A Study To Explore The Mechanism Of Zhigan Mixture In The Treatment Of Non-alcoholic Fatty Liver Disease Based On ER Stress/NLRP3 Cascade

BackroundNonalcoholic fatty liver disease(NAFLD)is a pathological syndrome characterized by excessive deposition of fat in hepatocytes.With the development of China’s national economy,NAFLD has become the most important chronic liver disease in China,which has brought serious threats to national health.At present,there is no specific drug due to the pathological mechanism of NAFLD that has not been completely explored.ER stress is believed to be involved in the development of NAFLD,which has attracted researchers’ attention in recent years.Chinese medicine has been used in the treatment of NAFLD for a long history and showed a significant beneficial effect.However,to explain the scientific connotation of TCM compound therapy for NAFLD is a challenge facing TCM researchers.Therefore,it is of certain practical significance to explore the pharmacological mechanism of Zhigan Mixture in the treatment of NAFLD based on endoplasmic reticulum stress.ObjectiveBased on the screening of potential active ingredients and the prediction of pharmacodynamic targets of Zhigan Mixture,we explored the pharmacological mechanism of Zhigan Mixture in the treatment of NAFLD,so as to determine whether the clinical efficacy of Zhigan Mixture is related to the restoration of hepatic endoplasmic reticulum functional homeostasis.Then we explored the molecular mechanism of Zhigan Mixture in the treatment of NAFLD,so as to clarify whether the clinical efficacy of Zhigan Mixture is related to the alleviation of hepatocyte endoplasmic reticulum stress and the inhibition of the activation of NLRP3 inflammasome.Methods1.The corresponding files were imported into Swiss,Super Pred and Stitch databases for target prediction after screening of potential ingredients of Zhigan Mixture in TCMSP platform."Non-alcoholic fatty liver diseases" and "NAFLD" were selected as the key words to search and predict the disease targets in OMIM,TTD,GAD and Pharm GKB databases.Finally,the interaction targets of Zhigan Mixture ingredients and NAFLD were imported into the String database.Then we constructed a single drug active ingredient target network model by using the correlation function of Cytoscape 3.4.0 software.2.Input the above interactive targets into David 6.8 database for GO biological function enrichment analysis.3.KEGG signal pathway enrichment analysis:The gene species are limited to human,and get the enrichment results by setting P<0.05.Finally,the results were visualized by Osmic share platform.4.Construction of NAFLD mice model:The male C57BL/6J mice were fed D12492 diet for 12 weeks to construct an in-vivo model of NAFLD.5.Grouping and intervention:The male C57BL/6J mice were randomly divided into 5 groups.The grouping details were recorded in Appendix 2 below.There was no significant difference in body weight between each group(P>0.05).6.Test:(1)OGTT and ITT were used to evaluate the improvement of Zhigan Mixture to peripheral insulin resistance of the mice induced by HFD;(2)Body weight dynamic test was used to evaluated the effects of Zhigan Mixture to improve the metabolic disorders of mice induced by HFD;(3)Oil Red O staining was used to evaluate the effect of Zhigan Mixture on hepatic fat deposition in NAFLD mice;(4)HE staining was used to evaluate the improvement of Zhigan Mixture on hepatic lipotoxic tissue damage in NAFLD mice;(5)The levels of TC,TG,LDL-C and HDL-C were measured to evaluate the effect of Zhigan Mixture on blood lipid levels of NAFLD mice;(6)The activities of ALT and AST were measured to evaluate the damage of liver function;(7)The mRNA levels of ChREBP-lc,SREBP-1c,PPAR a and PPAR y in the liver tissues of NAFLD mice were measured by qPCR to evaluate the regulation of Zhigan Mixture on fat metabolism related genes of NAFLD mice induced by HFD;(8)Western blot was used to detect the expression of ER stress/NLRP3 cascade related proteins;(9)To observe the activity of PERK autophosphoiylation and the activation of NLRP3 inflammasome in the liver tissues of mice by immunofluorescence,so as to evaluate whether the significant clinical efficacy of Zhigan Mixture is related to the restoration of liver endoplasmic reticulum function homeostasis and inhibition of NLRP3 inflammasome.Results1.The 146 interactive targets are mainly involved in the regulation of intracellular insulin and glucose and lipid metabolism,adaptive changes of stimulation signals and nutritional