Changes Of Gut Microbiota In Rats With D-gal-induced Vascular Aging And Interventional Effect Of GSC Extracts

Vascular aging is a degenerative change of vascular structure and function,a common pathological basis for the occurrence and development of various diseases,and a key factor affecting the health status of the elderly.Found in recent years,gut microbiota is important "physiological organ" in the body.It can affect the incidence of many diseases and t is closely associated with cardiovascular disease and aging.It was found that D-galactose（D-gal）can induce subacute aging models of animal in short time.The results are stable and easy to operate,and the aging mechanism and characteristics of D-gal-induced are similar to those of natural aging.Previous studies by the research group have proved that ginseng,sanqi and chuangxiong（GSC）extracts,a traditional Chinese medicine for Tonifying Qi and Activating Blood,can delay the natural aging process of blood vessels in rats or mice,and the vascular aging of mice induced by high glucose and high fat.Therefore,it is of great significance to establish D-gal-induced subacute senescence rats model,to explore the relationship between gut microbiota and vascular aging,and to find out the specific flora affecting vascular aging,and to observe the intervention effect of GSC extracts on gut microbiotaand vascular aging.The first section:D-gal induced vascular aging of rats and intervention of GSC extractsObjective:To observe the vascular aging in D-gal-induced subacute aging rats model and the interventional effect of GSC extracts.Methods:D-gal-induced subacute aging rat model was constructed by subcutaneously injecting 5%D-gal solution（125mg/kg）into the neck and back of SD rats for 10 consecutive weeks.The experiment is divided into youth group,blank control group,model group,Chinese medicine low dose group,high dose group,berberine group and metformin group to research the aorta of rats After 10 weeks,giving rats drug intervention for 10 weeks in a row.The model group was given the corresponding volume of pure water every day.The blank control group rats were fed normally,and did not carry out modeling experiment.The youth group is normal SD rats of 3-month-old.Behavioral test to observe the autonomous activity,exploration ability and learning and memory ability of rats;The morphology and lumen size of rat carotid artery wall were observed by high resolution small animal ultrasound imaging system;Blood glucose,serum total cholesterol,triglyceride,low density lipoprotein cholesterol and high density lipoprotein cholesterol levels in rats were detected by automatic biochemical analyzer;HE staining and Masson staining were used to observe the morphology of the aortic tube wall,and to analyze the changes in the thickness of the aorta intima and intima and the collagen content of the aortic tube wall in each group of animals;Immunohistochemical staining was used to observe and detect the expression levels of advanced glycationend products（AGE）ang matrix metalloproteinases 2（MMP-2）in the aorta;The expression of p16 and p53 proteins in the aorta was detected by Western blot;The levels of IL-1β,IL-6,IL-17A and TNF-α and the activity of superoxide dismutase（SOD）in serum were determined by Elisa.Results:（1）Compared with the blank control group.the number of grid penetration of rats in the model group were significantly reduced（P<0.05）;In the navigation test of positioning.the latency period is prolonged,the average speed is reduced,and in the space exploration test,the proportion of time in the target quadrant is reduced,and the times of crossing the platform is reduced（P<0.05）;The levels of GLU,TC and LDL-c increased（P<0.05）;Carotid wall significantly thickened（P<0.01）;Abnormal changes in the morphology of the aorta,hyperplasia and rupture of the intima,disorder and hyperplasia of the mediummembrane structure,and significantly increased intima thickness（P<0.01）;Collagen fiber content of vascular significantly increased（P<0.01）;The expression levels of AGE,MMP-2,p16 and p53 were significantly increased in the aorta（P<0.01）;The levels of IL-6,IL-17A and TNF-αwere significantly increased in Serum（P<0.05）.