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Back to Journal »Neuropsychiatric Diseases and Treatment» Volume 17

Shenma Yizhi Decoction improves brain mitochondrial structure of VCI rats through AMPK/UCP2 signaling pathway and improves cognitive impairment

Authors: Sun C, Liu Ming, Liu Jie, Zhang Tian, ​​Zhang Li, Li Hua, Luo Zhi

Published on June 16, 2021, the 2021 volume: 17 pages 1937-1951

DOI https://doi.org/10.2147/NDT.S302355

Single anonymous peer review

Editor who approved for publication: Dr. Jun Chen

Sun Chengcheng, 1 Liu Meixia, 1 Liu Jiangang, 2 Zhang Tingting, 3 Zhang Lei, 4 Hao Li, 1 Luo Zenggang 5 Department of Geriatrics, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, 100091; 2 Department of Cardiovascular Internal Medicine, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091 ;3Department of Geriatrics, First Clinical School of Shandong University of Traditional Chinese Medicine, Jinan, Shandong; 4Department of Emergency, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091; 5 Medical Management Office of Beijing Administration of Traditional Chinese Medicine, Beijing, 100053 Corresponding Authors: Liu Meixia; Liu Jiangang mailbox [email protected]; [email protected] Background: Shen Ma Yizhi Decoction (SMYZD) is an effective Chinese medicine prescription for the treatment of vascular dementia (VD). Modern research methods have clearly identified its active ingredients as gastrodin, ferulic acid, ginsenosides and β-sitosterol. Chronic cerebral hypoperfusion is a driving factor or risk factor of VD, leading to mitochondrial structure and dysfunction. Purpose: To observe whether SMYZD improves cognitive impairment by improving mitochondrial structure and function. Methods: Forty adult rats with vascular cognitive impairment (VCI) caused by bilateral common carotid artery ligation were randomly divided into 4 groups, including model group, donepezil group, low-dose and high-dose SMYZD group, 10 rats in each group. In each group. Another sham operation group was set up, and 10 rats served as the control group. The treatment group was given donepezil and two different doses of SMYZD. The donepezil group was given 0.45 mg/kg/d of donepezil, and the SMYZ-L group was given 2.97 g/kg/d of SMYZ, which was equivalent to the clinical dosage. The SMYZ-H group was given 11.88 g/kg/d SMYZ, which was 4 times the clinical equivalent dose. The sham operation group was used as the control group, and an equal volume of distilled water was given. The rats were treated by intragastric administration for 8 weeks. Perform Morris Water Maze (MWM) test to assess learning and memory abilities. Mitochondria from brain tissue are extracted from the brain for further testing. Detection of mitochondrial morphology and AMPK/PPARα/PGC-1α/UCP2 signaling pathways in mitochondria. Results: Through the intervention of SMYZD, the behavioral performance of rats and the pathological changes of brain mitochondria were significantly improved. The activities of SOD, GSH-Px, and GSH in serum increased, and the content of MDA decreased. In addition, SMYZD also reversed AMPK, PPARα, PGC-1α, UCP2 and ATP5A mRNA and protein expression levels. Conclusion: SMYZD may improve mitochondrial structure and energy metabolism by activating AMPK/PPARα/PGC-1α/UCP2 signaling pathway, thereby alleviating vascular cognitive impairment, providing a potential treatment strategy. Keywords: Vascular Cognitive Impairment, Shenma Yizhi Decoction, Mitochondrial Energy Metabolism, Bilateral Common Carotid Artery Occlusion

Vascular dementia (VD) is caused by cumulative damage to brain tissue mediated by vascular factors. It is an acquired, chronic and progressive mental retardation syndrome, which is characterized by progressive impairment of memory and cognitive abilities, and is generally considered second only to Alzheimer's disease (AD) in all types of dementia. 1,2 With the increase of global aging, the incidence of dementia is increasing at an alarming rate all over the world, which makes dementia the focus of public health. 3 The main pathogenesis of VD includes chronic cerebral hypoperfusion, 4 hippocampal neuron loss, 5 synaptic disorders, 6 axon abnormalities, 7 and white matter vascular changes. 8 Vascular cognitive impairment (VCI) includes the whole process of vascular cognitive impairment from mild to severe. The pathological changes of blood vessels significantly affect cognitive status, leading to cognitive impairment and dementia. Insufficient blood perfusion/vascular risk factors aggravate memory impairment, accelerate the progress of memory impairment, and have a negative impact on treatment results. 6 However, due to the many causes of VD and the limited treatment methods, the main clinical treatment for VD is to actively control risk factors to prevent stroke recurrence. 9

As the center of cell metabolism and the main regulator of redox balance, mitochondria are also the main organelles involved in ischemia and are very important for the regulation of energy metabolism, signaling pathways and cell death. 10 The brain is the most organ with active energy metabolism in the body. Stable and coordinated brain energy metabolism is very important for maintaining the normal state of the brain. 11 Therefore, the normal function of mitochondria is vital to the maintenance of the brain. Evolutionary studies have shown that the emergence of advanced cognitive functions is related to the increase in glucose utilization and the expression of genes related to energy metabolism. 11 When the blood supply to the brain is interrupted, brain cell damage is caused, and cognitive functions are closely related. To energy metabolism. Energy supply is very important for effective treatment of ischemic diseases. 12 Based on these findings, brain mitochondrial dysfunction also plays an important role in the pathogenesis of VCI.

