Mechanism Of Di’ao Xinxuekang Improving Nonalcoholic Fatty Liver By Regulating AMPK/SREBP-1c/ACC Signaling Pathway To Reduce Lipid Synthesis

Background:Non alcoholic fatty liver disease（NAFLD）refers to the condition caused by factors other than alcohol and other clear liver damage factors.The lesion is mainly in the liver lobules,with diffuse hepatic cell bullous steatosis and fat storage as pathological characteristics.Its pathogenesis is complex,and there is no drug approved for the treatment of this disease.The excessive accumulation of liver lipids is one of the important factors for the occurrence and development of NAFLD.AMP-activated protein kinase（AMPK）and its downstream target proteins sterol regulatory element-binding protein-1c（SREBP-1c）and acetyl coenzyme A carboxylase（ACC）play an important role in lipid synthesis.The regulation of AMPK/SREBP-1c/ACC signal pathway to reduce lipid synthesis may reduce the damage of lipotoxicity to tissue cells induced by excessive accumulation of lipids.Compound C as an AMPK specific inhibitor is often the first choice for mechanism research in experimental research.Di’ao Xinxuekang（DXXK）is a steroidal total saponin extracted from dioscorea panthaica prain et burk or the rhizome of dioscorea nipponica.It is the first traditional Chinese patent medicines and simple preparations to enter the EU market,which can significantly improve nonalcoholic steatohepatitis induced by high-fat diet in mice and steatosis in rats.Whether DXXK plays a role in the treatment of NAFLD by activating AMPK and its downstream target protein is still unknown.Therefore,this paper proposes that DXXK may play a role in improving nonalcoholic fatty liver（NAFL）by regulating AMPK/SREBP-1c/ACC signal pathway to reduce lipid synthesis.Objective:（1）To construct the disease characteristic model of NAFL in vivo and in vitro,and to clarify the improvement effect of Di’ao Xinxuekang on NAFL;（2）To explore whether Di’ao Xinxuekang can reduce lipid synthesis and improve NAFL by regulating AMPK/SREBP-1c/ACC pathway.Methods:（1）The therapeutic effect of DXXK on NAFL induced by high-fat and high-cholesterol diet and its effect on AMPK/SREBP-1c/ACC signal pathway in rats:67 healthy male SD rats were randomly divided into normal group and model group.The model group was fed with high-fat and high-cholesterol diet for 8 weeks.At the end of the 8th week,5 rats were randomly executed in the model group and 2 rats in the normal group were randomly executed for liver pathological staining,it was found that the liver of 5 rats in the model group had more than 5 fields of fatty changed and the area was more than 1/3.It was confirmed that the model was successfully established.The rats in the model group were randomly divided into model group,atorvastatin group(2.0 mg·kg^-1),DXXK high,medium and low(100,30,10 mg·kg^-1)dose group,n=10,gavage for 8 weeks.The level of blood lipid（TC,TG,LDL-C,）and liver function（ALT,AST,ALP,LDH and GGT）were detected by automatic biochemical analyzer;The levels of TC,TG and FFA in liver were detected with the test kit;Calculate liver index;Non-alcoholic fatty liver disease activity score（NAS）quantified pathological examination results;The fatty area of liver was observed by oil red O staining;Ultrastructural changes of hepatocyte nuclei and contents were observed by transmission electron microscopy;The expression of AMPK,p-AMPK,SREBP-1c,ACC,p-ACC protein in liver was determined by Western blot.（2）The improvement of DXXK on the lipid accumulation model of Hep G2 cells induced by oleic acid-palmitic acid and its effect on AMPK/SREBP-1c/ACC signal pathway:In the pharmacodynamics study,Hep G2 cells in logarithmic growth phase were inoculated into 6-well plates,and Hep G2 cells were divided into control group,model group,atorvastatin group（1μM）,Di’ao Xinxuekang high,medium and low concentration group(10,3,1μg·ml^-1);In the part of mechanism research,Hep G2 cells were divided into control group,model group and Di’ao Xinxuekang group(10μg·ml^-1),Compound C（10μM）group,Compound C（10μM）+Di’ao Xinxuekang group(10μg·ml^-1),the cells were incubated with drugs for 2 hours according to their groups.Except for the control group,the other groups were stimulated with 1m M oleic acid-palmitic acid（2:1）for 24 hours to establish a lipid accumulation model,and the levels of cell lipids and transaminases were measured;Oil red O staining was used to observe the level of cell lipid accumulation in each group;Western blot was used to detect the expression of AMPK,p-AMPK,ACC,p-ACC and SREBP-1c protein.Results:（1）Compared with the normal group,the levels of serum TC,TG,LDL-C,ALT,AST,ALP,LDH and GGT in the model group were significantly increased（P<0.01）,The expression levels of p-AMPK and p-ACC protein activation in liver were significantly decreased（P<0.01）,and the expression levels of ACC and SREBP-1c were significantly increased（P<0.01）.Compared with the model group,the levels of serum TC,TG,LDL-C,ALT,AST,ALP,LDH and GGT in DXXK group were significantly lower（P<0.05）;The liver index decreased and the levels of TC,TC and FFA decreased significantly;NAS score and fat positive area significantly decreased（P<0.05）;The ultrastructure of liver was improved;The activated expression levels of p-AMPK and p-ACC protein in liver were significantly increased（P<0.05）,and the expression levels of ACC and SREBP-1c were significantly decreased（P<0.05）.（2）In the pharmacodynamic study,compared with the control group,the levels of TC,TG and transaminase in the model group were significantly higher（P<0.01）;the expression levels of p-AMPK and p-ACC protein activation were significantly decreased and the expression levels of ACC and SREBP-1c were significantly increased（P<0.01）;compared with the model group,the levels of TC,TG and transaminase in DXXK and atorvastatin treated cells were significantly lower（P<0.05）;the level of lipid accumulation decreased significantly;the expression levels of p-AMPK and p-ACC protein activation were significantly increased,and the expression levels of ACC and SREBP-1c were significantly decreased（P<0.05）.In the part of mechanism study,compared with the control group,TC and TG of model group and Compound C cells were significantly increased（P<0.01）;the activated expression level of p-AMPK protein in model group and Compound C group decreased significantly（P<0.01）;Compared with the model group,the levels of TC and TG in DXXK group were significantly lower（P<0.01）;the expression level of p-AMPK protein activation was significantly increased in DXXK group,and the levels of TC and TG were significantly increased in Compund C group（P<0.05）.The expression level of p-AMPK protein was significantly decreased in Compund C group（P<0.05）;compared with DXXK group,the levels of TC and TG in DXXK+Compound C group increased;the expression level of p-AMPK protein decreased significantly（P<0.05）;it shows that Compound C can weaken the activation of DXXK on AMPK,and when AMPK is inhibited the degree of lipid accumulation increases.Conclusion:（1）DXXK can improve NAFL;（2）DXXK can improve NAFL by activating AMPK and regulating the AMPK/SREBP-1c/ACC pathway to reduce lipid synthesis.