Study On The Mechanism Of In Vitro Hypoglycemic Action Of Zhuojiangtang Tablet And Its Chemical Composition Analysis

Object:To explore the possible mechanism of FuZhu-JangTang Tablets to improve insulin resistance in cells,to identify the chemical constituents of FuZhu-JangTang Tablets,to verify its related targets for improving insulin resistance by using network pharmacology.Provide scientific basis for the ameliorate of diabetes by FuZhu-JangTang Tablets.Methods:Using high-resolution liquid chromatography-mass spectrometry to test chemical composition analysis of FuZhu-JangTang Tablets;18 compounds were selected by network pharmacology verify its possible hypoglycemic target;HepG2cells were induced with high insulin as a model of insulin resistance,and C2C12cells were induced with palmitic acid as a model of insulin resistance.CCK-8 method was used to detect the effect of FuZhu-JangTang Tablets on HepG2 cells and C2C12 cells in order to screen appropriate drug concentration;The glucose oxidase-peroxidase method(GOD-POD method)was used to measure the glucose consumption in HepG2 cells and C2C12 cells in each group;Annexin V-FITC/PI double staining method was used to detect the modeling method and effect of FuZhu-JangTang Tablets on apoptosis of HepG2cells;Real-time PCR was used to detect the expression of InsR?Tyr1135/1136,PI3Kp85,AKT and GLUT4 mRNA in insulin signaling pathway of HepG2 cells;Western Blot assay was used to detect the expression levels of key proteins such as InsR ?Tyr1135/1136 and IRS1 in HepG2 cells and C2C12 cells;Results:1.Chemical composition analysis of FuZhu-JangTang tablets:By comparing the mass spectrometry cleavage rules of the compound standards and the cleavage information of the reference literature and database mass spectrometry,this experiment quickly identified 37 compounds,there are 8 flavonoids,9 flavonoid glycosides,8 saponins,9 phenolic acids and 3 other classes.The chemical composition of the auxiliary hypoglycemic tablets is analyzed comprehensively.2.Network pharmacology results:The target pathways predicted by 18 compounds were associated with IR disease genes in five pathways,and there were 312 IR-related targets,of which four were associated with the PI3K/AKT insulin signaling pathway.3.The effect of FuZhu-JangTang tablets on insulin-resistant hepatocytes:1)Determination of glucose content by GOD-POD method:Different concentrations of FuZhu-JangTang Tablets can increase HepG2 cell glucose consumption in different degrees(P<0.05 or P<0.01).2)Annexin V-FITC/PI double staining assay:The selected drug concentration had no effect on the apoptosis of HepG2 cells.3)Real time PCR detection:Compared with the normal group,the expression of InsR?Tyr1135/1136,PI3Kp85,AKT and GLUT4 mRNA was significantly decreased(P<0.01).Compared with the model group,the experimental group was able to up-regulate InsR,PI3Kp85,AKT.GLUT4 mRNA expression(P<0.001or P<0.05).4)Western Blot assay:Compared with the normal group,the InsR?Tyr1135/1136,IRS1,PI3Kp85,PDK1,AKT,GLUT4,p-GSK-3? and their phosphorylation levels were significantly decreased(P<0.01),compared with the model group,the experimental group can up-regulate the expression of p-GSK-3? and PI3Kp85 and the levels of protein of InsR?Tyr1135/1136,IRS1,PDK1,AKT and their phosphorylation were up-regulated(P<0.05or P<0.01).4.The effect of FuZhu-JangTang tablets on insulin-resistant myocytes:1)Establishment of a model of myocyte insulin resistance:C2C12 cells differentiated into mature myocytes after induction of high glucose medium containing 2%horse serum for7 days,with 0.5mM palm Sodium has established a stable insulin resistance model for C2C12cells.2)Determination of glucose content by GOD-POD method:Different concentrations of FuZhu-JangTang Tablets can increase C2C12 cell glucose consumption in different degrees(P<0.05 or P<0.01).3)Western Blot assay:Compared with the normal group,the expression of p-InsR?Tyr1135/1136,p-AKT Thr308,p-PDK1 Ser241,PI3Kp85,GLUT4 and p-GSK-3?were significantly decreased(P<0.01),compared with the model group,the experimental group can up-regulate the expression of p-InsR?Tyr1135/1136,p-AKT Thr308,p-PDK1 Ser241,PI3Kp85,GLUT4 and p-GSK-3?(P<0.05 or P<0.01).Conclusions:1.A total of 37 compounds were rapidly identified and the target associated with IR was determined by network pharmacology.2.FuZhu-JangTang tablets can promote the consumption of glucose by hepatocytes and myocytes,and improve insulin resistance.The mechanism may up-regulate the expression of InsR?,PI3Kp85,AKT and GLUT4 genes in insulin PI3K/AKT signaling pathway and up-regulate p-InsR?.Tyr1135/1136,p-IRS1Try895,p-PDK1 Ser241,p-AKT Thr308 protein phosphorylation levels and PI3Kp85 protein levels were correlated.