AMP-activated protein kinase (AMPK) is generally regarded as a cell energy sensor and adenine nucleotide signal sensor. It is regulated by various metabolic stresses and plays a major role in regulating cell energy balance. 13 AMPK regulates the content and oxidation of mitochondria. 14 As a ligand-activated transcription factor, PPARα plays a key role in regulating lipid metabolism, inflammation and energy homeostasis, and mediates the improvement of hippocampal synaptic plasticity. Transgenic mice with cognitive impairment. 15,16 PGC-1α is the hub of energy metabolism, plays a key role in mitochondrial biogenesis and energy metabolism, and is the main regulator of mitochondrial function. 17 UCP2 is an ion transporter located in the inner mitochondrial membrane and is involved in maintaining mitochondrial function and regulating immune response and oxidative stress under physiological and pathological conditions. 18 According to recent studies, PGC-1α is involved in the pathophysiology of VD because it increases the expression of mitochondrial antioxidants and uncoupling protein (UCP), improves neuronal energy metabolism, and synthesizes the protease ATP-5A for 19 mitochondria It is not only the core hub of mitochondrial structure and function, but also promotes the formation of mitochondrial cristae. In addition, it is the main center of cell homeostasis. 20

Traditional Chinese medicine has unique advantages in the clinical treatment of VD due to its multi-effect and multi-target characteristics. Shen Ma Yi Zhi Tang (SMYZD) is composed of ginseng, gastrodia elata, and gastrodia elata. (Tianma), Daye Euonymus. (Thunb.) (Guijianyu) and Ligusticum chuanxiong Hort. (Chuan Xiong). The main active ingredient of ginseng is ginsenoside, which is a variety of steroidal saponins and has the ability to target multiple tissues and produce multiple pharmacological reactions. 21 Ginseng also contains polysaccharides, polypeptides, polyalkynes, fatty acids, etc.22. The chemical composition of gastrodia. It's gastrodin. 23 Previous studies have reported several biologically active and structurally interesting components from Euonymus alatus Sieb. (Thunb.), 59 exists in the form of sesquiterpenes, including sesquiterpene alkaloids, triterpenes, flavonoids, and phenolic compounds, such as β-sitosterol, aromatic compounds, and aramine. 24,60 Many compounds have been isolated from Ligusticum chuanxiong, 61 including Ligusticum chuanxiong lactone, tetramethylpyrazine (TMP) and ferulic acid. 25

Previous animal experiments conducted by our research team have shown that SMYZD has a significant impact on the treatment of dementia. 26-28 Nevertheless, the therapeutic mechanism of SMYZD through AMPK signaling pathway in mitochondrial biogenesis remains to be elucidated. Therefore, we studied the regulation of SMYZD on mitochondrial biogenesis through the AMPK/PPARα/PGC-1α/UCP2 signaling pathway in the brain tissue of VCI rats to further determine the underlying mechanism of this study.

70 SPF Sprague-Dawley (SD) rats, 10 weeks old, weighing 300±20 g, male and female, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. License number: SCXK (Beijing) 2016 –0006 . The rats were adaptively fed in the experimental animal room at a temperature of 22-26°C, a humidity of 40%-70%, and a barrier environment with free drinking and eating. The experimental study was carried out one week later. Animal modeling and experiments are carried out in strict accordance with the recommendations in the National Institutes of Health Laboratory Animal Care and Use Guidelines. This experiment was approved by the Animal Experiment Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (No. 2018XLC004-2).

Hematoxylin-Eosin (HE, Fluka (USA)) is imported from Shanghai Chemical Reagent Co., Ltd. and packaged separately, batch number: 46160. Donepezil hydrochloride, 5mg/tablet (trade name: Anlishen) was purchased from China Defensive Materials Company. (Lot number: 1702012). Ginsenoside Rg1 reference substance, purity 96.3%, ginsenoside Re reference substance, 92.9%, ginsenoside Rb3 reference substance, purity 89.1%, provided by Shanghai Yuanye Biotechnology Co., Ltd.; Gastrodin reference substance, purity 98.0%, 4-pair Hydroxybenzyl alcohol was provided by Shanghai Shidande Biological Technology Co., Ltd. Ferulic acid reference substance purity 90.6%, protocatechuic acid reference substance, purity 95.0%, nifedipine/phyrazine (alatamin) reference substance, purity 90.0%, β-sitosterol reference substance, purity 98%, provided by Chengdu Plant Standard Chemical Pure Biotechnology Co., Ltd.

