Paeonol Regulates Bile Acid Metabolism And Exerts Anti-atherosclerotic Effect Based On Intestinal Flora

BACKGROUND:Atherosclerosis（AS）is the common pathological basis of cardiovascular events such as myocardial infarction and cerebral infarction.Excessive blood lipid levels and abnormal cholesterol metabolism are important causes of cardiovascular diseases.Bile acids（BA）are the main downstream products of cholesterol catabolism.They can act as signal molecules to activate the nuclear receptor FXR.The downstream effects can regulate liver lipid production,plasma lipid clearance and intestinal cholesterol absorption.fat metabolisim.The intestinal flora is essential for the conversion of bile acids.The intestinal flora converts bound bile acids into free bile acids through bile salt hydrolase,and regulates nuclear receptors to participate in the conversion of liver cholesterol into bile acids and bile acids in the intestine.The process of reabsorption,and then regulate lipid metabolism to play an anti-AS effect.Pae is one of the main active ingredients in the dried root bark of the peony plant of the peony family.It has antibacterial,antioxidant,hypolipidemic,anti-atherosclerosis and other pharmacological functions.Most of Pae’s oral administration enters the body.The drug is excreted with feces through the intestine.Based on the above studies,we speculate that the reason why Pae has a better lipid-lowering effect may be that Pae excreted in the feces through the intestine affects the intestinal flora to regulate bile acid metabolism,thereby affecting lipid metabolism,improving blood lipid levels,and playing anti-AS effects..This study aims to explore whether Pae’s anti-AS effect regulates lipid metabolism through intestinal flora and bile acid metabolism,and to provide ideas for further revealing the new way of Pae’s anti-AS effect.OBJECTIVE:Replicate the ApoE-/-mouse AS model to explore whether Pae can affect the FXR/FGF15 signaling pathway by regulating the intestinal flora and bile acid metabolism of ApoE-/-mice,thereby affecting lipid metabolism to play an anti-AS effect.METHODS:ApoE-/-mice were fed with high-fat diet to establish an AS model;gross aorta and liver tissues were oil red O staining method to observe aortic plaque formation and liver tissue morphological changes;enzymatic method to detect serum total cholesterol（TC）and glycerol Triester（TG）,high-density lipoprotein cholesterol（HDL-C）,low-density lipoprotein cholesterol（LDL-C）and liver TC and TG levels;extraction of fecal bacterial DNA and analysis of bacterial 16S r DNA sequence,using ultra-high liquid phase Chromatographic tandem mass spectrometry UPLC-MS/MS method was used to detect the changes of bile acid components in the intestine of mice,and the total bile acid level in feces was detected by enzymatic method;the expression of CYP7A1,ileum FXR and FGF15 proteins in mouse liver group was detected by Western blot.RESULTS:1.Pae significantly inhibit the plaque formation and the increase of liver coefficient in ApoE^-/-miceHigh-fat diet can significantly increase the area of AS plaques in the aorta of ApoE-/-mice.Pae administration can significantly improve the extent of AS lesions,reduce the area of aortic plaques,improve the liver shape and size of AS mice,and reduce the liver of mice.coefficient.The use of antibiotics to inhibit the intestinal flora of mice can significantly weaken the effect of Pae on improving the pathological changes of AS mice,suggesting that the intestinal flora plays an important role in it.2.Pae significantly change the lipid metabolism level of ApoE^-/-micePae intervention can significantly reduce the increase in TG,TC,LDL-C levels induced by high-fat diet,and reduce HDL-C levels（P<0.01）.Pae can effectively regulate the blood lipid levels of AS mice.Antibiotic intervention can also reverse the lipid levels induced by a high-fat diet to varying degrees.Further analysis found that when antibiotics were used to inhibit the intestinal flora of mice,the effect of Pae on improving blood lipids was significantly inhibited.The combination of antibiotics and Pae was not as effective as Pae alone in regulating blood lipid levels,and Pae reduced the aortic plaque area of AS mice.It may be related to the intestinal flora.3.Pae changes the structure and composition of the intestinal flora of ApoE^-/-miceAfter the induction of high-fat diet,the diversity and richness of the intestinal contents of ApoE-/-mice were reduced.Pae intervention significantly increased the diversity and richness of the intestinal flora of AS mice,which could significantly reverse the high-fat diet The above-mentioned intestinal flora changes at the phylum level and the genus level,increasing the abundance of Bacteroides and Lactobacillus,which are closely related to BSH.4.Pae changes the intestinal flora of ApoE^-/-mice and affects bile acid metabolismPae intervention can significantly increase the ratio of free bile acid/bound bile acid in the ileum of AS mice（P<0.05）.By increasing the abundance of Bacteroides and Lactobacillus,which are closely related to BSH,increase the activity of BSH enzymes and convert bound bile acids into free bile acids.At the same time,it reduces the content of total bile acid in the ileum and promotes the efflux of total bile acid in feces.5.Pae inhibits enterohepatic FXR/FGF15 pathway,promotes bile acid synthesis and regulates lipid metabolismPae can increase the content of FXR antagonist TαMCA and TβMCA,and reduce the content of FXR agonist CDCA and LCA.After Pae intervention,the ileum FXR and FGF15 protein expression levels of AS mice were significantly reduced,and the liver tissue CYP7A1 protein expression level was significantly increased.It shows that Pae can reduce the expression of ileal FXR and FGF15 protein,increase the expression of liver CYP7A1 protein,and then promote the synthesis of cholesterol to bile acid in the liver.IN CONCLUSION:1.Pae can significantly reduce the aortic plaque area of AS mice,improve the blood lipid level and hepatic lipid level disorders induced by high-fat diet,so as to play the role of treating AS.2.Pae can significantly improve the structure of the intestinal flora of mice.At the phylum level,it is mainly manifested in increasing the ratio of Bacteroidetes/Firmicutes and reducing the abundance of Proteobacteria;at the genus level,Pae can significantly reverse the decrease in Bacteroides and Lactobacillus and the increase in Helicobacter abundance caused by high-fat diet.3.Pae may regulate bile acid metabolism by affecting the structure and composition of the intestinal flora,increase the conversion of bound bile acids into free bile acids,and promote fecal total bile acid efflux;at the same time increase the content of FXR antagonists TαMCA and TβMCA,Reduce the content of FXR agonists CDCA and LCA,inhibit the ileal FXR-FGF15 axis,promote liver bile acid synthesis,reduce liver and serum cholesterol content,and regulate lipid metabolism to play an anti-AS effect.