChIP)

Cultivate PC12 cells in a 150 mm culture dish containing 20 mL of growth medium to a density of 80% to 90%, and stir at room temperature with 1% formaldehyde for 15 minutes. Add glycine (125 mmol · L-1 final concentration) to terminate fixation. Wash the tissue in PBS and suspend the precipitate in SDS lysis buffer (50 mmol · L-1 Tris, 10 mmol · L-1 EDTA, 1% SDS). 6 × Chromatin was ultrasonically treated on ice for 10 s to obtain fragments of about 200~400 bp. Save 1/10 of the cracked product as input for ChIP standardization. Add polyclonal antibody samples targeting acetylated histone H3 or HDAC2 antibodies to each group and incubate them gently at 4 ℃ overnight, using rabbit or mouse IgG as a negative control. Incubate the immune complex with magnetic beads (life technologies) for 4-6 hours, then wash with 150 mmol · L-1 NaCl low salt buffer and 500 mmol · L-1 NaCl high salt buffer, resuspended in 120 mL of 1% SDS containing 0.1 mol · L-1 NaHCO3 and incubated overnight at 65 ℃. The cross-linked samples were treated with RNase and protease K (Sigma Aldrich, USA), and DNA (Qiagen) was purified using a QIQuick PCR purification kit. Quantitative real-time PCR was performed on immunoprecipitation DNA samples. PCR amplification of the transcriptional control region of the target gene (approximately 200 bp). The primer design is as follows: bdnf exon IV (5 '- TCAGGAGTACATATCGGCCACCA-3', 5 '- GTAGGC CAAGTGCCTTGTCCGT-3') GAPDH (5 '- AGACAGC CGCATCTTCTTGT-3', 5 '- CTGCGGAGAAGAAGT CAG-3').

10 Statistical processing

All data are expressed in x ± s. SPSS software was used for statistical analysis, and single-factor analysis of variance was used to compare multiple experimental groups. P<0.05 indicates a statistically significant difference.

result

1 CDPS vs. A β Protective effect of 25-35 induced injury

The results show that A β After 48 hours of treatment with 25-35 cells, the cell survival rate decreased to (42.91 ± 4.59)%, with a statistically significant difference compared to the control group (P<0.05). The positive drug ginsenoside (GSrd) has a protective effect on cells, with a statistically significant difference compared to the model group (P<0.05); CDPS (50 and 150 μ G · mL-1) has a protective effect on AD model cells, and is dose-dependent, with a statistically significant difference compared to the model group (P<0.05).

The results of MTT method are shown in Table 1.

Table 1  CDPS for A β The effect of 25-35 on the cell survival rate of PC12 induced by n=6, x ± s

Compared with the normal group, a: P<0.05; Compared with the model group, b: P ＜ 0.05

2 The Effect of 2 CDPS on the Apoptosis of Damaged Cells

2.1 Flow cytometry detection of cell apoptosis rate

From Figure 1, it can be seen that the apoptosis rate of the normal group is 0.0%, all of which are living cells; Compared with the normal group, the apoptosis rate of the model group significantly increased (33 7%), the difference was statistically significant (P<0 05), Description A β 25-35 causes apoptosis of PC12 cells; Compared with the model group, the cell apoptosis rate significantly decreased to (0.8%) after administration of GSrd. Give CDPS (50 and 150 μ After the intervention of group g · mL -1, the apoptosis rate of cells was significantly reduced in a dose-dependent manner, with 18.1% and 12.1%, respectively, indicating that CDPS can inhibit A β 25-35 induces apoptosis in PC12 cells, as shown in Table 2.

Figure 1  Flow cytometry analysis of CDPS on A β Protective effect of 25-35 induced apoptosis in PC12 cells

Table 2  CDPS for A β The effect of 25-35 on the apoptosis rate of PC12 cells n=4, x ± s

Compared with the normal group, a: P<0.05; Compared with the model group, b: P ＜ 0.05

2.2 Effect of CDPS on HATs and HDAC2 content in PC12 cells

The results showed that compared with the HATs activity model group, the blank control group significantly decreased; The HDAC2 activity model group showed a significant increase compared to the blank control group; After administration of GSrd, the activity of HATs significantly increased, while the activity of HDAC2 significantly decreased. Give CDPS (50 and 150 μ After g · mL-1 intervention, compared with the model group, the activity of HATs was significantly increased, while the activity of HDAC2 was significantly reduced, and there was a dose-dependent relationship. Explanation A β Histone acetylation modification is involved in the apoptosis process of PC12 cells induced by 25-35, and changes in enzyme activity of HATs and HDAC2 have an impact on the survival of PC12 cells. The results are shown in Figure 2.

