| Research backgroundDiabetes mellitus(DM)is one of the most common chronic metabolic diseases and type 2 diabetes mellitus(T2DM)is the most common type,accounting for 90%.Disorders of glycolipid metabolism are the main clinical manifestations of metabolic diseases such as T2DM,obesity and non-alcoholic fatty liver,and can induce cardiovascular and cerebrovascular complications[1].The treatment of diabetes includes drug therapy and lifestyle intervention and other means,which have achieved certain therapeutic effects in clinic.However,drug side effects such as gastrointestinal reaction,urinary tract infection,hypoglycemia are common.At present,the pathogenesis and pathogenesis of diabetes have not been completely clarified.In recent years,it has been found that the metabolism of intestinal flora-bile acids(BAs)can regulate the homeostasis of glycollipic metabolism,affect the synthesis and secretion of intestinal hormones,regulate glucolipid metabolism by transmembrane G-protein-coupled receptor 5(TGR5)and farnesoid X receptor(FXR)[2-5].Our hospital prescription Jiangtang Sanhuang tablet comes from one of the four classics,namely Zhang Zhongjing’s "Treatise on Febrile Diseases",and the application of Jiangtang Sanhuang tablet has accumulated more practical experience in clinical practice.Its role of purging heat and channeling fu organs,nourishing qi and Yin,promoting blood circulation and dispersing Jie plays an significant role in the regulation of glycolipid metabolism of T2DM.The hepato-intestinal axis is a key link in regulating glucose and lipid metabolism,and bile acid plays a central role in the hepato-intestinal axis.Therefore,it is of great academic value and practical significance to further understand and explore the intervention effect of Jiangtang Sanhuang tablets on intestinal microbiota-bile acid metabolic axis in the prevention and treatment of T2DM,which may provide new strategies for the pathogenesis and prevention of T2DM.Meanwhile,it provides more theoretical support and scientific research basis for clinical application of Jiangtang Sanhuang tablets.Research purposeJiangtang Sanhuang tablets have been used for a long time in the clinical treatment of T2DM,but the mechanism of its anti-diabetes is still not completely clear.Currently,gut microbiome and bile acids(BAs)metabolism are believed to mediate host metabolism and influence disease progression in T2DM.The mechanism may be that the biotransformation of bile acids by intestinal flora leads to changes in bile acid profile,which in turn leads to signal transduction of related nuclear receptor FXR and membrane receptor TGR5,thus improving glycolipid metabolism of T2DM.Therefore,in this study,we for the first time investigated the effect of Jiangtang Sanhuang Tablets on T2DM and its possible mechanism through the interaction between intestinal flora and BAs metabolism.Research methodsThis study induced T2DM rat model by SPF male SD rats with high sugar and high fat diet(HFD)joint streptozotocin(STZ)injection.Different doses of Jiangtang Sanhuang tablets(0.27g/kg,0.54g/kg,1.08g/kg)were administered for 4 weeks,and metformin tablets were used as the positive control group to observe the changes of glucose and lipid metabolism in T2DM model rats.Changes in intestinal microbiota and bile acid profiles were detected by 16S rRNA gene sequencing and UPLC-MS/MS methods.In addition,quantitative Real Time PCR and Western Blot were also performed to detect the mRNA and protein expression levels of FXR,FGF15,TGR5 and GLP-1 in the gut and CYP7A1 and CYP8B1 in the liver,which are involved in bile acid metabolism and hepato-intestinal circulation.Research resultsOur results showed that the intervention of Jiangtang Sanhuang tablet significantly improved hyperglycemia,hyperlipidemia,insulin resistance(IR),and pathological changes of pancreas,liver,kidney and ileum in T2DM rats,and reduced serum levels of proinflammatory cytokines.16S rRNA gene sequencing and targeted metabolomics UPLCMS/MS demonstrated the presence of intestinal flora and bile acid metabolism disorders in T2DM rats.The intervention of Jiangtang Sanhuang tablets effectively regulated the structure of intestinal micro flora.Meanwhile,the abundance of bacteria related to bile salt hydrolase(BSH)activity(such as Bacteroides,Lactobacillus,Bifidobacterium,etc.)was increased.This results in the change of bile acid profile(such as CDCA;DCA),and