Based on the result 62 of the previous stage, this experiment improved the preparation method of SMYZD and optimized the preparation process. SMYZD medicinal slices (Ingredients: Ginseng (Ginseng), Gastrodia (Gastrodia), Thunb.) (Thunb.) (Thunb.), Ligusticum chuanxiong (Ligusticum chuanxiong) 3:3): 3:2) All in Xiyuan Hospital of China Academy of Chinese Medical Sciences Preparation room preparation, Beijing Hospital Preparation, Beijing Pharmaceutical Preparation: Z20200005000. Decoction of ginseng slices in water is divided into two times: first decocting with 12 times of water for 2 hours, and then decocting with 10 times of water for 2 hours. Filter the decoction and combine. Gastrodia (Pegasus), Euonymus vulgaris. (Thunb.) (Guijianyu) and Ligusticum chuanxiong Hort. (Ligusticum chuanxiong) decocted in water for 3 times, 2 times with 10 times of water for 2 hours, and 8 times of water for 1 hour. The filtrate and ginseng filtrate were combined, and the filtrate was concentrated to obtain an extract with a relative density of 1.10±1.15 (50°C). Each gram of extract contains 2.44 grams of crude drugs.

The main chemical components of SMYZD are analyzed by liquid chromatography-mass spectrometry (LC-MS). The chromatographic column is a SymmetryLuna C18 column (150mm×2.1mm, L×ID), 025331036119 15 (Waters Company, USA), the pre-column is a 0.2cm sieve plate, batch number 06222010 (ESA Company, USA). The column temperature was 20°C, the injection volume was 10 μL, the autosampler was at room temperature, and the running time was 9.5 min. The mobile phase is an aqueous solution containing 0.1% formic acid, and the organic phase is a methanol-acetonitrile isobaric mixture (containing 0.1% formic acid), with a flow rate of 0.23 mL/min, room temperature, and an injection volume of 5 ul. Accurately weigh ginsenoside Rg1, ginsenoside Re, ginsenoside Rb3, gastrodin, 4-p-hydroxybenzyl alcohol, ferulic acid, protocatechuic acid and harringtonine (alatamin), β-sitosterol , Use 50% methanol aqueous solution to dissolve ultrasonically. And gradually dilute into a solution with a concentration of 5 ug/mL. Pass through a 0.22 μm filter membrane.

The animal modeling method is improved based on the literature. The 63 rats were fasted for 12 hours before the operation and anesthetized intraperitoneally with 2% sodium pentobarbital (0.2 mL/100 g). Then the rat was fixed in a supine position, the neck skin was disinfected with iodophor and cut 1.5 cm, subcutaneous tissue and muscle were bluntly separated, and bilateral common carotid arteries were exposed and ligated. Suture the wound and maintain the animal's body temperature until the animal can eat. In the sham operation group, bilateral common carotid arteries were bluntly separated without ligation. After surgery, penicillin was given 40000U/day (intramuscular injection) for 3 days to prevent infection, and the wound healing was closely observed. Morris water maze test was performed two weeks later, and 40 VCI rats were selected for comparison with the sham operation group. The rats were randomly divided into 4 groups according to their body weight, each with 10 rats: model control group (Model), donepezil hydrochloride group (Donepezil, 0.45 mg·kg-1), SMYZD high-dose group (SMYZ-H, 11.88 g·kg- 1) and SMYZD low-dose group (SMYZ-L, 2.97 g·kg-1). The dose given to animals is based on the clinically equivalent dose used in humans. The research group conducted an acute toxicity test of SMYZD, and the mice did not produce obvious symptoms of poisoning64. According to the conversion of human and animal body surface areas, each group was given the same amount of medicine once a day. In addition, 10 rats in the sham operation group (Sham) were given the same amount of pure water. After eight weeks, relevant tests and indicators were detected.

The Morris Water Maze Test (MWM) is used to test the spatial learning and memory ability of rats. Tests include position navigation and space exploration tests. The pool is divided into four quadrants, and a platform is placed in the center of the first quadrant. After pouring clean water on the top of the platform (1 cm), keep the water temperature at 21±2℃, and add an appropriate amount of ink to hide the platform. Location navigation experiment: training twice a day, at a fixed time. According to the principle of randomization, two different water entry points were randomly selected for the rats to enter the water, and the rats were gently put into the water from the water entry point down to the wall of the pool. At the same time, the time from entering the water to discovering and climbing on the platform (escape latency) and swimming distance are recorded. Navigate the location every day for 4 days. Space exploration test: This test is carried out 24 hours after the position navigation test. The platform was dismantled, and the activity time of the first quadrant and the number of times of crossing the original platform were recorded. The MWM test performed refers to Cao's method. 65

After Morris water maze test and fasting for 12 hours, blood was collected from the abdominal aorta of the anesthetized rats (injection of 2% sodium pentobarbital (0.2mL/100g) into the abdominal cavity), and the serum was separated the next day. After centrifugation at 4°C and 3000r/min for 15 minutes, the relevant indicators were detected. The brain tissue was quickly and completely removed and randomly grouped. The hemispheres of 6 rats in each group were fixed (placed in 4% neutral paraformaldehyde solution), and the hemispheres of the other 6 rats were quick-frozen in liquid nitrogen and stored at -80°C for further analysis.