Compared with the normal group, a: P<0.05; Compared with the model group, b: P ＜ 0.05

Figure 2  CDPS vs. A β Effects of 25-35 induced HATs and HDAC2 in PC12 cells

3 Effect of 3 CDPS on the expression of P300/CBP and HDAC2 proteins in PC12 cells

P300/CBP is currently discovered as a HAT related to learning and memory, while HDAC2 is recognized as an HDAC related to learning and memory. In this study, Western Blot was used to analyze the expression of P300/CBP and HDAC2 in each group of cells. The results showed that compared with the blank control group, the model group showed a decrease in P300/CBP expression (P<0 05) ; After administration of GSrd, the expression of P300/CBP increased (P<0 05) ; Give CDPS (50 and 150 μ After g · mL-1 intervention, the expression of P300/CBP increased (P<0 05), and it is dose-dependent. The results are consistent with the enzyme activity detection results (see Figure 3).

Compared with the normal group, a: P<0.05; Compared with the model group, b: P ＜ 0.05

Figure 3  Effect of CDPS on the expression of P300/CBP (A) and HDAC2 protein (B) in PC12 cells

4 Effect of 4 CDPS on BDNF secretion in PC12 cells

BDNF plays an important role in the survival, differentiation, growth, and development of neurons, preventing neuronal death from injury, improving the pathological state of neurons, and promoting the regeneration and differentiation of injured neurons. To explore the impact of CDPS on A β The protective mechanism of 25-35 induced apoptosis in PC12 cells. We used ELISA to detect the secretion of BD NF protein in the cell culture medium of each group. The results showed that compared with the blank control group, the model group had a decrease in BDNF secretion (P<0.05); After administration of GSrd, BDNF secretion increased (P<0.05); Give CDPS (50 and 150 μ After intervention with g · mL-1, the secretion of BDNF increased (P<0.05) in a dose-dependent manner. The neuroprotective effect mediated by CDPS is enhanced with the increase of BDNF secretion, as shown in Table 3.

5 The effect of 5 CDPS on H3 acetylation level in PC12 cells

Studies have shown that the secretion of BDNF is influenced by histone acetylation modifications. To determine whether the histone acetylation modification of BDNF in PC12 cells in this study is affected by A β 25-35, we used the chromatin immune coprecipitation method to analyze the levels of AC-H3 and HDAC2 in cells of each group. ChIP analysis shows in A β After 25-35 induction, the HDAC2 levels (Figure 4A) and H3 acetylation levels (Figure 4B) of BDNF exon IV were significantly reduced, consistent with the downregulation of BDNF expression (see Table 3). After administration of CDPS, the H3 acetylation level in the BDNF exon IV region increased (Figure 4B), showing a significant difference compared to the model group. The increase in the H3 acetylation level of BDNF exon IV region in the GSrd group is consistent with the report in the literature [17]. From the above results, it can be seen that the neuroprotective process mediated by CDPS is accompanied by a significant increase in BDNF secretion, which is regulated by Ac-H3 and HDAC2, as shown in Table 4.

Table 3  CDPS vs. A β The effect of 25-35 induced BDNF secretion in PC12 cells n=5, x ± s

Compared with the normal group, a: P<0.05; Compared with the model group, b: P ＜ 0.05

Compared with the normal group, a: P<0.05; Compared with the model group, b: P ＜ 0.05

Figure 4  Effect of CDPS on the acetylation levels of HDAC2 (A) and Accty-H3 (B) in PC12 cells

Table 4  CDPS vs. A β Effect of 25-35 induced Acety-H3 on PC12 cells n=5, x ± s

Compared with the normal group, a: P<0.05; Compared with the model group, b: P ＜ 0.05

