Study On The Mechanism Of Gegen Qinlian Decoction In The Treatment Of Hepatic IR Via MiR-146b/SIRT1 Pathway

Background:Type 2 diabetes has become a global public health problem that seriously affects human health and quality of life.Type 2 diabetes is characterized by insulin resistance and relative lack of insulin secretion,so it is important to improve insulin resistance to delay the progression of type 2 diabetes.Gegen Qilian Decoction(GGQLD)is a traditional chinese medicine.Previous studies showed that GGQLD could improve insulin sensitivity in newly diagnosed newly diagnosed type 2 diabetes mellitus,but its mechanism is still unknown.Objective:To explore the chemical composition of GGQLD and its potential mechanism by using network pharmacology combined with mass spectrometry to indicate the direction for fturther research.This study focused on the regulation of GGQLD on hepatic insulin resistance.In vitro and in vivo studies of GGQLD by miR-146b regulate SIRT1/FoxO1 to reduce oxidative stress and improve hepatic insulin resistance,and strive to elucidate the mechanism of action of GGQLD from liver treatment and improve insulin resistance.Methods:(1)Using the network pharmacology method,combined with the authoritative bioinformatics database to analyze the chemical composition of the herb-derived drugs in GGQLD and its potential therapeutic targets and signal pathways involved.From the perspective of the network,the mechanism of action of GGQLD was comprehensively analyzed.(2)High-fat-induced C57BL/6J insulin-resistant mice were used as models.Different doses of GGQLD,intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were used to evaluate insulin resistance.The biochemical and insulin levels in the serum of mice were detected by Elisa method.HE staining was performed on liver sections.Western blotting combined with qPCR was used to detect the mRNA and protein expression in SIRT1/FoxO1 signaling pathway.(3)HepG2 cells were used to induce oxidative stress with palmitic acid,and a HepG2 cell model of insulin resistance was established.The content of glycogen was determined by using the serum containing GGQLD,and the mRNA and protein expression of SIRT1/FoxO1 signaling pathway were detected by Western blotting combined with qPCR.(4)To detect the expression of miR-146b in high-fat C57BL/6J insulin resistant mice after intervention with GGQLD.HepG2 cells were transfected with miR-146b mimics and miR-146b inhibitor respectively,interfered with GGQLD containing serum and the protein expression of SIRT1/FoxO1 signaling pathway were detected by Western blotting combined with qPCR.Results:(1)A total of 389 compounds were obtained by network pharmacology analysis,involving 191 signaling pathways.Among the top 10 pathways of P value,the pathways associated with this study included AGE-RAGE signaling pathway in diabetic complications and endocrine resistance signaling pathways for diabetic complications.(2)UPLC/LTQ-Orbitrap Determination of the most monomeric components contained in the water extract and medicated serum of GGQLD include:Puerarin,wogonin,baicalin,jatrorrhizine,daidzein,palmatine,berberine,baicalin,baicalein and glycyrrhizic acid.(3)GGQLD can significantly reduce the body weight of mice induced by high fat,reduce ALT,AST,TC,TG,INS and HOMA-IR,and improve glucose tolerance and insulin tolerance.Hepatic HE staining showed that the vacuolar degeneration of liver was significantly decreased in the GGQLD group,and the lipid-deposited higher lipid control group was significantly reduced.The expression of SIRT1 mRNA in the GCGQLD group was significantly higher than that in the control group.Western blot results showed that compared with the normal control group,the expression levels of SIRT1 and PPARy protein in the high-fat control group were significantly decreased,and the expression levels of Ac-FoxO1 and FABP4 were increased.After drug intervention,the expression levels of SIRT1 and PPARy proteins in each group were significantly increased,and the expression of Ac-FoxO1 and FABP4 was decreased.At the same time,the expression of Ac-FoxO1 protein was reduced.(4)The glycogen content of HepG2 cells decreased significantly after palmitate induction,the expression of SIRT1 and PPARy was significantly decreased,and the expression of FABP4 and acetylated FoxO1 was increased.The serum containing GGQLD can reverse the palmitate-induced HepG2 cells with decreased glycogen synthesis ability and up-regulated the expression of SIRT1 and PPARy,inhibited the acetylation level of FoxO1.After siRNA transfection of SIRT1,there was no significant change in the level of acetylated FoxO1 in GGQLD group compared with the negative control group.(5)Compared with the normal diet group,the expression of miR-146b in the liver of the high-fat model group was significantly increased.The expression of miR-146b was significantly decreased after the intervention with GGQLD.The serum of GGQLD can reverse the palmitate-induced HepG2 cells with decreased glycogen synthesis ability.Compared with the control group,the expression of miR-146b in the model group was significantly increased,and the serum containing GGQLD could significantly inhibit the expression of miR-146b.Weston blot showed that overexpression of miR-146b inhibited the expression of SIRT1 protein,and silencing of miR-146b enhanced SIRT1 expression.Conclusions:(1)Network pharmacology analysis found that Gegen Qinlian Decoction was mainly involved in signaling pathways including AGE-RAGE signaling pathways and endocrine resistance signaling pathways in KEGG.(2)Experiments have shown that GGQLD can improve hepatic insulin resistance by inhibiting miR-146b,up-regulating SIRT1 levels,reducing FoxO1 acetylation.