After the brain tissue is fixed, it is dehydrated, transparent, embedded, sectioned, dewaxed, and hydrated, and stained with hematoxylin-eosin (HE), using ASP200S dehydrator, EG1150 embedding machine, RM2245 paraffin microtome (Leica, Germany).

The content of GSH, MDA and SOD was determined by colorimetry, and the content of GSH-PX was determined by non-enzymatic immunoassay. Firstly, the GSH antibody is coated on the microtiter plate to prepare a solid phase antibody to form an antibody-antigen-enzyme-labeled antibody complex. Finally, under the action of horseradish peroxidase (HRP), the color of the tested serum and the complex changed to yellow, indicating the GSH content. Using the colorimetric method, the amount of MDA in the lipid peroxide degradation product condensed with thiobarbituric acid (TBA) forms a red color, with the maximum absorption peak at 532 nm. SOD is detected by colorimetry. The self-oxidation of pyrogallol produces superoxide anion radicals. The ability of SOD to decompose O2 is proportional to the activity of SOD, which is converted into SOD content. GSH-PX was analyzed using a non-enzymatic immunoassay. The purified GSH-PX antigen is coated on the microtiter plate to form a solid-phase antigen, and the test sample forms an antibody-antigen-enzyme-labeled antibody complex. After washing, the substrate TMB is used for color development and is converted to blue under the catalysis of HRP enzyme. The color depth is positively correlated with the content of GSH-PX. Use a microplate reader to measure the absorbance (OD) at a wavelength of 450 nm to determine the GSH-PX concentration. The above steps strictly follow the instructions of the kit (GSH-PX-20181010, GSH-20181110, MDA-20181100 and SOD-20181111, Nanjing Jianshe, China).

Paraffin sections were deparaffinized (RM2035 Pathology Microtome, Shanghai Leica Instruments Co., Ltd.) and heated in EDTA (G1206, Solarbio, pH 8.0) for antigen retrieval for 8 minutes. Add hydrogen peroxide, dry the section slightly, and draw a circle around the tissue with a histochemistry pen (WG1066, Servicebio) (to prevent the antibody from flowing away). The sections were blocked with serum (bovine serum albumin (VI), G20191212, Solarbio), and then incubated overnight at 4°C with one or two of the following primary antibodies diluted in PBS: ATP5A1 mouse monoclonal antibody (lot number: 10002127, 1:300) or UCP2 rabbit polyclonal antibody (lot number: 00069126, 1:200, Proteintech Group, Inc. (USA)). Rinse the sections and incubate for 50 minutes at room temperature with a secondary antibody conjugated to HRP of the corresponding species. The nucleus was stained with DAPI. Tissue autofluorescence (autofluorescence quencher, G1221, Servicebio) is quenched. The sections were slightly dried and sealed with anti-fluorescence quenching sheets (G1401, Servicebio). Place the slice under a microscope to take an image (XSP-PM21AY fluorescence microscope imaging system, Shanghai Optical Instrument Co., Ltd.). The nucleus stained with DAPI was blue under ultraviolet excitation, and the positive staining was red and green in the section labeled with the corresponding fluorophore.

The brain tissue was fixed with 4% neutral glutaraldehyde and 1% osmium acid. The sample is eluted with acetone. After embedding the porous rubber template, the sample is dried in an oven to form a retention block. The sample was sliced ​​into 70 nm thick slices with a Leica ultra-fine instrument (model EMUC6, Leica, Germany). The sections were stained with uranyl acetate and lead citrate. A transmission electron microscope (H-7650, Hitachi Co., Ltd., Japan) was used to observe the section and capture the image.

1. Prepare brain tissue homogenate, take 100-200 mg of brain tissue and rinse with saline, blot dry with filter paper, cut into small pieces, and place in a small-capacity glass homogenizer. Add 1 mL of ice-cold Lysis Buffer to an ice bath at 0 ℃, and grind the microstructure 20 times. 2. Transfer the sample to a centrifuge tube and centrifuge at 1000×g at 4°C for 5 minutes. 3. Extract the supernatant and centrifuge at 1000×g for 5 minutes at 4°C. 4. Centrifuge the supernatant at 12000×g for 10 minutes at 4°C, and then transfer to a new centrifuge tube. Mitochondria will settle at the bottom of the tube. 5. Then, add 0.5 mL of washing buffer to the mitochondrial pellet, resuspend the mitochondrial pellet, and centrifuge at 1000×g at 4°C for 5 minutes. 6. Transfer the supernatant to a new centrifuge tube and centrifuge at 12,000×g at 4°C for 10 minutes. The supernatant was discarded, and high-purity mitochondria were deposited on the bottom of the tube. 7. Suspend the mitochondrial pellet in 50-100 μL Storage Buffer and store at -70 °C. Strictly follow the mitochondrial extraction kit instructions (SM0020, Beijing Sunshine Biotechnology Co., Ltd.) for operation.