changes.DNA damage repair,and expression of inflammatory factors.2.GO gene enrichment analy sis showed that:There were 360 biological processes(BP),accounting for 55%,mainly involving nutrition,oxidative stress and immune inflammation.The results of cellular component(CC)showed that 138 sites(22%)were located in membrane rafts,membrane micro domains and transmembrane regions composed of cholesterol and phospholipids;152 sites(23%)were located in molecular functions(MF),which were closely related to ER protein folding,including the regulation of DNA transcription factor activity and specific RNA polymerase Ⅱ activity.3.KEGG pathway enrichment analysis:The 146 interaction targets were significantly enriched in the advanced glycation end products pathway and blood shear stress signal transduction pathway which are closely related to endoplasmic reticulum stress.In addition,endoplasmic reticulum stress-induced c-Jun,IL-1β,IL-6 and Caspase 3 activation are also involved.4.Zhigan Mixture improved peripheral insulin resistance of the NAFLD mice:In OGTT,compared with HFD group,Zhigan Mixture significantly reduced blood glucose levels at each time point(P<0.05).Compared with Low-dose Zhigan Mixture group,High-dose group showed a significantly reduced blood glucose levels at 15 min(P=0.009,P<0.01),30 min(P=0.000,P<0.001),60 min(P=0.001,P<0.01),90 min(P=0.003,P<0.01).In ITT,compared with HFD group,the percentage of blood glucose decrease in Low-dose Zhigan Mixture group at each time point was significantly increased.The decrease percentage of blood glucose in High-dose Zhigan Mixture group was significantly increased at 15 min,30 min,60 min and 90 min(P<0.01).5.Zhigan Mixture improved obesity of the NAFLD mice:After 9 weeks of administration,compared with HFD group the weight of Low-dose Zhigan Mixture group decreased significantly(P<0.05),the weight of High-dose Zhigan Mixture group decreased significantly(P<0.01).6.Oil O red staining showed that Zhigan Mixture could alleviate liver fat deposition in the NAFLD mice.7.HE staining showed that Zhigan Mixture could alleviate the injury of liver tissues and restored the normal structure of liver in the NAFLD mice.8.Compared with HFD group,the levels of TC,TG and LDL-C were significantly decreased in Low and High-dose Zhigan Mixture groups and Statins group(P<0.05),and the levels of HDL-C were significantly increased(P<0.001).But Statins showed a better efficacy than Zhigan Mixture in TG levels(P<0.001)and HDL-C and LDL-C levels(P<0.05).9.Compared with HFD group,the levels of ALT and AST in Low and High dose Zhigan Mixture groups were significantly decreased(P<0.001,P<0.001).Compared with HFD group,Statin group did not show improvement of liver function damage(P>0.05,P>0.05).10.Compared with HFD group,the mRNA levels of CHREBP-1c and SREBP-1c of High-dose Zhigan Mixture group and Statin group were significantly decreased and PPAR a mRNA level was increased.11.Compared with HFD group,Zhigan Mixture significantly increased the expression of PDI:Low dose group(P<0.05),High dose group(P<0.01),Statins group(P<0.05);Zhigan Mixture significantly decreased the expression of BiP:Low dose group(P<0.01),High dose group(P<0.001),Statins group(P<0.001);Zhigan Mixture significantly decreased the expression of CHOP:Low dose group(P<0.05)Zhigan Mixture significantly inhibited the autophosphorylation expression of PERK:Low dose group(P<0.01),High dose group(P<0.001),Statins group(P<0.001).12.Compared with HFD group,Zhigan Mixture significantly inhibited the expression of NLRP3:Low dose group(P<0.001),High dose group(P<0.001),Statins group(P<0.001);Zhigan Mixture decreased the expression of ASC,but there was no statistical significance:Low dose group(P=0.331,P>0.05),High dose group(P=0.158,P>0.05),Statins group(P=0.148,P>0.05);The cleavage of Caspase-1 was significantly decreased by administration:Low dose group(P>0.05),High dose group(P<0.05),Statins group(P<0.01);Zhigan Mixture significantly inhibited the expression of IL-1β mature body:Low dose group(P<0.01),High dose group(P<0.01),Statins group(P<0.01).Conclusion1.Zhigan Mixture has a comprehensive efficacy to treat NAFLD because of its deep interaction with NAFLD disease targets,and this efficacy was closely related to the improvement of the endoplasmic reticulum functional steady.2.Zhigan Mixture could significantly improve the metabolic disorders and lipotoxic damage in the NAFLD mice,and the mechanism may be related to the improvement of ER stress/NLRP3 pathological cascade by inhibiting p-PERK/NLRP3/IL-1β pathway.