Study On The Mechanism Of Shouwu Pill Delaying Brain Aging In D-galactose Model Rats Based On The Gut Brain Axis

Objective:Brain aging is the main risk factor of neurodegenerative diseases.Due to its complex pathogenesis,the effect of a single treatment strategy is not ideal.Traditional Chinese medicine treatment has the advantage of all-around and multi-target regulation.Therefore,it is of great significance to seek a classic prescription to prevent and treat brain aging.Shouwu Pill is first seen in Shouwu Longevity Pill in Shibuzhai Medical Book.It has the effect of tonifying liver and kidney,and prolonging life.Traditional Chinese medicine enters the human body through oral administration and is absorbed through the intestine.The function of traditional Chinese medicine is closely related to the intestinal flora.In this study,we chose to explore the mechanism of Shouwu Pill in delaying brain aging of D-galactose model rats from the perspective of micro-organism-intestin-brain axis theory.In experiment 1,we selected the relevant target and signal pathway to explore from the perspective of hippocampal nerve cell damage,apoptosis,synaptic plasticity and mTOR signal pathway changes in D-galactose aging rats.The second experiment is to construct a pseudosterile rat model.The third experiment is to prepare fecal bacteria solution from the feces of rats in the first group,and then transplant the fecal bacteria to the pseudosterile rats.The effect of intestinal flora of rats in the first group on the relevant targets and signal pathways of pseudosterile rats is observed,with a view to clarifying the mechanism of Shouwu pill in delaying brain aging.Method:1.Theoretical discussion:carry out literature research,comprehensively sort out the understanding of TCM on brain and related viscera,the understanding of TCM on brain aging and its etiology and pathogenesis,and the relevant research on gut and brain axis,summarize and sort out the theoretical basis of Shouwu Pill in preventing and treating brain aging,and verify the mechanism of Shouwu Pill in treating brain aging with animal experiments.2.Experimental study:In experiment 1,40 SD rats were randomly divided into 4 groups:normal group,model group,vitamin E group and Shouwu pill group,with 10 rats in each group.In addition to the normal group,the other three groups used D-galactose(120mg·kg-1)to establish the aging model.At the same time,Shouwu pill group(1.08 g/kg)was used for intervention.Vitamin E group(0.018 g/kg)was given vitamin E by gavage once a day for 6 weeks.In the 7th week,fresh feces of 6 rats in each group were randomly taken and stored in a refrigerator at-80℃,and Morris water maze test was conducted at the same time.After completion,blood was taken from the abdominal aorta.Six hippocampus and jejunum samples were randomly taken from each group of rats,and the rest rats were taken from the whole brain.16SrRNA sequencing was used to analyze the changes of intestinal flora diversity and species abundance,and detect the hippocampus β-Galactosidase(β-GAL)activity,hematoxylin eosin(HE)staining to observe the pathomorphological changes of jejunum tissue,enzyme-linked immunosorbent assay(ELISA)to detect the content of trimethylamine oxide(TMAO)in serum and hippocampus of rats in each group,Spearman correlation coefficient method to analyze the correlation between the different bacterial groups based on genus level and serum TMAO,Nissl staining to observe the morphological changes of hippocampal neurons,and in situ end labeling(TUNEL)to detect the apoptosis of hippocampal cells,Golgi staining was used to observe the changes of hippocampal dendritic spines,immunohistochemistry was used to observe the changes of synapse-related proteins SYN and PSD-95,Western blot was used to detect the changes of the expression of related proteins in the hippocampus,and real-time fluorescence quantitative PCR was used to detect the expression level of genes related to the hippocampus.In experiment 2,50 rats were randomly divided into two groups:control group(10 rats)and pseudosterile rat model group(GF)(40 rats).The control group(Control)drank normal water,while the pseudosterile rat model group drank sterile water containing four kinds of antibiotics,with the concentrations of ampicillin 1 g/L,metronidazole 1 g/L,vancomycin 500 mg/L,and neomycin 1 g/L.The antibiotic drinking water was renewed every two days for 4 weeks.All rats were fed with sterile common feed.After 4 weeks,fresh feces of 6 rats in each group were taken for 16SrRNA sequencing