The Mechanism Of Improving Insulin Resistance By Metformin Treatment Based On FXR-MAFG-CYP8B1 Signaling Pathway

Objective 1.HepG2 cells were cultured in vitro and treated with metformin.The expression of cholesterol 12?hydroxylase?CYP8B1?,farnesoid X receptor?FXR?and V-Maf avian fibrosarcoma oncogene homolog G?MAFG?were observed by different concentrations of metformin at different times.2.To observe the expression of FXR,MAFG,CYP8B1 and the content of cholic acid?CA?in the liver tissue of type 2 diabetic rats,and the effect of metformin on it,we investigated the mechanism of improving on glucose metabolism,lipid metabolism and insulin sensitivity by metformin,which provides more theoretical support for the clinical application of metformin.Methods 1.HepG2 cells were used as experimental subjects for culture.HepG2 cells were placed in complete medium containing 10%fetal bovine serum and 1%cyan/streptomycin in high glucose-DMEM,and cultured in a 37°C,5%CO ₂ incubator until the cells were in good condition,then inoculated in a six-well plate.The cells were cultured in DMEM high glucose medium containing different concentrations of metformin?0,0.5mmol/L,1 mmol/L,2 mmol/L,5 mmol/L?for 24 h and were cultured in DMEM high glucose medium containing 2 mmol/L metformin for different time?0,12h,24h,48h?.Protein expression at the cellular levels of FXR,MAFG and CYP8B1 were detected by Western Blot.The cells were seeded in 96-well plates,and different concentrations of metformin were added to measure cell viability using a CCK-8 kit.2.After 1 week of adaptive feeding by giving an ordinary feed.Sixty 8-week-old male wistar rats were randomly divided into three groups:normal control group?CON,n=20?,diabetic rats group?DM,n=20?,and metformin treatment group?DM+MET,n=20?.The CON group were fed normal standard diet until the end of the experiment.The DM group and the DM+MET group were fed with high fat diet?HFD?and were injected low-dose streptozotocin?STZ?35 mg/kg to induce T2DM model.The DM+MET group was given oral metformin(500 mg·kg^-1·d^-1)after induction by diabetes model,and the treatment period was12 weeks.The body weight,food and water intake of the rats were recorded.The serum biochemical indexes of the rats in each group were determined at the end of the experiment.The content of cholic acid?CA?in liver tissue was determined by enzyme-linked immunosorbent assay?ELISA?.The mRNA and protein expression of FXR,MAFG and CYP8B1 at the animal levels was examined by RT-PCR and WB.Results In HepG2 cells.1,the cell viability of metformin-treated HepG2 cells decreased with the increasing concentration of metformin?P>0.05?.2.The protein expression of FXR and MAFG in HepG2 cells increased with the increasing concentration of metformin?P>0.05?,and the protein expression of CYP8B1 decreased gradually?P>0.05?.The protein expression of FXR and MAFG in HepG2 cells increased?P>0.05?,and the protein expression of CYP8B1 decreased gradually?P>0.05?.In the T2DM rat model.1,Body weight,water and food intake.After the successful modeling of high-fat diet combined with low-dose STZ injection,the food and water intake in DM group were significantly increased compared with CON group?P>0.05?,while the body weight was significantly reduced compared with CON group?P>0.05?.After metformin treatment,the food and water intake of DM+MET group were significantly reduced compared with that of DM group?P>0.05?,but it was still higher than that of CON group,and weight of DM+MET group was significantly reduced compared with that of DM group?P>0.05?.2,General biochemical indexes.Total cholesterol?TC?,triglyceride?TG?,low-density lipoprotein?ldl-c?,total bile acid?TBA?,serum fasting blood glucose?FBG?,fasting insulin?FIN?and insulin resistance index?homa-ir?in DM group were significantly higher than those in CON group?P>0.05?,while the levels of high-density lipoprotein?hdl-c?in DM group were significantly lower than those in CON group?P>0.05?.Total cholesterol?TC?,triglyceride?TG?,low-density lipoprotein?ldl-c?,total bile acid?TBA?,serum fasting blood glucose?FBG?,fasting insulin?FIN?and insulin resistance index?homa-ir?in DM+MET group were significantly lower than those in DM group?P>0.05?,while the levels of high-density lipoprotein?hdl-c?in DM+MET group were significantly higher than those in DM group?P>0.05?.3.Expression of FXR,MAFG and CYP8B1 in liver tissues.Compared with CON group,mRNA and protein expression of FXR and MAFG were significantly decreased in DM group?P>0.05?,while mRNA and protein expression of CYP8B1 were significantly increased?P>0.05?.Compared with the DM group,the mRNA and protein expressions of FXR and MAFG were significantly increased in the DM+MET group?P>0.05?,while the mRNA and protein expressions of CYP8B1 were significantly decreased?P>0.05?.4.Content of cholic acid?CA?in liver tissues.Compared with CON group,CA content in DM group was significantly increased?P>0.05?.Compared with DM group,CA content in DM+MET group was significantly reduced?P>0.05?.Conclusions In HepG2 cells.1.1.The activity of HepG2 cells was reduced in a dose-dependent manner.2.Metformin treated HepG2 cells up-regulated the protein levels of FXR and MAFG in a dose-dependent manner,and down-regulated the protein expression of CYP8B1.In the T2DM rat model.1.Metformin improved glucose and lipid metabolism disorder,insulin resistance,and bile acid metabolism in diabetic rats.2.Metformin up-regulates the mRNA and protein levels of FXR and MAFG in the liver tissues of diabetic rats,while down-regulates the mRNA and protein expression of CYP8B1,and reduces the level of cholic acid.3.Metformin may reduce the level of cholic acid by activating the FXR-MAFG-CYP8B1 signaling pathway,thereby regulating glucose and lipid metabolism and improving insulin resistance.