may further upregulate the FXR/FGF15 and TGR5/GLP-1 signaling pathways through activation of bile acid-related receptors,thus improving glycolipid metabolism in T2DM rats.General information:In the course of this experiment,compared with the model group,the SD rats in the control group had bright fur color,pure white and glossy,good mental state,lively and active,agile and responsive.The mental state of the rats in the model group was general,lazy,yellow fur,more urine and feces,sticky,smelly.All the rats in the model group showed typical symptoms of T2DM.In terms of body weight:After 8 weeks of intraperitoneal injection of HFD combined with low-dose STZ,the body weight of rats in the model group began to decrease.Statistical analysis showed that there was a significant statistical difference in body weight between the model group and the Control group(P<0.01).After 2 weeks of administration,the body weight of Meformin group and JTSH low-dose,medium-dose and high-dose groups no longer showed a decreasing trend,but there was no statistically significant difference compared with T2DM group(P>0.05).After 3 weeks of administration,the body weight of T2DM group was significantly lower than that of Metformin group and JTSH low,medium and high dose groups(P<0.05).After 4 weeks of administration,there was a statistically significant difference between the Control group and the T2DM group(P<0.01).The body weight of the T2DM group was significantly lower than that of the Metformin group,and the JTSH low-dose,medium-dose and high-dose groups(P<0.01).There was no significant difference in body weight among the administration groups(P>0.05).Fasting blood glucose(FBG):After 8 weeks of HFD combined with low-dose STZ intervention,the blood glucose in the model group showed a rapid increase trend,which had a statistically significant difference compared with the Control group(P<0.01).After 1-2 weeks of administration,FBG in Metformin group and JTSH group showed a downward trend compared with T2DM group,but the difference was not statistically significant(P>0.05).After 3 weeks of administration,FBG level in all administration groups was significantly lower than that in T2DM group,but the difference was not statistically significant(P>0.05).After 4 weeks of intervention,FBG level in Metformin group and JTSH medium and high dose groups was significantly lower than that in T2DM group(P<0.05).Oral glucose tolerance test(OGTT):Statistically,the blood glucose level of the Control group at OGTT 0min was significantly lower than that of the T2DM group(P<0.01).After drug intervention,the blood glucose level in all drug administration groups was significantly lower than that in T2DM group at 0min(P<0.05).After glucose intragastric administration,the blood sugar of rats in each group began to rise and reached the peak value in 30min.Statistical analysis showed that:The blood glucose level of the Control group and the T2DM group at OGTT 30min was significantly different(P<0.01),and the blood glucose level of the T2DM group at OGTT 30min was significantly higher than that of the JTSH lowmedium-high dose group and Metformin group(P<0.05).The blood glucose level of each group began to decrease 30min later.Statistical analysis showed that although the blood glucose level of T2DM group also decreased,it was still significantly higher than that of Control group(P<0.01).The blood glucose level in all administration groups was significantly lower than that in T2DM group,but there was no statistical significance between groups(P>0.05).It can be seen from the chart that the blood glucose level of each group still showed a downward trend at OGTT 90min.Statistical analysis showed that the blood glucose level of T2DM group was significantly higher than that of Control group at OGTT 90min(P<0.01).After 4 weeks of drug intervention,the blood glucose level of all drug administration groups at 90min OGTT was significantly lower than that of T2DM group,but there was no statistically significant difference(P>0.05).At OGTT 120min,statistical analysis showed that there were significant differences among groups(P<0.01),and paired analysis between groups showed that:There were still significant differences in blood glucose level between the Control group and T2DM group at OGTT 120min(P<0.01),and the blood glucose level in Metformin group and high-dose JTSH group at OGTT 120min was significantly lower than that