（2）After drug intervention,the ability of rats increased in autonomous activities and exploration behaviors.Their learning and memory ability increased,blood glucose levels decreased,blood lipid levels did not change significantly,carotid artery wall thickness was significantly reduced,vascular endothelium damage was reduced,and endometrium was relatively smooth and compact connection.The arrangement of the median membrane structure was acceptable,the thickness of the medial intima was reduced,the content of collagen fibers was reduced,the expression levels of AGE,MMP-2,p16 proteins were significantly reduced,the levels of serum IL-1β,IL-6,TNF-α reduced,and SOD activity improved（P<0.05）.Conclusion:Vascular aging can be found in the rats of D-gal-induced subacute senescence,that is to say,D-gal can induce vascular aging of rats.GSC extracts can improve D-gal-induced vascular aging in rats and delay the process of vascular aging.The second section:Changes of gut microbiota in rats with vascular aging and interventional effect of GSC Extracts.Objective:To observe the changes of gut microbiotain in D-gal-induced vascular aging rats and the intervention of GSC extracts.Methods:Using D-gal-induced fecal samples and serum from vascular aging rats as the research object.Elisa method was used to detect the content of D-lactic acid in the serum of the rats,colorimetric methods were used to detect the serum of the rats diamine oxidase（DAO）activity,to determine the integrity and damage of the intestinal mucosa;GC-MS method was used to detect the content of short chain fatty acids（SCFAs）in the feces of rats,to observe the changes of SCFAs;By high-throughput sequencing platform detect the 16S rDNA sequence in rat stool samples.Analyze Alpha diversity,composition and structure,and the differential bacteria between groups of rats and to observe the changes of gut microbiota in vascular aging rats that induced by D-gal and the effect of GSC extracts on gut microbiota.Results:（1）Compared with the blank control group,the serum d-lactic acid content and DAO activity were significantly increased in the model group（P<0.01）;Propionic acid,butyric acid and valeric acid in feces significantly decreased（P<0.01）;In the analysis of Alpha diversity,there was no significant difference between Chao1 index and Shannon index.No significant change in the number of OTU;PCoA analysis showed that samples of the model group and the blank control group were distributed in parallel,with a certain distance.At the level of phylum,Firmicutes and Bacteroidetes were the main gut microbiotain in the two groups,and relative abundance was respectively 59.78%and 35.85%in the control group,and 48.02%and 46.62%in the model group.The B/F value in the model group was significantly increased（P<0.01）;From the heat map analysis,there is a large difference in the composition of gut microbiota between the model group and the blank control group;LEfSe analysis shows that the differential bacteria of the two groups with LDA>4.The difference in the blank control group is mainly g_Prevotellaceae UCG 003、s_uncultured bacterium_gPrevotellaceae UCG 003 and g_Fusicatenibacteretc,the differential bacteria of model group include f_Prevotellaceae、c Bacteroidia、p_Bacteroidetes,etc.There are large differences between the two groups;ANOVA analysis of the significance of different species showed that the level of ruminococcaceae_ucg-013 significantly increased in the blank control group and the level of Alloprevotella significantly increased in the model group（P<0.05）.（2）After drug intervention,D-lactic acid content and DAO activity in serum of rats were significantly decreased（P<0.01）;The contents of acetic acid and propionic acid in the Chinese medicine high dose group increased（P<0.05）;PCoA and heat map analysis showed that the composition of gut microbiotain the Chinese medicine groups was close to that in the blank control group;At the level of phylum,the gut microbiotaof the Chinese medicine groups was also dominated by Firmicutes and Bacteroidetes,and their relative abundances were respectively 58.58%and 36.33%in the Chinese medicine low dose group,62.46%and 31.88%in the Chinese medicine high dose group,and the B/F value Significantly reduced（P<0.01）,tending to the normal control group;LEfSe analysis of the differential bacteria in the two groups of LDA>4 showed that the differential bacteria in the Chinese medicine low dose group was g_Erysipelotrichaceae_UCG₀03,and the differential bacteria in the Chinese medicine high dose group mainly included c_Bacilli、o Lactobacillales、f_Lactobacillacea,etc.ANOVA analysis showed that there were no difference species significantly higher than other groups in the Chinese medicine groupsConclusion:In D-gal-induced vascular aging rats,we can find abnormal changes of gut microbiota in the composition,structure and SCFAs of metabolites.In model group,the ratio of B/F and the abundance of Alloprevotella increased significantly,and we can see that the intestinal mucosa of rats damages severely and intestinal permeability increases,which may be an important gut microbiota affecting the vascular aging process;GSC extracts can regulate the composition and structure of gut microbiota,and make it close to normal,increase intestinal SCFAs content,reduce the ratio of B/F and the abundance of Alloprevotella,alleviate the damage of intestinal mucosal,decrease intestinal permeability,and improve the intestinal environment.