TRIzol reagent was used to extract total RNA from gastrocnemius tissue samples. All qRT-PCR analyses were performed using the ABI7500 real-time PCR system (Applied Biosystems). Use PrimeScript™ RT Reagent Kit (TaKaRa, Japan) with gDNA Eraser to generate cDNA template by reverse transcription. Perform real-time PCR in a CFX96 instrument after reverse transcription using the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 40 s. The relative mRNA expression was calculated using the 2-ΔΔCt comparison method. See Table 1 for the primer sequence of each target gene. Table 1 Real-Time Quantitative PCR primers

Table 1 Primers used for real-time quantitative PCR

The supernatant was collected, and the concentration of rat brain mitochondrial protein was determined by the bicinchoninic acid (BCA) method. The extracted cell protein was subjected to SDS-PAGE, transferred to NC membrane (0.45 µm), and then blocked with 3% BSA in TBST for 30 minutes. Then use anti-AMPK antibody (dilution 1:2000, rabbit monoclonal antibody, Abcam), anti-PPARα antibody (dilution 1:1000, mouse monoclonal antibody, GeneTex), anti-PGC-1α antibody (dilution 1:4000, rabbit more Clonal antibody, Abcam), anti-UCP2 antibody (1:2000 dilution, rabbit polyclonal antibody, Proteintech), or anti-p-AMPK antibody (1:5000 dilution, rabbit polyclonal antibody, Abcam) at 4°C overnight. After washing the membrane with TBST 6 times, 3 minutes each time, the membrane was incubated with the secondary antibody (CWBiotech, China) for 90 minutes. The exposed film is scanned directly, and the ImageJ software changes the image format from JPEG to Tif. Total Lab Quant V11.5 (Newcastle upon Tyne, UK) is used to calculate the integrated optical density (IOD) value of protein bands.

All data were processed using SPSS 22.0 statistical software and expressed as mean ± standard deviation (SD). Assuming homogeneity of variance, one-way analysis of variance (ANOVA) was used to compare multiple groups, and two-way ANOVA and LSD test were used to analyze MWT data. If the variance is not uniform, use Dunnett's T3 method. P <0.05 or P <0.01 was defined as a significant difference.

The main active ingredients are ginseng CA Mey, Gastrodia, Euonymus alatus Sieb. (Thunb.) and Ligusticum chuanxiong Hort. Use LC-MS detection. These compounds are ginsenosides, gastrodin, β-sitosterol and ferulic acid, as shown in Figure 1. Figure 1 SMYZD 1. LC-MS chromatogram of the aqueous extract of alanine; 2. β-sitosterol; 3. ferulic acid; 4. protocatechuic acid; 5. gastrodin; 6. 4-hydroxybenzyl alcohol ; 7. Ginsenoside Rg1; 8. Ginsenoside Re; 9. Ginsenoside Rb3.

Figure 1 LC-MS chromatogram of SMYZD 1 water extract. 2. β-Sitosterol; 3. Ferulic acid; 4. Protocatechuic acid; 5. Gastrodin; 6. 4-hydroxybenzyl alcohol; 7. Ginsenoside Rg1; 8. Ginsenoside Re; 9. Ginsenoside Rb3 .

As shown in Figure 2, the Morris water maze test was performed at the 8th week after the intervention to determine the effect of SMYZD on CCH-induced cognitive impairment. Starting from day 3 (days 3 and 4 of the experiment, P <0.05), the escape latency of the model group was significantly prolonged (40.29±36.54 vs 17.52±21.42 s), and the rats stayed in the target quadrant for less time , The number of platform crossings is less than that of sham rats (19.31 ± 3.98 vs 28.15 ± 5.21 s) (P <0.01) (2.7 ± 2.36 vs 7.3 ± 3.2 s) (P <0.05). After 8 weeks of SMYZD treatment, the escape latency of the SMYZ-H group was improved (18.36±22.42 48.02±36.59 s), and the SMYZ-H group rats stayed in the target quadrant for a longer time (25.44±7.31 vs 19.31 ± 3.98 s) ( P <0.01), and crossed the platform position more times than the model group (7.1 ± 4.12 vs 2.7 ± 2.36 s) (P <0.05), indicating that SMYZD may partially improve spatial memory and learning disabilities caused by CCH. Figure 2 The Morris water maze is used to assess spatial memory and learning ability. The escape latency (A) is the average value of each platform test for 4 consecutive days, and the time spent by the rat in the target quadrant (B) and the number of platform crossings (C) is measured during the probe test. (D) Typical swimming paths of rats in the sham, model, donepezil, SMYZ-H, and SMYZ-L groups in the MWM during learning (top panel) and memory probe testing (bottom panel). *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group. **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group.

Figure 2 The Morris water maze is used to assess spatial memory and learning ability. The escape latency (A) is the average value of each platform test for 4 consecutive days, and the time spent by the rat in the target quadrant (B) and the number of platform crossings (C) is measured during the probe test. (D) Typical swimming paths of rats in the sham, model, donepezil, SMYZ-H, and SMYZ-L groups in the MWM during learning (top panel) and memory probe testing (bottom panel). *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group. **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group.