of fecal bacteria to evaluate the effect of modeling.In experiment 3,40 pseudosterile rats were randomly divided into 4 groups,namely,FMT normal group,FMT model group,FMT vitamin E group,and FMT Shouwu pill group,with 10 rats in each group.In experiment 2,10 normal rats in the control group were included in the blank group.The feces of rats in each group in experiment 1 were prepared into a fecal bacteria suspension with a concentration of 100mg/ml.The rats in the FMT normal group,the FMT model group,the FMT vitamin E group and the FMT Shouwu pill group were respectively given the corresponding fecal bacteria suspension prepared by gavage,2ml/rat,and the rats in the blank group were given the same amount of normal saline by gavage,once every other day,a total of 8 weeks.After the completion of fecal bacteria transplantation,the indexes in experiment 1 were repeated to observe the effect of FMT on the intestine and brain of pseudosterile rats.Result:1.Experiment Ⅰ results(1)Morris water maze resultsCompared with the model group,the latency of the upper platform in vitamin E group was shortened(P<0.05),and the number of times of crossing the platform was increased(P<0.05).The average swimming speed of the rats in Shouwu pill group was increased(P<0.01),the latency of the upper platform was shortened(P<0.01),and the number of times of crossing the platform was increased(P<0.01).Compared with vitamin E group,the average swimming speed of Shouwu pill group was faster(P<0.01).(2)Hippocampus β-GAL test resultsCompared with the model group,vitamin E group and Shouwu pill group β-GAL activity decreased(P<0.01).(3)Detection results of intestinal flora 16SrRNACompared with the model group,the Chaol index of vitamin E group increased(P<0.05).The otus index increased(P<0.05),Shannon index increased(P<0.05),and Simpson index decreased(P<0.05).Beta diversity analysis,each group of samples can be distinguished.At the phylum level,compared with the model group,the rats in vitamin E group had an increase in pachyderma(P<0.05),a decrease in Proteobacteria(P<0.05),and a decrease in cyanobacteria(P<0.01).The rats in Shouwu pill group had an increase in pachyderma(P<0.01),an increase in Bacteroides(P<0.01),an increase in actinomycetes(P<0.01),and a decrease in cyanobacteria(P<0.01).At the genus level,the abundance of the model group rats of Prevotella,Lactobacillus,Spirillum,Bacteroides,Paravolotella,Brutella,and Parabacteroides were decreased,but after the intervention of Shouwu Pill,they all increased to different degrees,the difference was statistically significant.(4)TMAO detection results in serum and hippocampusCompared with the model group,the content of TMAO in serum and hippocampus of vitamin E group and Shouwu pill group decreased(P<0.01).(5)Correlation analysis of intestinal microflora and serum TMAO based on genus levelThere is a negative correlation between the abundance of the genera of Prevotella,Lactobacillus,Para-Prevotella and Broughter and the content of TMAO in serum(6)HE staining results of jejunumCompared with the model group,the striated border of the ileum in the Shouwu pill group was more regular,and the mucosal epithelial cells shed less.(7)Apoptosis of hippocampal neuronsCompared with the model group,the apoptotic positive cells in each treatment group decreased,the apoptotic index in vitamin E group decreased(P<0.05),and the apoptotic index in Shouwu pill group decreased(P<0.01);The apoptosis index of Shouwu pill group was lower than that of vitamin E group(P<0.05).(8)Nissl staining results of hippocampal neuronsCompared with the model group,the loss of neurons in vitamin E group and Shouwu pill group was improved,and the number of nissl bodies was increased,and the arrangement was denser.(9)Immunohistochemical results of hippocampal synaptic protein PSD-95 and SYNCompared with the model group,the average optical density of PSD-95(P<0.05)and SYN(P<0.01)protein in hippocampus CA1 area of rats in vitamin E group increased,and the average optical density of PSD-95 and SYN protein in hippocampus CA1 area of rats in Shouwu pill group increased(P<0.01).(10)Golgi staining results of hippocampal CA1 regionAt a concentric distance of 100 from the cell body μm、140 μm、180 μm、200 μAt m,compared with the model group,the number of dendritic and concentric circle intersections of hippocampal neurons in vitamin E group and Shouwu pill group increased(P<0.05,P<0.01);Compared with vitamin E group,the number of dendrites and concentric circles of hippocampal neurons in Shouwu pill group increased(P<0.01).Compared with the model group,the number,length and density of dendritic spines in vitamin E group and Shouwu pill group increased(P<0.01);Compared with vitamin E group,the number,length and density of dendritic spines in Shouwu pill group increased(P<0.01).