Discussion

A β Abnormal generation and deposition are early events and core pathological changes in AD. Mediate toxic effects such as cell apoptosis [18], inflammatory cascade response [19], oxidative stress, and mitochondrial dysfunction, and exacerbate the development of AD. Both in vivo and in vitro studies have shown that A β 25-35 with A β 1-42 has similar coagulation properties and toxic effects, which can cause neuronal damage, impaired learning, and memory abilities, and is widely used in A due to its advantages such as easy dissolution β Research on damage and toxic effects [20]. The MTT results indicate that A β 25-35 concentration 20 μ PC12 cells cultured under the condition of mol · L-1 for 48 hours exhibit significant apoptotic characteristics, making it an ideal AD cell model. V-FITC and PI dual staining flow cytometry was used to detect the apoptosis of PC12 cells in different experimental groups. A β The apoptosis rate of PC12 cells significantly increased after treatment with 25-35 and decreased significantly in a dose-dependent manner after administration of CDPS. This indicates that CDPS has the effect of inhibiting cell apoptosis, so the cell model in this study can be used to evaluate the effect of CDPS on A β An ideal cell model for inducing protective effects against PC12 cell damage by 25-35.

Cistanche deserticola polysaccharides--Anti Alzheimer's disease

GSrd is a monomeric component isolated from natural plants and has active biological activity. Research [21] indicates that GSrd can reduce the levels of reactive oxygen species in PC12 cells, and resist hydrogen peroxide-induced cytotoxic damage by reducing malondialdehyde content and enhancing the activity of antioxidant enzymes. In vivo experiments have found that GSrd can improve and promote neuronal survival by rebuilding the balance between Ac-H3 and HDAC2, increasing BDNF expression, and improving chronic brain injury [17]. CDPS, similar to GSrd, is isolated from natural plants of the tonifying class and has both neurotrophic and neuroprotective effects. Research has found that CDPS can improve the learning and memory abilities of AD model rats by increasing the expression of Bcl-2, reducing the expression of caspase-3, and inhibiting hippocampal neuronal apoptosis. Our previous experimental results [22-26] showed that CDPS could significantly increase the expression of synaptophysin growth-related protein 43 (GAP-43), increase the number of synapses in the hippocampus, enhance the plasticity of synapses, and alleviate the performance of learning disabilities in mice with scopolamine-induced learning and memory disorders. It was observed that CDPS could increase the activity of SOD in brain tissue of aging mice induced by D-neneneba galactose, reduce the content of MDA, improve neuronal damage in brain tissue, and activate the cAMP/PKA/CREB signaling pathway to improve learning and memory impairment. Therefore, GSrd was selected as a positive drug in this study.

This study explores the upregulation mechanism of BDNF mediated by CDPS. Studies have shown that BDNF plays an important role in synaptic treeing, neurogenesis, and cell survival [27]. However, these regulatory mechanisms gradually retreat towards epigenetic mechanisms, and through epigenetic modifications, gene expression plays an important role in various human diseases. Histone lysine residues are acetylated under the action of histone acetyltransferases HATs, thus loosening the structure of chromatin, exposing the binding sites of transcription factors and DNA, and starting the transcription mechanism. Histone deacetylase HDAC is related to transcriptional inhibition. HDAC2 is one of the most highly expressed Class I HDACs in the brain [28]. The increase of HDAC2 can accompany the cognitive decline of the human neurodegenerative brain [28-29], and HDAC2 also negatively regulates memory and synaptic plasticity in the brain of healthy mice [30-31]. This study reveals the mechanism by which H3 acetylation levels are significantly reduced and HDAC2 is significantly increased in the AD cell model. This study suggests that BDNF is regulated by epigenetic pathways.

Currently, promising results have been achieved in pharmacological treatments aimed at increasing histone acetylation in reversing cognitive impairment through the use of non-selective HDAC inhibitors [32-33]. This study shows that the level of HDAC2 increases and the secretion of BDNF decreases in the AD cell model; Blocking HDAC2 with GSrd enhances the expression of BDNF, and the results are similar after treatment with CDPS. Based on the above in vitro experiments, our research team will further investigate the effect of CDPS on the acetylation level of histones in an AD pathological state, and conduct subsequent experiments.

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