"Gualou-Xiebai" Resists Dyslipidemia By Regulating Microbiota-Derived Short-Chain Fatty Acids That Inhibit Gut Absorption Of Cholesterol In Atherosclerotic Mice

Atherosclerosis is a progressive vascular disease,which is the pathological basis of cardiovascular diseases such as coronary heart disease and myocardial infarction.The pathological process of AS was accompanied by complex lipid metabolism disorders in the organism.High cholesterol level is one of the main markers of lipid metabolism disorders.The abundance of harmful bacteria such as Collinaceae,Enterobacteriaceae,Streptococcaceae and Klebsiella in the gut microbiota of AS patients is significantly increased,which is positively correlated with cholesterol level.Studies have shown that the disorder of gut microbiota interferes with the level of its metabolites,affecting the synthesis,absorption,transport and excretion of cholesterol,leading to the accumulation of cholesterol in blood vessels and promoting the occurrence of AS.Plasma cholesterol is mainly derived from intestinal cholesterol absorption and hepatic cholesterol synthesis.Therefore,limiting cholesterol absorption is an important strategy to reduce blood cholesterol level."Gualou-Xiebai"（GLXB）herb pair,a classical Chinese herbal formula,was first proposed by Zhang Zhongjing（in the Eastern Han Dynasty,third century China）in the treatise“Jin Kui Yao Lue”.Modern pharmacological studies have shown that GLXB effectively regulates blood lipid levels and inhibits lipid accumulation in the blood of Apo E^-/-mice,However,whether GLXB intervention can regulate the intestinal microbiota to alleviate dyslipidemia remains unknown.Objective:This study aims to investigate whether the effect of GLXB on dyslipidemia in AS mice is related to the regulation of gut microbiota,and to identify the key bacteria and metabolites of gut microbiota regulated by GLXB.To further clarify whether GLXB affects intestinal cholesterol absorption by regulating gut microbiota and its metabolites,thereby exerting anti-dyslipidemia effect.This study provides a new idea for the treatment of AS with GLXB based on the“gut-vascular axis”.Method:An AS mouse model was established by administering a high-fat diet to apolipoprotein-E deficient(Apo E^-/-)mice.The Serum lipid levels of mice were measured by enzymatic method;The effect of GLXB on gut microbiota composition in mice with AS was investigated by 16Sr DNA-sqe,to identify the key gut bacteria regulated by GLXB.Targeted metabolomics of short-chain fatty acids（SCFAs）,a metabolite of gut microbiota,in cecal contents of AS mice was detected by gas chromatography-mass spectrometry,to identify the key microbial metabolites regulated by GLXB.The expression of proteins involved in cholesterol absorption in AS mice intestinal and intestinal epithelial cells was examined by Western Blot.Results:1.Pharmacodynamic observations:GLXB improved dyslipidemia and gut microbiota disorder in AS miceGLXB significantly reduced the serum levels of TC,TG and LDL-C,and significantly increased the level of HDL-C in mice.HFD-induced gut microbiota disorder in AS mice,and the richness and diversity of gut microbiota in AS mice were significantly reduced.The structure of gut microbiota was significantly changed,and 12 species were significantly different.GLXB significantly increased the richness and diversity of gut microbiota and improved the structure of gut microbiota in AS mice.It mainly increased the abundance of SCFAs producing bacteria Lachnospiraceae,Rikenella,Roseburia,Alloprevotella,Ruminiclostridium and Ruminococcaceae＿UCG-014.2.Omics screening:GLXB increased the content of propionic acid and butyric acid in gut microbiotaThe SCFAs levels in cecal contents of AS mice were altered,and propionate and butyric acid were significantly decreased,while GLXB could significantly increase propionate and butyric acid.Literature has shown that butyrate has a certain regulatory effect on intestinal cholesterol absorption related proteins.Therefore,we chose butyrate for the next mechanism discussion.3.Mechanistic study:GLXB inhibits intestinal cholesterol absorption by increasing the level of butyrateIn vitro,butyrate（0.1,0.2 and 0.4 mmol/L）significantly reduced cholesterol level in Caco-2 cells and inhibited the expression of cholesterol absorption-related proteins NPC1L1,MTP and Apo B48.Animal experiments:The intestinal cholesterol level of AS mice was significantly increased,and the expression of cholesterol absorption related proteins NPC1L1,ACAT2,MTP and Apo B48 was significantly increased.GLXB significantly reduced intestinal cholesterol levels and significantly inhibited the expression of the above proteins in AS mice.These results suggest that GLXB may inhibit the intestinal absorption of cholesterol by increasing the level of butyrate and inhibiting the expression of cholesterol absorption-related proteins.Conclusion:GLXB can inhibit the expression of intestinal cholesterol absorption proteins and reduce intestinal absorption of cholesterol by regulating gut microbiota and increasing the level of butyric acid,a metabolite of gut microbiota,to play an anti-dyslipidemia role in AS mice.