in T2DM group(P<0.05).Area under the curve(AUC)of OGTT:Statistical analysis showed that the area under OGTT curve was significantly different among different groups(P<0.01).Paired analysis between groups showed that AUC in T2DM group was significantly higher than that in Control group(P<0.01).After drug intervention,AUC of JTSH high-dose group and Metformin group was significantly lower than that of T2DM group(P<0.01).Fasting insulin(FINS)level:Compared with the Control group,FINS level in T2DM group was significantly increased(P<0.01).After 4 weeks of drug intervention,compared with T2DM group,FINS levels in JTSH groups and Metformin group were significantly decreased(P<0.05).The results of HOMA-IR index analysis showed that there were significant differences in HOMA index among all groups(P<0.01),and pin-to-group analysis showed that the HOMA index of T2DM group was significantly higher than that of Control group(P<0.01).The HOMA index of all drug administration groups was significantly lower than that of T2DM group(P<0.01),and the HOMA index of Metformin group was significantly lower than that of JTSH low-dose group(P<0.05).There was no statistically significant difference between Metformin group and JTSH medium-dose and high-dose groups(P>0.05).Fasting blood lipid level:Total cholesterol(TC)level:The results of statistical analysis showed that the total cholesterol level in T2DM group was significantly higher than that in Control group(P<0.01).After 4 weeks of drug intervention,TC levels in different drug administration groups showed a decreasing trend.Compared with T2DM group,TC levels in Metformin group and JTSH medium and high dose groups were significantly decreased(P<0.05).Triglyceride(TG)level:The TG level in T2DM group was significantly higher than that in Control group(P<0.01).TG levels in Metformin group and JTSH medium-dose and highdose groups were significantly decreased compared with T2DM group(P<0.05),while TG levels in JTSH low-dose group were decreased,but there was no statistical significance compared with T2DM group(P>0.05).Low density lipoprotein cholesterol(LDL)level:The level of LDL-C in T2DM group was significantly higher than that in Control group(P<0.01).After 4 weeks of drug intervention,the levels of LDL-C in different drug administration groups were decreased to varying degrees.Compared with T2DM group,LDL-C in Metformin group and JTSH lowdose,medium-dose and high-dose groups were significantly decreased(P<0.05).High density lipoprotein cholesterol(HDL)level:the HDL-C level in T2DM group was significantly lower than that in Control group(P<0.01).After intervention,the level of HDL-C was increased in different administration groups.Compared with T2DM group,the level of HDL-C in Metformin group and JTSH medium and high dose groups was significantly increased(P<0.05).The level of free fatty acid(FFA)in T2DM group was significantly higher than that in Control group(P<0.01).After 4 weeks of drug intervention,FFA levels in different drug administration groups were decreased,and FFA levels in Metformin group and JTSH medium and high dose groups were significantly decreased compared with T2DM group(P<0.05).Serum proinflammatory factor ELISA test results:Tumor necrosis factor(TNF-α)levels:Univariate analysis of variance showed that there were significant differences in TNF-α levels among all groups(P<0.01),and the level of TNF-α in T2DM group was significantly higher than that in Control group(P<0.01).After 4 weeks of drug intervention,TNF-α levels in Metformin group and JTSH low-dose,medium-dose and high-dose groups were significantly lower than those in T2DM group(P<0.05).Interleukin-6(IL-6)level:The level of proinflammatory factor IL-6 in T2DM group was significantly higher than that in Control group(P<0.01).After intervention,IL-6 levels in Metformin group and JTSH group were decreased,and the difference was significant compared with T2DM group(P<0.01).Interleukin-1β(IL-1β)levels in T2DM group were significantly higher than those in Control group(P<0.01).After drug intervention,IL-1β levels in Metformin and JTSH groups were significantly lower than those in T2DM group(P<0.01).Histopathology:H&E staining showed that,compared with the Control group,the structure of hepatocytes in T2DM group was disordered,with extensive hepatic adipose vacuolar degeneration,hepatocyte swelling and necrosis.Islet morphology is irregular,the boundary