Under the light microscope, the pyramidal cells, granular cells and neurons in the cerebral cortex of the sham-operated group were arranged normally, with tight connections, and no inflammatory cells or chronic ischemic changes were seen. In the model group, pyramidal cells, granular cells and neurons were sparsely scattered, the number of cells was significantly reduced, and inflammatory cells and chronic ischemic changes were seen. Compared with the model group, the number of cells in the three-drug group is significantly increased, the cell structure is more complete, and the above-mentioned cells are arranged better. Therefore, SMYZD ameliorated the pathological changes of the cerebral cortex of VCI rats caused by bilateral common carotid artery ligation, as shown in Figure 3. Figure 3 The effect of HE staining on the pathological morphology of rat cerebral cortex after 8 SMYZD intervention. Inflammatory cells and chronic ischemic changes are marked with black arrows (bar = 50 μm).

Figure 3 The effect of HE staining on the pathological morphology of rat cerebral cortex evaluated by HE staining 8 weeks after SMYZD intervention. Inflammatory cells and chronic ischemic changes are marked with black arrows (bar = 50 μm).

Compared with the sham operation group, the serum GSH and GSH-PX concentrations in the model group were significantly reduced, and the MDA content was significantly increased (P <0.05, P <0.01). Compared with the model group, the contents of GSH and GSH-PX in the donepezil hydrochloride group and the high-dose SMYZD group were significantly increased, the content of MDA was significantly decreased (P <0.05), and the content of SOD was significantly decreased in the high-dose SMYZD group. The group increased significantly (P <0.05, P <0.01). Based on these results, SMYZD reduced mitochondrial damage in VCI rats caused by oxidative stress (Figure 4). Figure 4 The contents of GSH (A), GSH-Px (B), SOD (C) and MDA (D) in the serum of VCI rats in each group. *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group. **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group. (n=10).

Figure 4 The contents of GSH (A), GSH-Px (B), SOD (C) and MDA (D) in serum of rats in each group of VCI. *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group. **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group. (n=10).

ATP5A, a mitochondrial marker, served as a negative control for the sham operation group. Fewer mitochondria were observed in the model group. After the intervention, the fluorescence image showed that the number of mitochondria increased to a certain extent, indicating that SMYZD increased the number of mitochondria in VCI rats caused by bilateral common carotid artery ligation, as shown in Figure 5. Figure 5 The effect of SMYZD on the expression of mitochondrial uncoupling protein 2 (UCP2) in the cerebral cortex of rats with chronic cerebral ischemia (×400). Note: Red fluorescence represents mitochondrial UCP2, and green fluorescence represents the expression of mitochondrial marker mitochondrial syncytial protease ATP5A; its expression is located in the inner or outer mitochondrial membrane. The blue fluorescence represents the nucleus. Answer: False; B: Model; C: Donepezil; D: SMYZ-H; E: SMYZ-L.

Figure 5 The effect of SMYZD on the expression of mitochondrial uncoupling protein 2 (UCP2) in the cerebral cortex of rats with chronic cerebral ischemia (×400).

Note: Red fluorescence represents mitochondrial UCP2, and green fluorescence represents the expression of mitochondrial marker mitochondrial syncytial protease ATP5A; its expression is located in the inner or outer mitochondrial membrane. The blue fluorescence represents the nucleus. Answer: False; B: Model; C: Donepezil; D: SMYZ-H; E: SMYZ-L.

The tissues of rats in each group were observed by transmission electron microscope (TEM). In the sham operation group, the mitochondrial structure of the brain tissue was basically normal, neatly arranged, the membrane shape was basically complete, the mitochondrial cristae was dense, and there was no obvious swelling or vacuole formation. The mitochondrial structure of the model group changed significantly, the mitochondrial membrane was blurred, part of the membrane was broken, the mitochondrial cristae broke loosely and dissolved, and the matrix particles were reduced or disappeared. In the drug group, the disorder of mitochondrial structure was significantly improved, the mitochondrial membrane was generally clear, and the number of mitochondrial cristae increased. The results are shown in Figure 6. Figure 6 The effect of SMYZD on the subcellular structure of mitochondria in the brain tissue of VCI rats (×20,000). TEM observation results of mitochondrial structure of rats in each group. Mitochondria are marked with blue arrows (bars = 500 nm).

Figure 6 The effect of SMYZD on the subcellular structure of mitochondria in the brain tissue of VCI rats (×20,000). TEM observation results of mitochondrial structure of rats in each group. Mitochondria are marked with blue arrows (bars = 500 nm).

