Experimental Study On Activation Of Hepatocytes MAFB/FXR Signaling Pathway In Obese Rats With Type 2 Diabetes Mellitus By Sleeve Gastrectomy

Objective:Sleeve gastrectomy and gastric bypass surgery are the mainstream surgery for weight loss and metabolic surgery at present.It was thought that sleeve gastrectomy should be performed for simple obesity patients,and gastric bypass for obese patients with type 2 diabetes mellitus.However,with the deepening of clinical practice and the progress of basic research,it is found that for obese patients with type2 diabetes mellitus,especially those with a shorter course and less degree of disease,sleeve gastrectomy is no less than gastric bypass to reduce weight and blood glucose.Moreover,the operation is simple,and it does not change the normal anatomy structure of gastrointestinal tract,and it has fewer postoperative complications.If the effect of the operation is not good,it can also be changed to the second stage revisional operation,which has been paid more and more attention and applied at home and abroad.The rate of sleeve gastrectomy is now more than 50% in the volume of bariatric surgery in China and the United States.Based on this,it is further confirmed that sleeve gastrectomy is a kind of bariatric procedure which is worthy of basic and clinical study.The weight reduction and hypoglycemic effect of sleeve gastrectomy is accurate,but the exact mechanism of the treatment is still not well known to the clinician.The mainstream view of the past considered the foregut theory and the hindgut theory.Both the two theories assumed changes in gastrointestinal hormones caused by changes of the secretory function of the endocrine cells in the gastrointestinal tract lead to the adjustment of the enteroinsular axis.However,these theories are still not good enough to reveal the exact mechanism.Recent studies have shown that bile acids play an important role after sleeve gastrectomy.A significant increase in plasma bile acid level after sleeve gastrectomy has been found.The bile acid receptor includes two forms: membrane receptor(TGR5)and nuclear receptor(farnesoid X receptor,FXR).After the sleeve gastrectomy,the weight loss and hypoglycemic effect of TGR5 knockout mice decreased significantly,suggesting that TGR5 mediated the weight loss and hypoglycemic step of sleeve gastrectomy.FXR is a nuclear receptor,the essence of which is a transcriptional regulator,which regulatesthe expression of the target gene in the downstream and is regulated by the upstream transcriptional factor.The activation of FXR may play a more important role in sleeve gastrectomy,but the existing clinical and basic studies have not been well explained.Previous molecular biology studies suggested that FXR regulates the expression of a series of genes downstream,the most important of which are the key genes involved in regulating carbohydrate and lipid metabolism,such as glucose-6-phosphatase(G6Pase),phospho-enolpyruvate carboxykinase(PEPCK),small heterodimer partner(SHP),cholesterol 7 alpha-hydroxylase(CYP1A1),sterolregulatory element binding proteins-1(SREBP-1),peroxisome proliferatorsactivated receptor-alpha(PPARa),etc.Similarly,as a nuclear receptor,FXR is also regulated by the related upstream transcriptional factors.The known related transcriptional factors include musculoaponeurotic fibrosarcoma oncogene family B(MAFB),forkhead box O3(FOXO3),early growth response protein 2(EGR2),etc.The animal experiments in this study were designed to investigate the expression changes of FXR and its downstream genes that regulate glucose and lipid metabolism after sleeve gastrectomy,and to find the expression changes of transcriptional factor MAFB in the upstream of FXR.The molecular biology experiments in this study were designed to investigate the regulation of MAFB expression to FXR and its downstream genes,and to explore the combination of MAFB to FXR promoter,and to make clear the activation of MAFB/FXR signaling pathway by sleeve gastrectomy,and then to make clear the new therapeutic mechanism of sleeve gastrectomy,and to present a theoretical basis and theoretical foundation for clinical practice.Methods:1.Establishment of a model of obese rats with type 2 diabetic mellitus.8-week old male SD rats were given a high-fat diet for 12 weeks and then single intraperitoneal injection of 2% STZ(treated at 65 mg/kg).After 72 hours,the random blood glucose was measured by a fast blood glucose meter in the caudal vein.The rats whose three times of random blood glucose > 16.7 mmol/L,and the body weight >395g were regarded as the successful models,which can be enrolled to the object of observation.2.20 obese rats with type 2 diabetes mellitus were divided into two groups randomly(sleeve gastrectomy group and sham operation group),each with 10,another 10 male SD rats with the normal-fat diet and the same week old asthe control group.The experimental animals were sacrificed on the liver tissue for 28 days after operation or control treatment.The weight,fasting blood glucose,fasting insulin,TG、TC、FFA、ALT and AST were measured before and after the operation.The lipid deposition in liver tissue was observed by oil red O staining.The relative expression of MAFB and FXR in hepatocytes was detected by Western blot.The expression of SHP,SREBP-1,PPARa,CYP7A1,PEPCK and G6 Pase in liver cells were detected by real-time quantitative PCR.3.We constructed MAFB overexpression plasmid and transferred MAFB overexpression plasmid to hepatocytes,which were randomly divided into no-load group(control group)and MAFB overexpression group.The relative expression of MAFB and FXR in hepatocytes was detected by Western blot.The expression of SHP,SREBP,PPARa,CYP7A1,PEPCK and G6 Pase in liver cells were detected by real-time quantitative PCR.4.We constructed a reporter plasmid and its mutant plasmid containing the FXR promoter region,and co-transferred to 293 T cells with MAFB overexpressed plasmids.The binding of MAFB and FXR promoter was detected by double