Study On The Mechanism Of "Bile Acid-FXR-Intestinal Hormones" Axis Regulating Blood Glucose Homeostasis In T2DM Based On The Theory Of Zhu-Pi-San-Jing

Objective:To study the possible mechanism of the classical prescription Ban-xia-xie-xin decoction regulating blood glucose homeostasis in type 2 diabetes mellitus(T2DM)through the axis of "Bile acid/FXR/intestinal hormone" based on the theory of "invigorating the spleen and dispelling essence".Based on the hypothesis that Ban-xiaxie-xin decoction regulates bile acid metabolic profile(MPBA),activates FXR signal transduction through "bile acid-FXR-intestinal hormone" axis,and then affects intestinal hormone(FGF15/GLP-1)secretion to maintain blood glucose homeostasis,the difference of bile acid metabolism is observed by means of molecular biology and metabonomics.It is verified that Ban-xia-xie-xin decoction affects blood glucose homeostasis by regulating bile acid metabolic profile,activating natural ligand FXR and mediating intestinal hormones(GLP-1 and FGF15).It is clarified that "invigorating spleen and dispersing essence" in traditional Chinese medicine is to promote the secretion(metabolism)of intestinal metabolites "bile acid" and "intestinal hormone".It is clarified that Ban-xia-xie-xin decoction can reduce the fluctuation of blood sugar and maintain the steady state of blood sugar through the way of " Bile acid/FXR/intestinal hormone ".Methods:In this study,SPF(Specific pathogen free)male SD(Sprague Dawley)rats without specific pathogen were used to establish T2DM model by intraperitoneal injection of streptozotocin(STZ)induced by high-fat and high-sugar diet.The experiment was divided into two parts: observation experiment and mechanism verification experiment,in which the observation experiment was divided into blank group(NC),T2DM model group(DG),low,middle and high dose of Ban-xia-xie-xin decoction group(low dose LBXD:3.47g/kg,medium dose MBXD:6.93g/kg,high dose HBXD:13.86g/kg)and metformin group(MG,0.16g/kg).To understand the overall mental status of rats by observing and recording the coat color,reaction,physical activity and exercise amount of rats in each group at a fixed time every day,and to observe and record the fasting body weight,24-hour food intake and 24-hour water intake of rats in each group every week.to understand the basic metabolism of rats.The changes of blood glucose were monitored at 8:00,12:00,14:00,16:00 and 18:00 every week,and the daily average blood glucose level(MBG),the standard deviation of blood glucose level(SDBG),the coefficient of variation of intraday blood glucose(CV)and the maximum range of blood glucose fluctuation(LABG)were calculated to quantify blood glucose variability.Oral glucose tolerance test(OGTT)was used for evaluate the modeling criteria and therapeutic effect of rats.The effects of insulin(INS)and glucagon(GC)in serum and pancreas were detected by ELISA method,and the effects of islet function and traditional Chinese medicine on blood glucose homeostasis were preliminarily evaluated.Biochemical analyzer was used to detect the changes of fasting serum glucose(Glucose,Glu)and blood lipids(TC,TG,LDL-C and HDL-C)in rats of each group after 8 weeks of treatment,and to evaluate Ban-xia-xie-xin decoction effect’s on glucose and lipid metabolism;HE staining and light microscope to observe the histomorphological changes of liver,ileum and pancreas;liquid chromatography-mass spectrometry(LC-MS)was used to detect the changes of bile acid metabolic profile(MPBA)in serum and feces of NC group,DG group and HBXD group,to explore the bile acid metabolism and effective bile acid components under the intervention of Banxia Xiexin decoction.Western blot and q PCR methods were used to detect the expression of bile acid receptor FXR and TGR5 in ileum of NC group,DG group and HBXD group.The levels of serum and pancreatic glucagon-like peptide-1(GLP-1),serum gastrin-like peptide(GIP),glucagon-like peptide-2(GLP-2),Peptide YY(PYY)and cholecystokinin(CCK)were measured by ELISA to observe the effect of Ban-xiaxie-xin decoction on intestinal hormones.In the part of mechanism verification experiment,using FXR inhibitor Gly-β-MCA,the experimental animals were divided into blank group(NC),T2DM