| Nonalcoholic fatty liver disease(NAFLD)is a clinical syndrome marked by the liver fatty infiltration,steatosis,hepatic steatohepatitis,cirrhosis or even hepatic carcinoma.The incidence of NAFLD is increasingly higher and gradually gets younger in recent years.There are approximately 15% to 30% adults who have NAFLD.Despite that the majority of non-alcoholic fatty liver(NAFL)patients at early stage have no liver injury,approximately one-fifth of them are at risk for developing into non-alcoholic steatohepatitis(NASH),cirrhosis,and even liver cancer.However,no effective and ideal managements for NAFLD have been covered.Therefore,NAFLD has become major public health problems that bring about great hazard to mankind.The causes and pathogenesis of NAFLD are controversial.The "multi-hit" hypothesis is dominant currently.This hypothesis holds that insulin resistance(IR)and high concentration of free fatty acids cause lipopexia in liver as first hit,causing hepatic steatosis and enhanced sensitivity to internal and exogenous damage factors.The second hit is oxidative stress injury caused by ROS and related events,causing liver inflammation and fibrosis.In the third hit,the oxide stress persists,inflammation and hepatocellular necrosis create a vicious cycle,resulting in cirrhosis,even hepatic carcinoma.It turns out that excessive fat accumulation is a necessary condition for the occurrence of NAFLD,and oxidative stress is pivotal to the development of NAFLD.For the past few years,using of slimming drug,insulin-sensitizing agents,lipid-lowering agents or antioxidants(such as vitamins E and C)and other treatments for NAFLD carried out lots of clinical research,while the therapeutic drugs with clear effect on NAFLD and with no remarkable side effects haven't been discovered.Majority of flavonoid-rich plant extracts and plant compounds have functions of anti-oxidation,improving IR(insulin resistance)and accommodating lipid metabolism,they are considered likely to have effect on NAFLD control.Dihydromyricetin(DHM)is a kind of natural flavonoids compound with high content in ampelopsis grossedentata.Researches have been reported that DHM has a positive effect and pharmacological function in regulating glucose and lipid metabolism,protecting liver from high fat and improving fatty liver.However,the potential mechanism is still poorly understood.In the development of NAFLD,the expression of key genes which regulate lipid synthesis such as sterol regulatatory element binding protein 1c(SREBP-1c),fatty acid synthase(FAS)and acetyl-CoA carboxylase(ACC)are increased significantly.Whether DHM can improve fatty liver injury and the specific mechanism in liver cells is required further study.Our laboratory conducted the population interventional investigation and found that DHM can improve lipid metabolism and inflammation in patients with NAFLD.The vitro studies have found that DHM improved insulin resistance in skeletal muscle cells by activating AMPK signaling pathways.It was reported that the activation of silent information regulator 1(SIRT1)and adenosine monophosphate activated protein kinase(AMPK)can improve liver steatosis.Thus,this study proposes the following hypothesis: DHM improves liver steatosis by activating AMPK / SIRT1 signaling pathways to regulate lipid metabolism,reduce fat accumulation in hepatic cells.DHM may be an ideal plant compounds for NAFLD prevention and treatment.This study further explored the possible molecular mechanisms that DHM improved hepatic steatosis,so as to find experimental evidence for the NAFLD prevention and drug discovery.In this study,Hep G2 cells were treated by 0.2 mmol/L palmitic acid(PA)for 24 h to establish a model of hepatic steatosis,then treated by DHM with different concentrations(0?5?10?20 ?mol/L)for 24 h.Firstly,the degree of lipid accumulation was observed by Oil Red O staining and triglyceride(TG)determination.The protein expression of Silence information regulator 1(SIRT1),AMP-activated protein kinase(AMPK),sterol regulatory element binding protein-1c(SREBP-1c),fatty acid synthetase(FAS),acetyl-CoA carboxylase(ACC)were detected by wenstern blotting.The m RNA levels of SIRT1,SREBP-1c,FAS and ACC were analysed by qRT-PCR.Finally,combined SIRT1 with AMPK inhibitor and siRNA,the current study futher detected the role of AMPK/SIRT1 signaling pathway in the improvement of DHM on hepatic steatosis.The main results are as follows:(1)Lipid droplets accumulation in HepG2 cells was enhanced by PA treatment,TG content and the lipid droplets accumulation in Hep G2 cells were reduced after treatment by DHM with different concentrations.These indicated that DHM has significant effects on improving hepatic steatosis.(2)DHM significantly reduced the expression of SREBP-1c,FAS and ACC,which are the genes related to lipid synthesis.Which showed that DHM down-regulated the expression and activity of lipid synthesis related genes,inhibited lipid synthesis and improved hepatocyte fatty degeneration in HepG2 cells.(3)The expression of SIRT1 and AMPK especially phosphorylated AMPK protein level dropped significantly in HepG2 cells treated by PA.DHM significantly increased SIRT1 and phosphorylated AMPK protein expression,which indicated that DHM can promote the phosphorylation of AMPK,increase the AMPK activity,and enhanced the SIRT1 expression to improve adipose accumulation in HepG2 cells.When the activity of AMPK was inhibited by Compound C or the activity of SIRT1 was inhibited by EX527 or the SIRT1 expression was silenced by SIRT1 siRNA interference,the role of DHM on improving fatty degeneration and inhibiting proterin expressiong of lipid synthesis related genes SREBP-1c,FAS,and ACC was weakened in HepG2 cells.These finding suggested that AMPK/SIRT1 signaling pathways played an important role in the process that DHM improved hepatocyte steatosisIn conclusion,these results indicated that DHM inhibited the expression of lipid synthesis related genes SREBP-1c,FAS,ACC to improve fat accumulation by regulating AMPK / SIRT1 signaling pathway in HepG2 cells. |
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