Mitochondria are the target organelles damaged by oxidative stress, and mitochondrial dysfunction leads to reduced ATP production. Activated AMPK, PPARα and PGC-1α are important regulators of mitochondrial biogenesis. UCP2 plays a key role in the regulation of oxidative stress, and ATP5A is the core hub that controls the structure and function of mitochondria. Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of AMPK, PPARα, PGC-1α, UCP2 and ATP5A in brain mitochondrial samples in each group to further verify this phenomenon. The mRNA expression levels of AMPK, PPARα, PGC-1α, and ATP5A in the model group were significantly lower than those in the sham operation group (P<0.05, P<0.01). After SMYZD intervention, the expressions of AMPK, PPARα, PGC-1α, and ATP5A mRNA were significantly higher than those in the model group (P <0.05, P <0.01). Compared with the sham operation group, the expression level of UCP2 mRNA in the model group was significantly increased (P <0.01). Compared with the model group, the expression levels of UCP2 mRNA in the donepezil, SMYZ-H and SMYZ-L groups were significantly reduced (P <0.05, P <0.01). Therefore, SMYZD promotes mitochondrial energy metabolism in VCI rats through AMPK, PPARα, PGC-1α and UCP2 pathways (Figure 7). Figure 7 AMPK (A), PPARα (B), PGC-1α (C), UCP2 (D) and ATP5A (E) mRNA expression in each group. *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group. **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group. (n=5).

Figure 7 AMPK (A), PPARα (B), PGC-1α (C), UCP2 (D) and ATP5A (E) mRNA expression in each group. *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group. **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group. (n=5).

We detected the expression of the above indicators at the mRNA level. Next, use Western blotting to further verify their expression at the protein level. Compared with the sham operation group, the protein levels of pAMPK (B), PPARα (C), PGC-1α (D), UCP2 (E) and ATP5A (F) in the model group decreased, and the difference was significant. After SMYZD treatment, the protein levels of pAMPK, PGC-1α, UCP2, ATP5A were significantly higher than those in the model group (P <0.05). The difference in AMPK protein expression levels is not significant, indicating that AMPK plays a role in this pathway after phosphorylation (Figure 8). Figure 8 WB protein expression bands in each group (A) AMPK (B), pAMPK (C), PPARα (D), PGC-1α (E), UCP2 (F), ATP5A (G) protein expression comparison groups in each group. *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group; **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group. (n=5).

Figure 8 WB protein expression bands in each group (A) AMPK (B), pAMPK (C), PPARα (D), PGC-1α (E), UCP2 (F), ATP5A (G) protein expression comparison groups in each group. *Compared with the sham operation group, P <0.05; #P <0.05 compared with the model group; **Compared with the sham operation group, P <0.01; ##P <0.01 compared with the model group. (n=5).

In this study, we found that SMYZD improves mitochondrial structure and energy metabolism to improve chronic cerebral hypoperfusion, which can lead to vascular cognitive impairment. First, it was detected that the expression of pAMPK, PPARα, PGC-1α and UCP2 in the brain mitochondria of VCI rats was reduced, accompanied by pathological changes in mitochondria. More importantly, we verified that the therapeutic effect of SMYZD is to improve the structure of mitochondria by activating the AMPK/PPARα/PGC-1α/UCP2 signaling pathway, and reduce the pathological damage of the brain of VCI rats and the damage caused by oxidative stress.

SMYZD contains a variety of active substances. These compounds include gastrodin, ferulic acid, ginsenosides and β-sitosterol. Gastrodin (4-(hydroxymethyl)phenyl-β-D-glucopyranoside) is a phenol glycoside extracted from Chinese herbal medicine (Gastrodia elata Bl.). It has anti-oxidant, anti-inflammatory and anti-apoptotic properties, and is widely used in the treatment of neurological diseases and cardiovascular and cerebrovascular diseases. GAS crosses the blood-brain barrier, thereby reducing the impact of different stress factors on the cognition of experimental animals. 29 GAS has been proven to regulate mitochondrial dynamics and maintain the structure and function of mitochondria to protect cells from oxidative damage. 30 Ferulic acid can reduce oxidative stress and prevent cell apoptosis by activating PPARγ. 31 In addition, ferulic acid can reduce oxidative stress associated with vascular damage and endothelial cells in mice, and improve mitochondrial biogenesis. 32 Ginseng has been used to treat various diseases, and it has been proven to have significant effects in treating various diseases. Control diseases of the central nervous system. These effects are exerted through its neurogenesis, anti-apoptotic and antioxidant properties, and inhibition of mitochondrial dysfunction. 33 The main ginsenosides Rb1, Rg1, Rb2, Rc, Rd and Re dominate the chemical characteristics of the genus in vivo and in vitro. 34 Rb1 or Rg1 increases the activity of complexes I, III and V, increases the level of ATP, and improves the function of mitochondria. 35 Two main types of ginsenosides may affect the kinetics of mitochondrial turnover. 36 It significantly improves the redox state of cells and improves mitochondrial function. The function of the glutathione system and the increase of SOD activity to protect mitochondria indicate that ginsenosides Rb1 and Rg1 have neuroprotective potential. 37 In addition, ginsenoside Rb1 regulates mitochondrial energy metabolism. 38 β-sitosterol is a biologically active compound naturally present in plants with a variety of biological functions, such as immune regulation, anti-inflammatory and antioxidant activities. 39 β-sitosterol protein protects the rat heart from oxidative damage by enhancing the redox cycle of mitochondrial glutathione. 40 β-Sitosterol increases the fluidity of the inner mitochondrial membrane, thereby enhancing the function of mitochondria. 41

Based on the above research on the pharmacological effects of the various components of SMTZD, we infer that the treatment may restore mitochondrial structure and improve mitochondrial function by inhibiting oxidative stress, and play a synergistic effect in improving the cognition of VCI rats.