model group(DG),high dose of Banxia Xiexin decoction group(HBXD),Gly-β-MCA group(Gly-β-MCA),high dose of Banxia Xiexin decoction + Gly-β-MCA group(HBXD+ Gly-β-MCA)and metformin group(MG).The levels of serum glucose and blood lipids were detected by blood biochemical analyzer to understand the effect of inhibitor on glucose and lipid metabolism and the effect of Ban-xia-xie-xin decoction on inhibitor;the effect of bile acid FXR receptor on intestinal hormones(INS,PYY,GLP-2,GIP,CCK)in T2DM rats was detected by ELISA method;the expression of FXR in liver and ileum,GLP-1 and FGF15/19 in ileum of T2DM rats was detected by immunofluorescence(IF).The effects of FXR on hepatic gluconeogenesis pathway(FXR,FOXO1,PEPCK and G6pc),FXR on intestinal glucose transport(FXR,SGLT1 and GLUT2),FXR on intestinal hormone GLP-1(FXR,TGR5)and FXR on intestinal hormone FGF15/19(FXR,SHP and FGF15/19)were detected by Western blot to reveal the regulatory effect of FXR receptor on intestinal hormones GLP-1 and FGF15/19.And the specific mechanism of regulating blood glucose homeostasis in T2DM rats.Result:1.Effect of Ban-xia-xie-xin decoction on glucose and lipid metabolism1.1 Effect of Ban-xia-xie-xin decoction on general condition of T2DM ratsAfter the intervention of Ban-xia-xie-xin decoction,the coat color,shape,range of movement,movement,stool color and dry humidity,urine color,dryness and taste of bedding in T2DM rats were improved in varying degrees.At the same time,Ban-xiaxie-xin decoction could improve the drinking water of T2DM rats,especially in the low and high dose group(P<0.05),but had no significant effect on the body weight and food intake of T2DM rats(P>0.05).1.2 Effect of Ban-xia-xie-xin decoction on histomorphology of T2DM ratsThe histomorphological changes of pancreas,liver and intestinal tract were observed.The results showed that Banxia Xiexin decoction could improve the morphology of islet cells,reduce the infiltration of inflammatory cells in ileum,reduce the dissolution of intestinal villi,and reduce the infiltration of inflammatory cells and steatosis in liver in T2DM rats.1.3 Effect of Ban-xia-xie-xin decoction on glucose and lipid metabolism in T2DM RatsBan-xia-xie-xin decoction could significantly reduce postprandial blood glucose,OGTT(2h)blood glucose and OGTT(AUC)in T2DM model rats,and play a certain role in reducing blood TC and LDL-C(P<0.01),but had no significant effect on blood TG and HDL-C(P>0.05).2.Effect of Ban-xia-xie-xin decoction on blood glucose variability2.1 Effect of Ban-xia-xie-xin decoction on Insulin and Glucagon in T2DM RatsAlthough serum and pancreatic insulin levels were significantly increased by Banxia-xie-xin Decoction(P<0.05),the effects of serum and pancreatic glucagon,insulin to glucagon ratio(I/G)were not statistically significant(P>0.05).2.2 Effect of Ban-xia-xie-xin decoction on MBG,SDBG,CV and LABGCompared with the DG,the group of LBXD,HBXD and MG could significantly improve the level of MBG(P<0.05),and Ban-xia-xie-xin decoction had a certain effect on improving the standard deviation(SDBG),coefficient of variation(CV)and maximum fluctuation range of blood glucose(LABG)of blood glucose.3.Effects of Ban-xia-xie-xin decoction on serum and fecal bile acid metabolic profiles and active components3.1 Effect of Ban-xia-xie-xin decoction on metabolic profile and active components of serum bile acidIn terms of serum bile acid metabolism,compared with NC group,serum TCA,TCDCA,TDCA,THCA,THDCA+TUDCA,CDCA-G and GCDCA in DG group were significantly increased(P<0.05).While HBXD could decrease serum TCA and THDCA+TUDCA levels and increase serum CDCA,CA and HDCA levels.than those in DG group(P<0.05).3.2 Effect of Ban-xia-xie-xin decoction on metabolic profile and active components of bile acid in fecesIn terms of fecal bile acid metabolism,compared with NC group,the levels of fecal bile acid alpha-MCA and beta-MCA in DG group were significantly increased(P<0.05).While the level of fecal bile acid HDCA in HBXD group was significantly lower than that in DG group(P<0.05).4.Effect of Ban-xia-xie-xin decoction on bile acid receptor FXR and TGR5In HBXD group,the expression of FXR protein were up-regulated in ileum(FXR/GAPDH ratio,P<0.05),and the relative expression