| Background:Insulin resistance(IR)is a key driving factor for type 2 diabetes(T2DM).However,the pathogenesis of IR is unknown and there is no effective treatment now.Liver is one of the key IR organs.Our previous research found that gegen qinlian decoction(GGQLD)can improve liver lipid deposition and IR in mice.The Forkhead box O1(FoxO1)was involved,but its specific target is still unclear.The latest research shows that membrane associated RING-CHI(MARCH1)can promote the ubiquitination,degradate insulin receptor and induce the occurrence of IR.It has been reported that there was a binding site for FoxO1 in the promoter region of MARCH1.The mechanism between GGQLD improving IR and FoxO1-MARCH1 signaling is still unclear.Objectives:(1)To explore the potential targets of GGQLD against T2DM through network pharmacology analysis.(2)To establish an IR animal model with high-fat diet feeding C57BL/6J mice,to investigate the mechanism of IR improvement through FoxO1-MARCH1 signaling by 8 weeks of GGQLD.(3)To establish a palmitic acid(PA)-induced HepG2-insulin resistance(IR)cell model and observe the effects of GGLQD on glucose metabolism and FoxO1-MARCH1 signaling pathway in HepG2 cells.Methods:(1)The effective components of GGQLD was screened and the candidate targets of GGQLD were predicted by network pharmacological analysis.Then the protein-protein interaction(PPI)network was established,the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment analyses were performed,and finally the core targets and potential signaling of GGQLD against T2DM were predicted.(2)An IR animal model was established that 6-8 weeks old C57BL/6J male mice was fed with 60%high-fat for 12 weeks.Then they were randomly divided into 6 groups after modeling:IR model group(HFD),GGQLD very low dose group(GGQLD-VL,GGQLD 5 g/kg/d by gavage),GGQLD low-dose group(GGQLD-L,GGQLD 10 g/kg/d by gavage),GGQLD medium dose group(GGQLD-M,GGQLD 15 g/kg/d by gavage),GGQLD high-dose group(GGQLD-H,GGQLD 20 g/kg/d by gavage)and pioglitazone group(PIO,PIO 20 mg/kg/d by gavage).Normal control group of mice(CON)with standard feed feeding was also set up.Both the CON and HFD groups were given normal saline(10 mL/kg/d)by gavage.After 8 weeks of feeding,the body weight,fasting blood glucose,insulin level,triglycerides(TG),total cholesterol(TC),and low-density lipoprotein cholesterol(LDL-C)were detected in each group,and homeostatic model assessment for insulin resistance(HOMA-IR)was calculated.Intraperitoneal glucose tolerance(IPGTT)was made and IPGTT area under the curve(IPGTT AUC)was calculated.HE staining was used to observe the morphology and structure of hepatocytes in mice;qRT-PCR and western blot technology were used to detect the expression levels of genes and proteins associated with the FoxO1-MARCH1 signaling pathway.(3)A cell model of palmitic acid(PA)-induced HepG2-insulin resistance(IR)was made,and the intervention doses of PA,GGQLD and PIO were determined by CCK-8 detection.Six groups were divided in the vitro research that included CON(only HepG2 cells),PA(PA and HepG2 cells),GGQLD(GGQLD and HepG2 cells),PIO(PIO and HepG2 cells),PA+GGQLD(PA,GGQLD and HepG2 cells)and PA+PIO(PA,PIO and HepG2 cells).The glucose consumption of six groups was observed by glucose oxidase detection test,and the protein expression levels of FoxO1-MARCH1 signaling pathway correlated factors were detected by westeren blot technology.Results:(1)140 active ingredients were obtained,204 common targets and 18 potential key targets of T2DM against T2DM were found by network pharmacology analysis.GGQLD against T2DM were closely related with ubiquitin-like protein ligase binding and transcription factor activity,etc.KEGG enrichment analysis suggested that the underlying mechanisms of GGQLD aginst T2DM was related to the signalling pathway of endocrine resistance and inflammation.