The Study Of Chaihu Shugan Powder Ameliorates Insulin Resistance In A Mouse Molde Of Liver Stagnation Type Metabolic Syndrome Via MiRNA-613/LXRα

Objective:To investigate the effects of chaihu shugan powder(CSP)on insulin resistance(IR)in a mouse model of the metabolic syndrome(MS)associated with liver stagnation,as well as the expression of the liver X receptor(LXR)in the liver tissues of MS associated with liver stagnation and the microenvironment of hepatocyte IR model induced by high glucose and high insulin.To validate the regulatory function of the miRNA-613/LXR signaling pathway in enhancing the metabolic regulation of IR by CSP and to confirm the efficacy and objectivity of dialectical categorization treatment in traditional Chinese medicine.Method:Contents 1:To examine the effects of CSP on MS with liver stagnation type.By feeding a high-fat,high-fructose diet to mice for 16 weeks and then subjecting them to chronic binding stress for two weeks,the mouse model of hepatic stagnation type MS was developed.Metformin and atorvastatin calcium pills served as the positive control.The clinical dose of CSP is a medium dose;a low dose is half the clinical dose;and a high dose is two times the clinical dose.Analysis was done on how CSP affected general health,sucrose water preference,body weight,visceral fat,liver mass,serum lipid profiles,fasting blood sugar(FBS),intraperitoneal glucose tolerance test(IPGTT),insulin resistance test(ITT),and other metabolic symptoms;H&E staining and oil red O staining were used to observe the pathological changes in the liver.The protein expression levels of insulin receptor substrate-1(IRS-1),serine/threonine-protein kinase(Akt),phosphorylated insulin receptor substrate-1(p-IRS-1ser307)and phosphorylated serine/threonine-protein kinase(p-Aktser473)was examined by Western blot analysis.Contents 2:To evaluate the effect of various serum CSP-concentrations on insulin resistance in the IR model of hepatocytes.High glucose(33.3mM)and high insulin(100nM)were added to mouse primary hepatocytes and human hepatocytes to induce the IR model in vitro.Cells were divided into Con group,IR group,2.5%,5%,and 10%bupleurum of CSP groups.Lipid drop precipitation of hepatocytes was detected by oil red O staining,glucose uptake ability of hepatocytes was detected by flow cytometry,and the protein expression levels of p-IRS-1Ser307,IRS-1,p-AktSer473 and Akt in the insulin signaling pathway were detected by Western blot.Contents 3:Network pharmacology and mass spectrometry were used to further explore the underlying molecular mechanisms of enhancing IR via CSP.The crucial targets of CSP on IR were predicted using network pharmacological analysis and mass spectrometry.The protein expression levels of LXR,sterol regulatory element-binding protein-1(SREBP-1),fatty acid synthase(FASN),and acetyl CoA carboxylase(ACC)were examined in vivo and in vitro using immunohistochemistry,fluorescence stain,and Western blotting.In the meantime,the contents of free fatty acid(FFA)and diacylglycerol(DAG)were detected by ELSA.Contents 4:To reveal the effect of LXRa silencing on lipid precipitation and insulin resistance in the hepatocyte IR model.Sh-LXRa and sh-NC(with plasmid transfection)were transfected into the IR model in vitro.And the transfected hepatocytes were stained with oil red O and detected by 2-NBDG.To verify CSP alleviates IR by reducing FFA synthesis and DAG accumulation via LXRα/SREBP-1 signaling pathway.The LXRαagonist T091317 was then administered to the cell to detect FFA synthesis,DAG accumulation,and glucose uptake.Meanwhile,the protein expression levels of LXRα,SREBP-1,FASN,ACC,p-IRS-1Ser307,IRS-1,p-Aktser473,and Akt were also detected.Contents 5:To determine whether CSP could improve IR by influencing miR-613 to regulate the LXRα/SREBP-1 signaling pathway and inhibit fatty acid de novo synthesis.The LXRα-bound miRNAs were predicted using the TargetScan database and then confirmed by PCR and dual luciferase reporter assay.miR-613 analogs(miR-613m)and inhibitors(miR-613i)were transfected into hepatocytes.The protein expression of the LXRα/SREBP-1 signaling pathway and IRS-1/Akt signaling pathway were detected.Result:Contents 1:The mice in the Model group were listless,slow,and sluggish,and the preferred rate of sucrose solution of liver stagnation type metabolic syndrome mice was significantly decreased(P<0.050),suggesting that Model group mice showed symptoms of liver stagnation.Body shape,body weight,visceral fat to body weight ratio,liver weight,serum TG,TC,LDL-C,and liver TC and TG levels in the Model group were significantly increased(P<0.050).After the administration of CSP,the mice recovered mentally and acted more flexibly,and there was a statistical difference between the mice and the Model group in the increased preference for sucrose solution(P<0.050).At the same time,the changes in body shape,body weight,visceral fat,and liver weight of mice were reduced,and the levels of TG,TC,and other lipid profiles in serum and liver were reduced,too.The most significant difference was found in the Chaihu Shugan powder high-dose group(P<0.050).FBG,the AUC area of IPGTT and