around the islet is blurred,the islet atrophy is serious,the islet cell damage;Mild hyperplasia of glomerular basement membrane,glycogen deposition,renal tubule dilatation;The terminal cells of the ileum are disordered and the epithelial cells are damaged.Compared with T2DM group,the histopathological changes of liver,pancreas,kidney and ileum were significantly improved after treatment with metformin and JTSH.Oil red O staining again proved that the degree of liver steatosis in T2DM group was more serious,and the number of lipocyte in T2DM group was significantly higher than that in Control group(P<0.01),while that in metformin group and JTSH low-dose,medium-dose and high-dose groups was significantly lower than that in T2DM group(P<0.05).qRT-PCR:The relative expression level of Fxr mRNA in ileum:Statistical analysis showed that the relative expression level of Fxr in T2DM group was significantly lower than that in Control group(P<0.05).After the intervention of Jiangtang Sanhuang tablets,the relative expression level of Fxr in JTSH group was significantly higher than that in T2DM group(P<0.05).Fgf15 mRNA relative expression level:The relative expression level of Fgf15 in T2DM group was significantly lower than that in Control group(P<0.05),and after the intervention of Jiangtang Sanhuang tablets,the relative expression level of Fgf15 in JTSH group was significantly higher than that in T2DM group(P<0.05).Tgr5 mRNA relative expression level in T2DM group was significantly lower than that in Control group(P<0.05).After the intervention of Jiangtang Sanhuang Tablets,the relative expression level of Tgr5 in JTSH high-dose group was significantly higher than that in T2DM group,and the difference was statistically significant(P<0.05).Glp-1 mRNA relative expression level:Statistically,Glp-1 relative expression level in T2DM group was significantly lower than that in Control group(P<0.05).After the intervention of Jiangtang Sanhuang tablets,Glp-1 level in JTSH high-dose group was significantly increased,and the difference was statistically significant compared with T2DM group(P<0.05).The relative expression level of liver Cyp7a1 mRNA in T2DM group was significantly higher than that in Control group(P<0.01).Compared with T2DM group,the relative expression level of Cyp7a1 in JTSH group was significantly decreased(P<0.01).Cyp8b1 mRNA relative expression level:The relative expression level of Cyp8b1 mRNA in T2DM group was significantly higher than that in Control group(P<0.01).After the intervention of Jiangtang Sanhuang tablets,the Cyp8b1 level in JTSH group was significantly decreased compared with T2DM group(P<0.01).Western blot:Statistically,compared with Control group,the expression level of FXR protein in intestinal tract of T2DM group was significantly decreased(P<0.05).The JTSH high-dose group reversed the downregulation of FXR expression,and the difference was statistically significant compared with T2DM group(P<0.01).The protein expression level of FGF15 in T2DM group was also significantly down-regulated compared with Control group(P<0.05),but high dose JTSH intervention reversed this change(P<0.01).Compared with the Control group,the protein expression levels of CYP7A1 and CYP8B1 in T2DM group were significantly up-regulated(P<0.05,P<0.01),while the protein expression levels of CYP7A1 and CYP8B1 in high-dose JTSH group were significantly down-regulated(P<0.05).In addition,compared with the Control group,the expression levels of TGR5 and GLP-1 in T2DM group were significantly decreased(P<0.01).After the intervention of Jiangtang Sanhuang tablets,the expression levels of TGR5 and GLP-1 in JTSH high-dose group were significantly up-regulated compared with T2DM group(P<0.05).Intestinal flora 16S rRNA gene sequencing:Alpha diversity:16S rRNA gene sequencing results showed that compared with the Control group,there was no significant difference in Shannon index and Simpson index in the T2DM group(P>0.05).Similarly,compared with the T2DM group,There was no significant difference in Shannon index and Simpson index in high-dose JTSH group(P>0.05).In terms of Chao1 and Observed species index,T2DM was significantly higher than Control group(P<0.01),while JTSH group was significantly lower than T2DM group(P<0.01).Beta diversity:Principal coordinate analysis(PCoA)showed that the distance from the Control group to the high-dose JTSH group was smaller than that from the