(11)Results of protein expression in hippocampusCompared with the model group,the relative table of p-PI3K,p-Akt,Caspase-3 protein and the ratio of p-PI3K/PI3K,p-Akt/Akt in vitamin E group and Shouwu pill group decreased(P<0.01),the relative expression of Fox03a,p-Fox03a,Bcl-2 protein and the ratio of p-FoxO3a/FoxO3a,Bcl-2/Bax increased(P<0.01),the expression of Bax protein decreased(P<0.01),and the expression of p-4EBP2 protein increased in vitamin E group(P<0.01),the expression of p-mTOR and p-p70S6K protein decreased(P<0.01),the expression of 4EBP2(P<0.05)and p-4EBP2(P<0.01)protein increased.Compared with vitamin E group,p-Akt(P<0.05),Caspase-3(P<0.01),the relative expression of Bcl-2 protein and p-Fox03a/FoxO3a,the ratio of Bcl-2/Bax increased(P<0.01),and the expression of Bax protein decreased significantly(P<0.01),the expression of p-mTOR and p-p70S6K protein decreased(P<0.01).(12)mRNA results of hippocampusCompared with the model group,the expression of Fox03a and 4EBP2 mRNA in vitamin E group and Shouwu pill group increased(P<0.01),and the expression of mTOR and p70S6K mRNA decreased(P<0.01);Compared with vitamin E group,the expression of Fox03a mRNA in Shouwu pill group increased(P<0.05),and the expression of p70S6K mRNA decreased(P<0.05).2.Experiment Ⅱ resultsDetection results of intestinal flora 16SrRNAAlpha diversity analysis index comparison,compared with the control group,the Chaol index,Faith index,Observed index of the pseudosterile group_The otus index,Shannon index and Simpson index all decreased(P<0.01);Beta diversity analysis,each group of samples can be distinguished.At the phylum level,compared with the control group,the relative abundance of Bacteroides in pseudoaseptic group decreased(P<0.01),the relative abundance of Proteobacteria increased(P<0.01),the relative abundance of Softwall bacteria decreased(P<0.01),and the relative abundance of Actinomyces decreased(P<0.01).3.Experiment Ⅲ results(1)Morris water maze resultsCompared with the FMT model group,the total swimming distance of the FMT Shouwu pill group was shorter(P<0.01),the latency of the upper platform was shorter(P<0.01),and the number of times of rats crossing the platform in the FMT vitamin E group and the FMT Shouwu pill group was increased(P<0.01);Compared with the FMT vitamin E group,the total swimming distance of the FMT Shouwu pill group was shorter(P<0.05).(2)Hippocampus β-GAL test resultsCompared with FMT model group,FMT vitamin E group and FMT Shouwu pill group β-GAL activity decreased(P<0.01).(3)Detection results of intestinal flora 16SrRNACompared with FMT model group,the Chao1 index and Shannon index of FMT Shouwu pill group increased(P<0.01).Otu index increased(P<0.01);Compared with the FMT vitamin E group,the Chaol index of the FMT Shouwu pill group increased.Beta diversity analysis,each group of samples can be distinguished.At the phylum level,compared with the FMT model group,the relative abundance of Bacteroides,Bacteroides and Actinomyces in the FMT vitamin E group and the FMT Shouwu pill group increased(P<0.01);Compared with FMT vitamin E group,the relative abundance of Bacterobacter and Bacteroides in FMT Shouwu pill group increased(P<0.01).At the genus level,the abundance of the genera of Prevotella,Lactobacillus,Spirillum,Bacteroides,Paravolotella and Parabacter di in the FMT model group decreased,but after the intervention of Shouwu Pill,they all increased to different degrees,the difference was statistically significant(P<0.05).(4)Test results of TMAO in rat serum and hippocampusCompared with FMT model group,the content of TMAO in serum and hippocampus of FMT vitamin E group and FMT Shouwu pill group decreased(P<0.01).(5)Correlation analysis of intestinal microflora and serum TMAO based on genus levelThe abundance of Lactobacillus,Spirillum,Paravolvulus,and Parabacter di was negatively correlated with the content of TMAO in serum.(6)HE staining results of jejunumIn the FMT model group,a large number of necrosis occurred in the jejunum mucosa layer,the mucosa layer narrowed,the lamina propria was accompanied by a large number of lymphocytes,plasma cell infiltration,a large number of mucosal epithelial hyperplasia,and the number of intestinal glands decreased and arranged in disorder.In the FMT Shouwu pill group,the structure of each layer of jejunum tissue is clear,the mucosa epithelium is complete,and the glands in the lamina propria are rich and orderly arranged.