(2)The weight of mice in GGQLD-H was decreased compared with that of HFD(P<0.05),and weight of other groups of GGQLD didn’t change.The levels of blood glucose,insulin and HOMA-IR in high-fat-induced IR mice were reduced significantly in the three groups of GGQLD-L,GGQLD-M and GGQLD-H compared with HFD group(P<0.05).There was no significant difference among the three groups,and no significant difference existed compared with CON and PIO groups.IPGTT AUC of all groups of GGQLD was improved greatly compared with HFD(P<0.05),and that in GGQLD-L、GGQLD-M、GGQLD-H and PIO groups was better than that of GGQLD-VL,no significant difference existed among each group and compared with CON.Compared with HFD group,the serum levels of TG,TC and LDL-C in high-fat-induced IR mice were significantly reduced in all groups of GGQLD(P<0.05),and there was no significant difference among each group of GGQLD and significance existed among the GGQLD and PIO.GGQLD and PIO intervention improved the fat vacuolar changes of liver cells,reduced lymphocyte infiltration on liver histopathology.GGQLD-L,GGQLD-M and GGQLD-H improved significantly,while GGQLD-VL had a weaker effect.Compared with the HFD group,the gene transcription levels of FoxO1 and MARCH1(P<0.05)were significantly reduced in each group of GGQLD,but higher than that in CON and PIO groups(P<0.05),and there was no significant difference among GGQLD groups.The expression levels of MARCH1 genes in GGQLD-L,GGQLD-M,GGQLD-H and PIO groups were lower than those in GGQLD-VL(P<0.05),and there was no significant difference between the four groups.Compared with HFD group,there were no significant differences in gene expression levels of INSR and IRS1 in the GGQLD-VL and GGQLD-L groups,while the levels of gene expression in the GGQLD-M,GGQLD-H and PIO groups increased significantly compared with the HFD group and GGQLD-VL groups(P<0.05),and there were no significant differences among the three groups.Compared with the HFD group,the gene transcription level of IRS2(P<0.05)was significantly increased in each group of GGQLD,but it was significantly lower than that in the CON group(P<0.05),while the GGQLD-L,GGQLD-M,GGQLD-H and PIO groups increased significantly than the GGLQD-VL group(P<0.05),and there were no significant differences between the four groups.The results of western blot in liver tissue showed that GGQLD downregulated Acetylated FoxO1(Ac-FoxO1)and MARCH 1,upregulated insulin receptor substrate1(IRS),phosphorylated IRS1(p-IRS1)and insulin receptor β(INSRβ)on protein expression.(3)The intervention concentrations of PA,GGQLD and PIO were 0.3 mmol/L,1 μg/mL and 10μmol/L,respectively.The glucose consumption in the PA+GGQLD group was higher than in the PA group(P<0.05),and there was no significant difference compared with PA+PIO.In the group of PA+GGQLD,the levels of Ac-FoxO1 and MARCH1 in HepG2 cells(P<0.05)were down-regulated and INSRα,INSRβ and p-IRS1 levels were upregulated(P<0.05).Conclusions:(1)GGQLD against T2DM by multiple targets,multiple pathways,and multiple mechanisms,were closely related to the regulation of ubiquitin-like protein ligase binding and transcription factor activity,the mechanisms were mainly related with endocrine resistance and inflammation signalling pathway.(2)The IR model was established in C57BL/6J mice with high fat feeding for 12 weeks,and GGQLD significantly improved the glycolipid metabolism and HOMA-IR,and reduced liver damage with intervention for 8 weeks.The mechanism of improving liver IR by GGQLD was found to be related with the FoxO1-MARCH 1 signaling.Ac-FoxO1 and MARCH1 levels were down-regulated,p-IRS and INSRβ levels were upregulated on protein expression by GGQLD.We speculated that GGQLD inhibited the translocation of acetylated FoxO1 into the nucleus,thereby suppressing the transcriptional activation of MARCH1 and its targeting of insulin receptor substrates,thus restoring insulin signaling pathways and improving liver insulin resistance.(3)In vitro research,we found that GGQLD can significantly increase the glucose consumption of PA-induced HepG2-IR,downregulate the protein levels of Ac-FoxO1 and MARCH1,and upregulate the protein levels of INSRα,INSRβ and p-IRS1.In summary,the mechanism of GGQLD to improve IR may be related to the regulation of the FoxO1-MARCH1 signaling pathway.We hypothesized that GGQLD may improve IR by inhibiting FoxO1 acetylation into the nucleus,which in turned inhibiting the initiation transcription of MARCH1 and its targeted ubiquitination degradation of insulin receptors. |