Oxidative stress is usually defined as the imbalance between oxidants and antioxidants at the cellular or individual level. Oxidative damage is one of the results of this imbalance, including cell apoptosis and tissue damage, which ultimately leads to various diseases. 42 Oxidative stress is the main mechanism of BCCAO-induced early brain injury, leading to vascular endothelial cell damage, intracellular calcium overload, and mitochondrial dysfunction. Mitochondrial dysfunction induced by oxidative stress in the hippocampus is believed to play a major role in the pathogenesis of VD. 43 Enzymes involved in antioxidant defense include superoxide dismutase (SOD) and glutathione peroxidase, as well as water-soluble and fat-soluble antioxidants, such as glutathione. 44 MDA is the product of lipid peroxidation and is a recognized indicator of oxidative stress. It is a toxic molecule that destroys the structure of cell membranes. 45 The content of SOD and MDA reflects the level of oxidative stress in brain tissue. The level of GSH is closely related to the steady state of the redox system. Monitoring changes in GSH levels can help assess the effect of treatment. 46 GSH-PX acts as an antioxidant directly or indirectly in the body. 47 At the same time, as an indicator of oxidative stress, its content represents the strength of antioxidant. The body's antioxidant capacity. Central nervous system damage is usually accompanied by damage to nerve tissues and cells, as well as decreased protein levels in cells, cerebrospinal fluid, and blood. In this study, serum MDA levels in VCI rats increased, while GSH, GSH-PX and SOD levels decreased. Consistent with previous pharmacological studies, SMYZD reduced the level of MDA in VCI rats and increased the activities of GSH, GSH-PX and SOD, indicating that it inhibited oxidative stress in VCI rats.

As the main regulator of energy homeostasis, AMPK regulates more aspects and functions of mitochondria. AMPK regulates at least three basic aspects of mitochondrial homeostasis: biogenesis, fission, and mitochondrial phagocytosis. Almost all types of mitochondrial damage or defects activate AMPK, so AMPK acts as the guardian of mitochondria. 48 AMPK combines with downstream effectors to ensure optimal mitochondrial function and help regulate mitochondrial biogenesis. The AMPK/PPARα pathway regulates the content and function of mitochondria. 49 AMPK/PGC-1α pathway not only enhances mitochondrial biosynthesis50, but also participates in the regulation of mitochondrial energy metabolism. 51 PGC-1α (Peroxisome Proliferator Activated Receptor γ Co-activator 1-alpha) is a transcriptional co-activator of many genes involved in energy management and mitochondrial biogenesis. 52 AMPK regulates the activity of UCP2 by increasing the expression of PGC-1α, and the upstream region of PGC-1α is regulated by AMPK. 53 AMPK activates PPARα and up-regulates UCP2, indicating that the upstream region of PPARα may be regulated by AMPK. 54 In addition, the expression levels of AMPK, pAMPK, PPARα, PGC-1α and UCP2 in the brain mitochondria of VCI rats decreased. The expression of UCP2 in real-time fluorescent quantitative PCR experiments is the opposite of Western blot analysis. Possible explanations are provided below. Mitochondria are not only the main place for energy production, but also the main organelle involved in ischemia. The oxidative capacity of mitochondria extracted from hypoxic and ischemic myocardium is reduced. Ischemia leads to a decrease in oxygen uptake, a slowdown in ATP consumption, a decrease in ATP synthesis, and an increase in UCP2,55 expression, indicating that BCCAO-induced hypoxia and ischemia leads to abnormal mitochondrial respiratory function And oxidative phosphorylation. In addition, the expression of UCP2 mRNA is regulated by many factors in the receptor, such as miRNAs56 and growth hormone. 57 In addition, genetic polymorphisms of UCP2 have been reported, which may lead to changes in the structure/function/activity of gene products.58

At the same time, SMYZD reversed the expression level of the above indicators. Therefore, we propose that SMYZD may improve mitochondrial structure by activating AMPK, PPARα, PGC-1α and UCP2 signaling pathways, thereby promoting mitochondrial biogenesis, thereby improving cognitive impairment in VCI rats.

Surprisingly, we found that SMYZD can not only improve the damage caused by oxidative stress, but also improve the damage of mitochondrial structure through AMPK/PPARα/PGC-1α/UCP2 signaling pathway, thereby improving the learning and memory ability of VCI rats. These results may help to understand the new mechanism related to the positive effects of SMYZD and further verify its therapeutic effect on VD.

This journal was funded by the G20 Engineering Innovation Research Project of Beijing Science and Technology Plan (No. Z171100001717016). National Science and Technology Major Project "Basic New Drug Research and Development" (No. 2019ZX09301114). I would also like to thank Professor Wei Yun from Beijing University of Chemical Technology for his help in pharmacy.

The authors report no conflicts of interest in this work.

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