Mechanism Of Baicalin Regulates Glucose And Lipid Metabolism In High-fat Diet Mice Through Gut Microbiota Maintenance

Background:Long-term high-fat diet can change the normal microecology of gut microbiota,leading to energy metabolism,intestinal mucosal barrier and immune system changes.It is one of the important risk factors for metabolic diseases such as diabetes and hyperlipidemia.Short chain fatty acids(SCFAs)are metabolites of microbiota,including acetic acid,propionic acid and butyric acid,which play an important role in maintaining the stability of the body environment and regulating energy balance.Insufficient production of SCFAs is associated with abnormal glucose and lipid metabolism.Baicalin is a flavonoid extracted from the root of dried Astragalus membranaceus.The previous research work in our group showed that oral administration of baicalin can improve the abnormal glucose and lipid metabolism caused by long-term high-fat diet,but its mechanism of action is still unclear.The aim of this study was to investigate whether baicalin improves glucose and lipid metabolism and correlates with regulation of gut microbiota and SCFAs metabolites.Objective:To study the mechanism of baicalin in improving glucose and lipid metabolism in high-fat mice by regulating gut microbiota.Methods:1.High fat diet induced mouse model and group administration60 C57BL/6J male mice were randomly divided into normal group and high fat group to establish a mouse model induced by a high-fat diet.After successful modeling,the mice were divided into normal diet group(ND),normal diet+baicalin group(NDB),normal diet+metformin group(NDM),high-fat diet group(HFD),high-fat diet+baicalin group(HFDB),High-fat diet+metformin group(HFDM),the mice in baicalin groups were given200mg/kg/day and the mice in metformin groups were given 250 mg/kg/day,normal and high-fat control group were given an equal volume of 0.5%sodium carboxymethyl cellulose solution for 15 weeks.2.Fecal microbiota transplantation(FMT)and modeling30 male C57BL/6J mice were randomly divided into non-sterilization group(10)and sterilization group(20).Non-sterilization components were normal control group(5 rats)and high-fat control group(5 rats);Sterilized with antibiotics and divided into 4 groups,and the bacteria collected and extracted by the previous batches of mice(ND,HFD,HFDB,HFDM)were sequentially divided into FMT-ND,FMT-HFD,FMT-HFDB and FMT-HFDM group,high-fat modeling was performed for 8 weeks after transplantation,we recorded body weight and blood glucose,and collected feces samples.3.Blood glucose and blood lipid and Western blot detectionDuring the administration period,the body weight and blood glucose of the mice were measured weekly,and the glucose tolerance and insulin tolerance tests were performed,the daily food intake was recorded,and the stool samples were collected and stored in a-80°C refrigerator.After the administration,the total cholesterol(TC),low density lipoprotein(LDL-C),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)were determined by automatic biochemical analyzer.The expression level of the energy metabolism pathway protein AMPK was detected by Western blot.4.Determination of gut microbiota abundance levelThe intestinal metabolites of mice were detected by 16S rRNA amplicon pyrophosphate,using the Illumina HiSeq4000 platform to sequence,and finally analyzed by bioinformatics.5.Determination of short chain fatty acidsEight SCFAs(Acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid,isovaleric acid,caproic acid and heptanoic acid)were detected by gas chromatography in intestinal metabolites of mice.Quantitative dissolution standards to prepare stock solutions and determine the peak positions of the eight SCFAs,the nitrile was selected as the internal standard,the concentration of short-chain fatty acids in the sample was calculated as the ratio of the peak area of the nitrile to the peak area of the desired short-chain fatty acid.Results:1.Baicalin improves abnormal glucose and lipid metabolism caused by high-fat dietBaicalin effectively reduced fasting blood glucose(P<0.05),improved glucose tolerance and insulin tolerance(P<0.01),decreased epididymal fat(P<0.01)and peripheral fat(P<0.05)in model group.Serum low-density lipoprotein(LDL-C)and total cholesterol(TC)in HFDB group were significantly decreased(P<0.05),and liver damage AST(P<0.05)and ALT(P<0.01)were alleviated.The expression of p-AMPK-related protein in the intestine was increased,these indicated that baicalin can improve the abnormal glucose and lipid metabolism in mice and regulate the energy metabolism of the intestinal system.No obvious adverse reactions were found in the normal drugs group.2.Transplantation of baicalin reduces the increase of fat in mice and improves blood sugar metabolismThe body weight(P<0.001)and fasting blood glucose(P<0.01)of the mice transplanted with baicalin group were significantly lower,and the proportion of epididymal fat(P<0.01)and perirenal fat(P<0.05)and body weight was decreased.3.Baicalin alters the composition of gut microbiota in mice fed a high-fat dietAnalysis of the sequencing results of the first batch of fecal metabolites in mice showed that baicalin increased the abundance of Akkermanisa,Coprococcus and Ruminococcus,and reduced the abundance of Odoribacter and Parabacteroides,indicating that baicalin can effectively regulate and restore the abundance of the microbiota to the level of normal(P<0.05).At the species level,50 OTUs in the baicalin group were consistent with the normal control group.After sequencing of the transplanted microbiota,the transplantation of baicalin increased the abundance of Akkermanisa,Coprococcus and Ruminococcus in mice,and reduced the abundance of Odoribacter and Parabacteroides at the genus level.At the species level,18 OTUs of transplanted baicalin were consistent with the normal control group.4.Baicalin increases the content of short-chain fatty acids in intestinal metabolitesThe content of acetic acid in SCFAs of baicalin group increased(P<0.05),propionic acid,butyric acid,isobutyric acid,valeric acid and heptanoic acid increased significantly,but the content of isovaleric acid and caproic acid decreased,but there was no statistical difference.Conclusion:The abundance of Akkermanisa,Coprococcus and Ruminococcus in mice were increased,the abundance of Odoribacter and Parabacteroides were decreased after baicalin intervention.Baicalin increases the production of SCFAs,which are primarily acetic acid.Baicalin may improve the abnormal glucose and lipid metabolism caused by high-fat diet by regulating gut microbiota disorder and increasing the production of SCFAs.This study provides a new target and experimental basis for baicalin to regulate glucose and lipid metabolism disorders.