T2DM group to the high-dose JTSH group.Cluster analysis showed that the microbiota in the Control group and the highdose JTSH group clustered more closely than that in the Control group and T2DM group.Phylum level:Through the analysis of the level of Bacteroidetes in the end ileum of each group,it was found that Firmicutes and Bacteroidetes were the main bacteroidetes in each group,and the ratio of Firmicutes to Bacteroidetes(F/B)in T2DM group was significantly higher than that in Control group(P<0.01).After high-dose intervention of Jiangtang Sanhuang tablets,F/B ratio was significantly decreased,and the difference was statistically significant compared with T2DM group(P<0.01).Genus Level:Statistical analysis showed that compared with the Control group,the relative abundance of Romboutsia and Bacteroides in T2DM group decreased significantly(P<0.01).After high dose treatment of Jiangtang Sanhuang tablets,The relative abundance of Romboutsia,Bacteroides,Lactobacillus,Turicibacter Bifidobacterium and Clostridium sensu stricto 1 increased significantly,and compared with T2DM group,The difference was statistically significant(P<0.01,P<0.01,P<0.05,P<0.05,P<0.05).Targeted metabolomics UPLC-MS/MS analysis:Based on the analysis of UPLC-MS/MS and serum BAs in the terminal ileum,the levels of total BA and total BA in the terminal ileum in T2DM group were significantly higher than those in Control group(P<0.01),while the levels of total BA and total BAs in ileum in highdose JTSH group were significantly lower.The difference was statistically significant(P<0.05).Major un-conjugated and conjugated BA levels:Statistical analysis showed that compared with Control group,the level of free bile acid(CDCA,β-MCA)in T2DM group was significantly decreased(P<0.01).Free bile acids(CDCA,DCA,β-MCA)in JTSH group were higher than those in T2DM group(P<0.01,P<0.05,P<0.05).In terms of conjugated bile acid levels,compared with Control group,TCA and TMCA(α+β)levels in T2DM group were significantly increased(F<0.01,P<0.05),while TCA levels in JTSH group were significantly lower than those in T2DM group(P<0.05).In addition,the level of TMCA(α+β)in high-dose JTSH group was decreased,but the difference was not statistically significant compared with T2DM group(P>0.05).Spearman correlation analysis:Through the correlation analysis of intestinal flora and biochemical indicators,it can beseen that Romboutsia,Bacteroides,Lactobacillus,Turicibacter and Bifidobacterium flora and biochemical parameters(e.g.FBG,FINS,HOMA-IR,AUC of OGTT,blood lipid,function of liver and kidney)and pro-inflammatory factors(TNF-α,IL-1β,IL-6)were negatively correlated,while Enterococcus was positively correlated with IL-6,ALT and TBA.The correlation analysis of intestinal flora and related indexes of bile acid spectrum shows that Romboutsia,Bacteroides,Lactobacillus and Turicibacter are positively correlated with free bile acids including DCA,CDCA,βMCA,ωMCA and γMCA.Bifidobacterium is negatively correlated with CA and TCA.Conclusion(1)The high-fat and high-glucose environment will lead to the disorder of glucose and lipid metabolism,insulin resistance and weight increase.(2)Jiangtang Sanhuang Tablet can significantly improve the level of hyperglycemia,hyperlipidemia,insulin resistance(IR),pathological changes of pancreas,liver,kidney and ileum in T2DM model rats,and can reduce the level of serum pro-inflammatory cytokines.(3)16S rRNA gene sequencing and targeted metabolomics UPLC-MS/MS analysis showed that there was obvious structural disorder in intestinal flora and BAs metabolism in T2DM rats.JTSH intervention can regulate the structure of intestinal flora and bile acid metabolism,increase the abundance of intestinal flora(such as Bacteroides,Lactobacillus,Bifidobacterium,etc.),that is,the flora with bile acid hydrolase(BSH)activity,which may increase the level of free bile acid(such as CDCA,DCA,MCA)at the end of the ileum.(4)By regulating the interaction between intestinal flora and bile acid axis,further upregulate the intestinal FXR/FGF15 and TGR5/GLP-1 signal pathways,thereby improving the glucose and lipid metabolism homeostasis of T2DM rats.(5)In terms of regulation of glucose and lipid metabolism in T2DM rats,the high dose group of JTSH has a better effect than the low and medium dose group of JTSH,and is equivalent to the positive control group of Metformin. |
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