(7)Apoptosis of hippocampal neuronsCompared with FMT model group,the apoptosis positive cells in FMT vitamin E group and FMT Shouwu pill group decreased,and the apoptosis index in FMT vitamin E group and FMT Shouwu pill group decreased(P<0.01).(8)Nissl staining results of hippocampal neuronsIn the FMT model group,hippocampal neurons were lost and sparsely arranged,the number of nissl bodies decreased,and nissl bodies were pyknotic and hyperchromatic;In the FMT vitamin E group and the FMT Shouwu pill group,the loss of neurons was improved,the number of nissl bodies was increased,and the arrangement was dense.(9)Immunohistochemistry of hippocampal synaptic protein PSD-95 and SYNCompared with the FMT model group,the average optical density of PSD-95 and SYN protein in the hippocampus CA1 region of rats in the FMT vitamin E group increased significantly(P<0.01),while the average optical density of PSD-95 and SYN protein in the hippocampus CA1 region of rats in the FMT Shouwu pill group increased significantly(P<0.01);Compared with the FMT vitamin E group,the average optical density of PSD-95 protein and the average optical density of SYN protein in the FMT Shouwu pill group increased(P<0.05).(10)Golgi staining results of hippocampal CA1 regionAt a concentric distance of 100 from the cell body μm、140 μm、180 μm、200 μ At m,compared with the FMT model group,the number of dendrites and concentric circles of hippocampal neurons in the FMT vitamin E group and the FMT Shouwu pill group increased;Compared with the FMT vitamin E group,the number of dendrites and concentric circles of hippocampal neurons in the FMT Shouwu pill group increased(P<0.01).Compared with FMT model group,the number,length and density of dendritic spines in FMT vitamin E group and FMT Shouwu pill group increased(P<0.01);Compared with the FMT vitamin E group,the number of dendritic spines in the FMT Shouwu pill group increased(P<0.01)and the length increased(P<0.05).(11)Results of protein expression in hippocampusCompared with FMT model group,the relative table of p-PI3K,p-Akt,Caspase-3 protein and the ratio of p-PI3K/PI3K,p-Akt/Akt in FMT vitamin E group and FMT Shouwu pill group decreased(P<0.01),the relative expression of FoxO3a,p-FoxO3a,Bcl-2 protein and the ratio of p-FoxO3a/FoxO3a,Bcl-2/Bax increased(P<0.01),while the expression of Bax protein decreased(P<0.01),the expression of p-4EBP2 protein increased in FMT vitamin E group(P<0.01),the expression of p-mTOR and p-p70S6K protein in FMT Shouwu pill group decreased(P<0.01),the expression of 4EBP2(P<0.05)and p-4EBP2(P<0.01)protein increased.(12)mRNA results of rats in each groupCompared with FMT model group,the expression of Fox03a and 4EBP2 mRNA in FMT vitamin E group and FMT Shouwu pill group increased(P<0.01),and the expression of mTOR and p70S6K mRNA decreased(P<0.01).Compared with FMT vitamin E group,the expression of Fox03a mRNA in FMT Shouwu pill group increased(P<0.05),and the expression of p70S6K mRNA decreased(P<0.05).Conclusion:(1)The pathogenesis of brain aging is yin deficiency of liver and kidney,and the treatment of delaying brain aging is to replenish yin essence and yin blood,and treat both liver and kidney;(2)Shouwu pill can improve the pathological morphology of jejunum in D-galactose-induced aging rats,increase the biological diversity of intestinal flora in D-galactose-induced aging rats,and regulate the structure of flora;(3)Shouwu pill can improve the learning and memory ability of D-galactose model rats,enhance the expression of synaptic plasticity related protein,improve the morphology of neurons,repair nerve function,reduce neuronal apoptosis,inhibit mTOR signal pathway,and delay brain aging;(4)Using fecal bacteria transplantation,the jejunum morphology and flora structure of rats in the FMT Shouwu Pill group were more similar to those in the blank group and the FMT normal group.The production of TMAO in the hippocampus and serum was reduced,the expression of synaptic plasticity related protein was increased,the morphology and function of neurons were restored,the apoptosis of neurons was reduced,and the mTOR signal pathway was down-regulated;(5)Shouwu Pill regulates the structure of intestinal flora,reduces the production of TMAO,inhibits neuronal apoptosis,enhances synaptic plasticity,and inhibits mTOR signal pathway by regulating the "gut brain axis",thus delaying brain aging.