of TGR5 has a certain upward(TGR5/GAPDH ratio,P<0.05).The relative expressions of FXR and TGR5 m RNA in ileum of T2DM rats were increased by HBXD(P<0.05).5.Effect of Ban-xia-xie-xin decoction on intestinal hormonesBan-xia-xie-xin decoction significantly increased the levels of GLP-1,GIP,PYY and CCK in serum and pancreas of T2DM rats(P<0.05),but there was no significant difference in GLP-2(P>0.05).6.Ban-xia-xie-xin decoction improves blood glucose homeostasis in T2DM rats by "bile acid-FXR-intestinal hormone" pathway.6.1 Bile acid FXR receptors on serum glucose and blood lipids in T2DM ratsHBXD could significantly reduce the levels of Glu,TC and LDL-C in T2DM rats(P<0.05).Compared with HBXD group and HBXD plus Gly-β-MCA group,it was found that Gly-β-MCA could inhibit the effect of HBXD on reducing the levels of Glu,TC.6.2 Effects of bile acid FXR receptor on intestinal hormones(INS,PYY,GLP-2,GIP,CCK)in T2DM ratsHBXD could significantly increase the levels of INS,PYY,GIP and CCK in T2DM rats(P<0.05).Serum INS in HBXD group was significantly higher than that in Gly-β-MCA group(P<0.05),PYY and CCK in HBXD group and MG group were significantly increased(P<0.05),and GIP in HBXD group and MG group had a certain upward trend.6.3 Effect of FXR receptor on the expression of GLP-1 and FGF15/19 in T2DM ratsThe fluorescence signals of FXR,FGF15/19 and GLP-1 in liver and ileum were significantly enhanced in HBXD group,but decreased in Gly-β-MCA group.Compared with Gly-β-MCA group and HBXD group,HBXD could enhance the signals of liver and ileum FXR,ileal FGF15/19 and ileal GLP1 weakened by FXR inhibitor.6.4 Study on the mechanism of Bile Acid FXR receptor regulating Blood glucose Homeostasis in T2DM RatsStudy on the effect of FXR on hepatic gluconeogenesis pathway: Compared with DG group,the expression of FXR and FOXO1 in Gly-β-MCA group were inhibited(P<0.05),while the expression of G6 pc tended to be up-regulated.Compared with Gly-β-MCA group,there were significant differences in FXR and G6 pc in HBXD,HBXD plus Gly-β-MCA and metformin group(P<0.01).The effect of FXR on ileal GLUT2: Compared with DG group,the expressions of FXR and GLUT2 in Gly-β-MCA group,HBXD group and MG group were significantly increased(P<0.05),but there was no significant difference in SGLT1(P>0.05).Compared with Gly-β-MCA group,the expressions of FXR and GLUT2 in HBXD,HBXD plus Gly-β-MCA and MG groups were significantly increased(P <0.01).Effect of FXR on intestinal hormone GLP-1: Compared with DG group,the expression of FXR and TGR5 protein in Gly-β-MCA group were significantly decreased(P<0.05),while FXR and TGR5 signal were significantly increased in HBXD group and MG group(P<0.05),and FXR and TGR5 protein expression was increased in HBXD group,HBXD+Gly-β-MCA group and MG group compared with Gly-β-MCA group(P<0.01).The effect of FXR on intestinal hormone FGF15/19: Compared with DG group,the expression of FXR,SHP and FGF15/19 in Gly-β-MCA group decreased(P<0.05),while the expression of FXR,SHP and FGF15/19 increased in HBXD group and MG group(P<0.05).Compared with Gly-β-MCA group,the expressions of FXR,SHP and FGF15/19 in HBXD group,HBXD+Gly-β-MCA group and MG group were all upregulated(P<0.05).Conclusion:1.There are certain similarities between bile acid,intestinal hormone and spleen "visceral essence" in traditional Chinese medicine.The so-called invigorating spleen and dispelling essence is to promote the secretion of bile acid and intestinal hormone by cultivating spleen "visceral essence".The regulation of invigorating spleen and dispersing essence on blood glucose homeostasis is to improve blood glucose homeostasis by means of bile acid and intestinal hormones.2.Ban-xia-xie-xin Decoction promotes serum bile acid metabolism,decreasing serum TCA,THDCA+TUDCA and fecal HDCA levels and up regulates serum CDCA,CA and HDCA contents.It also exerts its effects on hepatic glucose metabolism and intestinal glucose transport by affecting the receptors FXR and TGR5,and on gut hormones GLP-1 and FGF15 / 19.3.Ban-xia-xie-xin decoction,represented by the method of "invigorating the spleen and dispersing essence",can improve the blood glucose homeostasis of T2DM through the pathway of "Bile acid-FXR-intestinal hormone".