Study On The Mechanism Of Erhuang Quzhi Formula In The Treatment Of Non-alcoholic Fatty Liver

Objective:Erhuang Quzhi Formula（EHQZF）is used clinically for the treatment of non-alcoholic fatty liver disease（NAFLD）,but its mechanism of action is unknown,the aim of this study is to investigate the key herbs,active ingredients,important targets,and core mechanisms of EHQZF for treating NAFLD,and to further investigate the efficacy of EHQZF compared with its streamlined formula for lowering blood lipids and treating fatty liver,to simplify the Formulation.Methods:Network pharmacology was used to analyze the herbal attribution,distribution of components,main therapeutic diseases of EHQZF and to study the main components and mechanism of EHQZF in the treatment of NAFLD;LC-MS was used to analyze and quantify key active ingredients;an in vitro model of NAFLD was established by FFA（OA:PA=2:1）induction of Hep G2 and WRL68 cells,and the effect of EHQZF on cellular lipid droplets was examined using Oil Red O staining;a high-fat diet was used to establish an in vivo model of NAFLD,the ICR mice were randomly divided into blank group（CON）,model group（HFD）,low-dose group(LEH,0.6 g·kg^-1),medium-dose group(MEH,1.2 g·kg^-1),high-dose group(HEH,2.4 g·kg^-1)and positive drug benzofibrate group(BZBT,60.67 m g·kg^-1),except the mice in the CON group which were fed standard chow and the rest groups were fed high-fat chow,and after 7weeks,each dosing group was gavaged once a day for 5 weeks,and the weekly body weights were recorded.At the 11th and 12th week,the mice were tested for glucose tolerance（IPGTT）,insulin tolerance（IPITT）and cold tolerance,and finally,after euthanasia,the mice were taken for testing.The efficacy and mechanism of EHQZF on NAFLD mice were investigated by IPGTT,IPITT,body weight change,liver and spleen coefficients,pathological changes in adipose tissue,UCP1 expression in adipose tissue,cold tolerance test in mice,serum index and pathological changes in liver.The effects of EHQZF on liver BCL2,BAD,P-AKT,P-P38 MAPK,P-JNK,P-ERK1/2,and P-STAT3 protein expression were investigated by Western Blot.Tyloxapol was used to induce hyperlipidemia in mice to investigate the effects of EHQZF and its condensed formulations QZF-Ⅰand QZF-Ⅱin lowering blood lipids and reducing hepatic lipid accumulation.Results:Network pharmacology and LC-MS results showed that EHQZF can treat NAFLD,HPL and other metabolic diseases,when EHQZF treats NAFLD,Glycyrrhizae Radix et Rhizoma,Salviae Miltiorrhizae Radix et Rhizoma,Nelumbinis Folium,Rhei Radix et Rhizoma,Curcumaelongae Rhizoma and Polygoni Cuspidati Rhizoma et Radix occupy an important position in the compound,flavonoids and phenolic acid compounds are the key active ingredients,EHQZF can regulate PI3K-AKT,apoptosis,JAK-STAT,MAPK and other signaling pathways against NAFLD,in which the core targets of AKT1,STAT3,MAPK14,c-JUN,BCL2,BAD and other key active ingredients are closely docked;in vitro and in vivo experiments of NAFLD showed that EHQZF at 0.39～1.56 mg·ml^-1and 0.78～3.125 mg·ml^-1could reduce lipid droplet accumulation in Hep G2 and WRL68 cells treated with 0.6 m M FFA（P<0.01,0.001）;EHQZF improved glucose tolerance and insulin tolerance,reduced body mass and liver and spleen mass in mice（P<0.01,0.001 or P>0.05）;adipose tissue data showed that EHQZF reduced epididymal adipose tissue（e WAT）mass and adipocyte hypertrophy,promoted e WAT browning,increased brown adipose tissue（BAT）mass,increased UCP1 expression in e WAT and BAT,and increased body thermogenesis（P<0.05,0.01,0.001,or P>0.05）;serum indices showed that EHQZF significantly increased HDL-c levels,decreased AST,LDL-c,TG,and TC levels in serum（P<0.05）;liver pathological sections showed that EHQZF could reduce hepatic lipid droplet accumulation and liver mitochondrial damage;Western Blot results showed that EHQZF significantly inhibited BAD,P-JNK,P-P38MAPK and P-ERK1/2 protein expression,and significantly promoted P-AKT,BCL2 and P-STAT3 protein expression.The comparison of the efficacy of EHQZF and its condensed formula in hyperlipidemic mice showed that QZF-II（Polygoni Cuspidati Rhizoma et Radix,Curcumaelongae Rhizoma,Salviae Miltiorrhizae Radix et Rhizoma,Nelumbinis Folium）was effective in increasing serum SOD levels,decreasing TC,ALT and AST levels,and reducing liver IL-1βand IL-6 levels（P<0.01、0.05、0.001）,and improved hepatic lipid droplet accumulation.Conclusion:EHQZF could alleviate NAFLD by inhibiting insulin resistance,improving adipose tissue status,reducing hepatic lipid accumulation,inhibiting MAPK pathway,regulating apoptosis pathway,reducing hepatic mitochondrial damage,inhibiting hepatocyte apoptosis and liver inflammation.