ITT in the Model group was significantly higher than that in the Control group(P<0.050),and the values of FBG,IPGTT,and ITT in the model group were significantly decreased after CSP administration,especially in CSPH group(P<0.050).Western blot results also showed that compared with the Control group,the level of p-IRS-1Ser307 in liver tissue protein in the Model group was increased while the level of p-Aktser473 was decreased(P<0.050).The results indicated that CSP could effectively improve the symptoms of liver depression and glucose and lipid metabolism in MS mice with liver depression,and had a significant effect on IR,and CSPH had the best effect(P<0.050).Contents 2:Lipid droplets were significantly increased in the IR models of mouse primary hepatocytes and human hepatocytes,while the fluorescence intensity of 2-NBDG was significantly decreased(P<0.050).After the administration of CSP-containing serum,lipid droplets in hepatocytes were decreased while 2-NBDG fluorescence intensity was increased,especially in the 10%CSP group(P<0.050).In addition,compared with the Con group,the protein expression of p-IRS-1Ser307 in the IR group was significantly increased(P<0.050),while the protein expression of p-AktSer473 was significantly decreased(P<0.050).After administration of CSP-containing serum,the trend was reversed,and the 10%CSP group showed the strongest effects(P<0.050).It is suggested that CSP intervention could lessen lipid precipitation and insulin resistance in the hepatocyte IR model.Contents 3:21 major active components of CSP were discovered by the use of mass spectrometry and network pharmacological analysis,and 244 gene targets were enriched as a result.The CSP targets were highly correlated with the targets of glucolipid metabolic disorder,and the protein interaction network was made for CSP-IR-related targets.The results showed that the fatty acid de novo synthesis signaling pathway SREBPF showed a strong correlation.Based on the findings of network pharmacological analysis,FFA and DAG levels were detected in vivo and in vitro,as well as expression levels of associated factors of de novo synthesis of lipids.FFA and DAG levels in the Model group liver tissues and hepatocyte IR models were both significantly increased(P<0.050).CSP could effectively decrease the levels of FFA and DAG levels in liver tissue and hepatocytes(P<0.050).The expressions of LXRα,SREBP-1,FASN,and ACC were also significantly increased in the Model group and hepatocyte IR model(P<0.050),and CSP intervention could effectively reduce the expressions of these expressions(P<0.050).Contents 4:Transfection of the sh-LXRa plasmid into the human hepatocyte IR model significantly inhibited the number and size of lipid droplets in the IR model,and improved glucose uptake by cells(P<0.050).These results suggest that LXRa can directly affect lipid synthesis and insulin resistance of hepatocytes under IR conditions.Then,the LXRa agonist T091317 was added to the 10%CSP group,and T091317 significantly inhibited CSP and improved the accumulation of lipid droplets,FFA,and DAG in the IR model of hepatocytes.The LXRα/SREBP-1 protein level of the fatty acid synthesis signaling pathway was significantly increased in the T091317+10%CSP group.At the same time,T091317 decreased the glucose uptake of cells in the 10%CSP group.In addition,T091317 inhibited the effect of CSP on IRS-1/Akt in the insulin pathway(P<0.050).It is suggested that LXRa is the main target of CSP inhibiting the fatty acid synthesis in the IR model of hepatocytes and improving IR.Contents 5:Bioinformatics analysis and double luciferase gene reporting assay confirmed the existence of binding sites between miR-613 and NR1H3(LXRα)3’-UTR.Transfection of miR-613m inhibited the expression of a fatty acid synthetic protein in the IR model of hepatocytes and improved insulin pathway signaling(P<0.050).PCR results showed that CSP intervention could increase the RNA level of miR-613 in the IR model of hepatocytes,and miR-613i could reduce the effects of CSP on fatty acid synthesis proteins and insulin pathway proteins in the IR model of hepatocytes.It is suggested that CSP inhibits fatty acid synthesis and improves insulin resistance in the IR model of hepatocytes through miR-613/LXRα.Conclusion:Liver stagnation-type MS mice fed high fat and high sugar for 16 weeks combined with chronic binding stress for 2 weeks showed symptoms of liver stagnation,glucose,and lipid metabolism disorders,and IR.CSP administration could effectively improve the above symptoms in Liver stagnation-type MS mice.Moreover,CSP-containing serum could improve de novo fatty acid synthesis and insulin resistance in the high glucose and insulin-induced hepatocyte IR model.In the IR model of hepatocytes,the expression of miR-613 was significantly decreased,the expression of its target gene NR1H3(LXRα)was increased,the regulation of fatty acid synthesis pathway LXRα/SREBP-1 was activated,the levels of FFA and DAG in hepatocytes were increased,and eventually blocked the signaling of insulin IRS-1/Akt pathway.CSP inhibits de novo fatty acid synthesis and improves insulin resistance via miR-613/LXRa.