| Non-alcoholic fatty liver disease(NAFLD)refers to factors other than alcohol and other clear injure of liver,due to diffuse hepatocellular big bubble fat into the main characteristics of clinical syndrome,including three stages that are simple fatty liver,non-alcoholic fatty steatohepatitis(NASH)and cirrhosis of the liver stage.The pathogenesis of NAFLD has not been studied clearly,and the more mature theory is "Two-Hit".Daphnetin(DAP)is an active ingredient extracted from the Daphne koreana of Daphne genera,which has good anti-oxidation and anti-inflammatory activity.With daphnetin rarely making the news affect NAFLD,therefore,this article with oleic acid(OA)treatment HepG2 cells as in vitro model of NASH,around the glucolipid metabolism and oxidative stress,research on the prevention and treatment of NASH daphnetin effect and its mechanism.First,we selected HepG2 cells as the experimental objects,and established two models of prevention model and treatment model.The prevention mode was used to treat HepG2 cells 24h with 0.5mM OA and DAP(5?20?50?M).In the treatment model,HepG2 cells were first treated with OA 24h to establish a model,and then the model cell was treated with DAP for 24h.Then we tested three biochemical indicators including the intracellular TG content,cell glucose uptake ability and the content of ROS in cells,also tested genes' mRNA and protein expression of the glucolipid metabolism and oxidative stress related genes in HepG2 cells(pAMPK,SREBP-1 c,PNPLA3,PPAR?,PI3K,pAKT,AKT and CYP4A).Finally,we took the statistical analysis.Experimental results are following:Under the prevention and treatment mode,(1)compared with control group,the intracellular TG of OA treatment group was obviously increased,SREBP-1 c,PNPLA3 mRNA and protein expression were up,and PPARa mRNA and protein expression and pAMPK level were down;Compared with the OA treatment group,the content of TG in the OA+DAP group was decreased,the mRNA and protein expression of SREBP-1 c and PNPLA3 were down-regulated,and the mRNA and protein expression of PPARa and the level of AMPK phosphorylation were up-regulated.(2)After adding the OA,HepG2 cells significantly lower absorbed the fluorescence glucose,and PI3K protein expression and pAKT/AKT were down;OA and daphnetin joint processing,cell glucose uptake ability was enhanced,PI3K protein expression and pAKT/AKT were up.(3)Compared with the control group,the ROS content in the cells of OA group increased significantly and the expression of CYP4A increased;Compared with the OA group,the OA+DAP group effectively reduced the content of ROS in the cells and the content of CYP4A.Experimental results show that DAP have prevention and treatment effect of NAFLD,its mechanism has:(1)through increasing AMPK levels of phosphorylated the key factor of glucolipid metabolism,inhibiting expression of SREBP-1 c and PNPLA3 the key genes of lipid synthesis and inducing PPARa expression the key genes of lipid oxidation,reduce liver lipid accumulation.(2)Improving the expression of PI3K and pAKT/AKT level to alleviate insulin resistance,thereby promotes the uptake of glucose of liver cells.(3)By down-regulating the expression of CYP4A,the key gene of ROS,alleviates the oxidative damage of liver cells.To summing up,the experimental study on daphnetin having effects on inducing of lipid accumulation,insulin resistance and oxidative stress injury by OA treating HepG2 cells.Therefor,DAP has certain preventive and treatment to NASH,and its mechanism is that DAP have be connected with regluting glucolipid metabolism key factors(AMPK),liver lipid metabolism key genes(SREBP-1c,PNPLA3 and PPARa),insulin signaling pathway PI3K/AKT pathway and oxidative stress key genes(CYP4A).This provides a useful reference for further studies on the anti-NAFLD/NASH expriment of coumarin compounds. |
PDF Full Text Request