luciferase experiments.Results : 1.Surgical results: In the sleeve gastrectomy group,2 cases died after operation,of which 1 case died of bleeding from the margins of gastric stump(first days after the operation),and 1 case died of gastric fistula(second days after the operation),and no death occurred in other groups.2.Body weight: The weight of the rats in the sham operation group and the sleeve gastrectomy group was significantly higher than the weight of the control group before the operation(P<0.01),which showed that the obese rat model was established successfully.After 1 week of the operation,the weight loss of group SG rats was significantly lower than that of the sham operation group(P<0.01),suggesting that the effect of weight reduction after SG was good.3.Fasting blood glucose(FBG): After 4 weeks,the level of FBG in the sleeve gastrectomy group was significantly lower than the level of FBG in the sham operation group(P<0.01),which showed that the effect of hypoglycemic effect was good after SG.4.Fasting insulin(FINS): After 4 weeks,insulin increased significantly in the sham operation group compared with the control group and the sleeve gastrectomy group(P<0.01),suggesting that the rats fed with high-fat diet caused significant insulin resistance,while SG significantly alleviated insulin resistance.5.TG,TC,FFA,ALT and AST concentrations: After 4 weeks,compared with the sham operation group,TG,TC,FFA,ALT and AST were significantly lower than sleeve gastrectomy group(P<0.01),and compared with the control group,TG,TC,FFA,ALT and AST increased significantly(P<0.01)than sham operation group,suggesting that SG can significantly reduce blood lipid and aminotransferase and the obese rats with type 2 diabetic mellitus have obvious hyperlipidemia.6.Liver tissue oil red O staining: Compared with the sham operation group,the liver oil red O staining of the rats in the sleeve gastrectomy group was obviously shallow,suggesting that SG could obviously reduce the lipid deposition of the liver.7.SHP,SREBP-1,PPARα,CYP7A1,PEPCK,G6 Pase mRNA expression(QRT-PCR): After 4 weeks,the expression of SHP and PPARa mRNA in SG group was significantly higher than the expression of SHP and PPARa mRNA in sham operation group(P<0.01),and the expression of SREBP-1,CYP7A1 and PEPCK mRNA in SG group was significantly lower than the expression of SREBP-1,CYP7A1 and PEPCK mRNA in sham operation group(P<0.01),while there was no significant difference between the two groups in G6 Pase mRNA expression.8.MAFB,FXR expression(Western Blot): After4 weeks,the expression of MAFB protein in SG group was significantly higher than the expression of MAFB protein in sham operation group(P<0.01),and the expression of FXR protein in SG group was significantly higher than the expression of FXR protein in sham operation group(P<0.05),suggesting that SG could significantly increase the expression level of MAFB and FXR protein in rat hepatocytes.9.Construction of MAFB overexpression plasmid: The target gene MAFB was amplified by PCR and identified by recombinant plasmid digestion.The recombinant plasmid was identified by sequencing and Blast comparison with the target gene.It was confirmed that MAFB gene was cloned into pcDNA3.0 vector successfully.10.MAFB,FXR expression(Western Blot): The expression level of MAFB and FXR protein in MAFB overexpression group increased significantly(P<0.01),indicating that the over expression plasmid was successfully constructed,and the downstream target gene FXR was overexpressed.11.SHP,SREBP-1,PPARα,CYP7A1,PEPCK,G6 Pase mRNA expression(QRT-PCR): The expression of SHP and PPARa mRNA in hepatocytes of MAFB overexpression group was significantlyhigher than that in the control group(P<0.01),while the expression of SREBP-1,CYP7A1,PEPCK and G6 Pase mRNA was significantly lower than that in the control group(P<0.01).12.Construction of a reporter plasmid containing the FXR promoter region: The target gene FXR promoter was amplified by PCR and identified by recombinant plasmid digestion.The recombinant plasmid was identified by sequencing and Blast comparison with the target gene.It was confirmed that the FXR promoter gene was cloned into PGL3 vector successfully.13.Construction of a mutant plasmid containing FXR promoter region: The target site of the promoter region of the FXR gene has been successfully mutated in the target mutation area,and the base from GCCGAAGG mutation to TGGTTCTA.14.Double luciferase experiments: For the wild type FXR promoter reporter plasmid,MAFB can be combined with its promoter.But for mutant FXR promoter plasmid,MAFB could not bind to its promoter.There was a significant difference in luciferase density between the wild group and mutant group(P<0.01),suggesting that MAFB was combined with FXR promoter to control downstream target gene expression.Conclusions: 1.4 weeks after SG,the body weight of the rats decreased obviously,and the fasting blood glucose decreased obviously,and the serum fasting insulin decreased obviously,and the insulin resistance was obviously relieved.2.4 weeks after SG,the serum TG,TC,FFA,ALT and AST tended to be normal in rats,and the lipid deposition in the liver was obviously reduced.3.SG can upregulate the expression of SHP and PPARa mRNA and downregulate the expression of SREBP-1,CYP7A1,PEPCK mRNA in rat hepatocytes,which improved the metabolism of glucose and lipid in liver.4.SG can upregulate the expression of MAFB and FXR protein in rat hepatocytes,and then affect the expression of regulated downstream genes,which improved the metabolism of glucose and lipid in liver.5.The overexpressed MAFB plasmids can promote the expression of FXR gene,and then affect the expression of the regulated downstream genes,which improved the metabolism of glucose and lipid in human Chang liver cells.6.Transcriptional factor MAFB can promote the expression of FXR protein by binding to the promoter of the FXR gene.7.SG can achieve its weight reduction and hypoglycemic effect by activating the MAFB/FXR signaling pathway.