Study On Improvement Of Ferulic Acid On Cholesterol Metabolism Disorder Induced By High Fat Diet In Mice And Its Mechanism

With the change of modern human dietary structure,the intake of a large amount of highfat and high-cholesterol foods leads to the disorder of lipid metabolism in the body.Among them,hypercholesterolemia,which is manifested by disorders of cholesterol metabolism,is the pathological basis of many cardiovascular diseases.The current clinical drugs for the treatment of hypercholesterolemia limit their application in the prevention of hypercholesterolemia due to their potential side effects.Therefore,the search for active substances of natural origin for the prevention of hypercholesterolemia has been of great interest.Ferulic acid is a phenolic acid compound commonly found in plant foods,such as whole grains,vegetables,and fruits.Numerous studies have shown that it has a preventive effect on hypercholesterolemia.However,its specific molecular mechanism has not been fully elucidated.Therefore,in this study,hypercholesterolemia mice induced by a high-fat diet were used as animal models to study the preventive effect of ferulic acid on hypercholesterolemia,and then RNA-seq was used to screen the key genes for improving cholesterol metabolism disorders with ferulic acid in high-fat diet mice.After that,the mechanism mediated by the key gene was verified by using non-targeted metabolomics.Finally,the molecular mechanism of the key gene activated by ferulic acid was deeply explored using technologies such as targeted metabolomics and 16 s r DNA amplicon sequencing.The study is expected to reveal the molecular mechanism of ferulic acid in improving hypercholesterolemia,and provide a theoretical basis for the development of functional foods to prevent and improve cardiovascular disease.The main research contents and results are as follows:1.Ferulic acid supplementation improves cholesterol metabolism disorder in mice fed a high-fat diet: The hypercholesterolemia animal model was established by feeding C57BL/6J male mice with high-fat diet for 12 weeks,and the preventive effect of 100 mg/kg/day ferulic acid supplementation on hyperlipidemia was studied in mice.The results showed that the serum total cholesterol of the mice in the ferulic acid group(HFD+FA)decreased by 13.2%(p < 0.05)and the excretion of bile acids in the feces increased by 37.7%(p < 0.05)as compared with the high-fat group(HFD).These results showed that ferulic acid supplementation could significantly improve the cholesterol metabolism disorder induced by a high-fat diet.2.Screening and validation of key genes for improving cholesterol metabolism disorder with ferulic acid: RNA-seq was performed on liver samples from three groups,and Cyp7a1,a key gene encoding cholesterol catabolism enzymes cholesterol 7α hydroxylase(CYP7A1),was screened.Compared with the HFD group,the hepatic m RNA level of Cyp7a1 of mice in the HFD+FA group was up-regulated 2.1-fold(p < 0.05).Subsequently,the hepatic expression of Cyp7a1 in mice from three groups was verified by q RT-PCR,Western Blot and immunohistochemistry.The results showed that ferulic acid supplementation could significantly increase the hepatic m RNA and protein levels of Cyp7a1 in mouse(p < 0.05).3.Validation of the mechanism that ferulic acid improving cholesterol metabolism disorder by regulating the key liver gene Cyp7a1: To explore the mechanism that ferulic acid improving cholesterol metabolism disorder by regulating the key liver gene Cyp7a1,nontargeted metabolomics were performed on the liver of mice from three groups and total cholesterol level were detected.The results showed that the hepatic primary bile acids CA andβ-MCA in the HFD+FA group increased by 2.4 times and 6.0 times(p < 0.05),respectively,and the total liver cholesterol decreased significantly by 28.7%(p < 0.05),as compared with the HFD group.It suggested that ferulic acid supplementation increased the conversion from cholesterol to bile acids by activating hepatic Cyp7a1 and decreased hepatic cholesterol levels.4.Ferulic acid supplementation increases intestinal bile acid reabsorption and serum bile acid levels by activating Cyp7a1 in mouse liver: Targeted metabolomics and q RT-PCR were used to detect serum bile acid profiles and the m RNA levels of bile acid reabsorption protein in ileum,respectively.The results showed that compared with the HFD group,the serum total bile acid of the mice in the HFD+FA group increased by 33.5%(p < 0.05),and the ileal m RNA levels of the reabsorption protein OST-α and ASBT of the mice in the HFD+FA group were respectively 8.4-fold and 9.3-fold increase(p < 0.01).It showed that ferulic acid supplementation increases the reabsorption of bile acid in the gut and bile acid levels in serum.5.Ferulic acid activate hepatic Cyp7a1 through the non-FXR pathway: To explore whether Cyp7a1 activation is mediated by hepatic or intestinal FXR signaling,mouse fecal16 s r DNA amplicon sequencing was performed and hepatic or intestinal FXR signaling pathway was detected by q RT-PCR.The results showed that ferulic acid supplementation did not change the abundance of fecal BSH enriched bacteria and the expression of intestinal FGF15 or liver SHP in high-fat diet mice,indicating that hepatic Cyp7a1 activation by ferulic acid through the non-FXR signalingp athway.6.Study on the activation law of CYP7A1 by ferulic acid in mouse liver: The hepatic m RNA levels of Cyp7a1 gene were detected at different time points after a single administration of 100 mg/kg ferulic acid to mice on a high-fat diet.The results showed that the m RNA level of Cyp7a1 gene was significantly increased(p < 0.05)within 4-12 h of intragastric administration of ferulic acid to mice.However,ferulic acid had no significant effect on the promoter activity of the Cyp7a1 gene in AML12 cell.It indicated that the activation of Cyp7a1 gene by ferulic acid is time-sensitive,and this process may require the participation of extrahepatic signals.The above results indicate that ferulic acid supplementation activates hepatic CYP7A1,the rate-limiting enzyme of the classical bile acid synthesis pathway through the non-FXR pathway,and increases the conversion of cholesterol into bile acids in the liver.Therefore,the cholesterol metabolism disorder induced by a high-fat diet in mice was improved,and the reabsorption of bile acids and excretion of total fecal bile acids were increased.It has laid a theoretical foundation for the in-depth elucidation of natural products such as ferulic acid for preventing hypercholesterolemia and cardiovascular disease induced by a high-fat diet.