Study On The Effect And Mechanism Of Ginsenoside Rc On HFD Induced Atherosclerosis In ApoE^-/- Mice

Cardiovascular diseases（CVDs）are leading cause of death worldwide.Atherosclerosis（AS）is the pathological basis of CVDs and characterized by the formation of fibrofatty lesions in the artery wall.The risk factors for AS include low-density lipoprotein cholesterol（LDL-C）,hypertension,cigarette smoking,obesity and diabetes mellitus.Numerous investigations owe pathogenesis of AS to disorder of lipid metabolism,inflammation,oxidative stress and vascular endothelial damage.Despite the advanced treatment applied to clinic,therapeutic challenges including side effects and nonadherence to pharmacological therapy are increasingly apparent.Shexiang Baoxin Pill（SBP）,a famous traditional Chinese medicine（TCM）formulation deriving from an ancient Suhexiang Pill,has been widely used to treat CVDs.Our previous investigation on material basis and mechanism of SBP against AS had found that ginsenoside Rc（GRc）was an active ingredient in alleviating AS.Recently,the association among CVDs,gut microbiota and metabolites have aroused increasing attention.This study aims to confirm the efficacy of GRc on high fat diet（HFD）-induced AS in apolipoprotein E-deficient(ApoE^-/-)mice and to investigate its potential mechanism from the perspective of gut microbiota and its metabolites,thus providing scientific theory support for treating AS.The detailed research steps and results are as follows:1.The effect of GRc on atherosclerotic plaque,serum lipid and inflammatory cytokines in ApoE^-/-mice fed a HFD was investigated.7-week-old male C57/BL6 ApoE^-/-mice were fed a HFD with GRc treatment（40 mg/kg/d GRc by gavage）for 12 weeks and samples were collected.The results showed that GRc significantly reduced aortic plaque area,atherosclerotic lesions and collagen in aortic sinus,improved lipid disorder and systemic inflammation in HFD-induced ApoE^-/-mice,implying its effective effect on alleviating AS.2.In addition,the effect of GRc on composition of gut microbiota in ApoE^-/-mice was investigated.The results of 16S r RNA sequencing showed that GRc significantly increased the abundance and diversity of gut microbiota.βdiversity analysis indicated different constructures of the bacterial communities among different groups.Besides,the gut microbiota related to CVDs were significantly changed by GRc at different taxonomic levels.At phylum level,GRc significantly regulated the relative abundance of Bacteroidetes and Firmicutes.At genus level,GRc markedly reshaped 8 intestinal floras related to CVDs.The above results implied the effective alteration of GRc on gut microbiota to beneficial composition that alleviated AS.3.Furthermore,the effect of GRc on fecal metabolites in ApoE^-/-mice was investigated.Cecal contents were analyzed by untargeted fecal metabolomics.The results showed that there was significant difference in fecal metabolic profile between GRc and HFD group.KEGG pathway enrichment analysis showed that 7 metabolic pathways related to AS were found out.Accordingly,GRc significantly changed 23 differential metabolites from above 7pathways.These results implied that GRc could modulate fecal metabolic profiles,regulate metabolic pathways and differential metabolites related to AS in ApoE^-/-mice.The potential biomarkers of AS might be novel targets for treating AS.4.Spearman correlation analysis was applied to examine the possible connection among gut microbiota,potential biomarkers in feces and atherosclerotic indexes based on the results of 16S r RNA sequencing and untargeted fecal metabolomics.The results showed that there was intensive connection among differential gut microbiota,potential biomarkers in feces and atherosclerotic indexes,which implied the potential mechanism of GRc against AS was probably via regulating gut microbiota,followed by altering fecal metabolites to beneficial profile that alleviated AS.In conclusion,the present study confirmed the efficacy of GRc in the treatment of HFD-induced AS in ApoE^-/-mice.In addition,differential intestinal floras,potential metabolic pathways and biomarkers of AS were investigated by 16S r RNA sequencing and untargeted fecal metabolomics methods.Spearman correlation analysis was applied to examine the possible connection among gut microbiota,potential biomarkers in feces and atherosclerotic indexes,providing that the potential mechanism of GRc against AS was probably comprehensive effects of regulating gut microbiota and differentially expressed fecal metabolites closely related to AS.This study firstly investigated the potential mechanism of GRc on antiatherosclerotic effect from perspective of gut microbiota and metabolites,provided potential novel targets and research methods for drug development to treat AS.