Study On The Hepatoprotective Effect And Mechanism Of Polysaccharides From Anoectochilus Roxburghii (ARP) On Non-alcoholic Fatty Liver Disease (NAFLD)

Objective: To investigate the lipid metabolism,hepatoprotective effect and possible mechanism by observing the ameliorating effect of Anoectochilus roxburghii polysaccharides(ARP)on fetal bovine serum(FBS)-induced steatosis in human liver cell line LO2 and high-fat emulsion(HFE)-induced oxidative stress in NAFLD rats and provide futher scientific evidence for development and clinical application of ARP.Methods: High-FBS medium(DMEM with 50% FBS)was used to induce steatosis of human liver LO2 cells,Oil red O staining,the hepatocytes lipid accumulation was observed by inverted microscope,and the TG level was detected by TG kit.Determination of the cytotoxicity effect of ARP on LO2 cells,and assigned control group,model group,high-dose group of ARP(ARP-H),medium-dose group of ARP(ARP-M)and low-dose group of ARP(ARP-L).The control group was cultured in growth medium(DMEM with 10% FBS),and others were cultured in high-FBS medium(DMEM with 50% FBS)for 24 h so as to induce steatosis of cells.In the next 24 h,the control group was cultured in growth medium,the model group was treated with high-FBS medium,and the treatment groups were treated with ARP(dissolved in growth medium).After that,the supernatant was collected for the dermination of TG,AST,ALT,SOD,GSH-Px,and MDA.The rats were randomly divided to six groups(eight for each): the control group,the model group,simvastatin group,high-dose group of ARP(ARP-H),medium-dose group of ARP(ARP-M)and low-dose group of ARP(ARP-L).To study the effect of ARP on NAFLD,the rats in normal group had a regular basic diet,while other groups were given daily via gavage to HFE to induce NAFLD rat model.In the period of modeling,rats in the ARP-H,ARP-M,ARP-L groups were given ARP crude extract 238,119,59.5 mg/kg via gavage and those in the control group and model group were orally treated with distilled water.Rats in simvastatin group were given simvastatin 8 mg/kg daily.At the completion of the experiment,rats were anaesthetized with 0.5% pentobarbital(50 mg/kg)by intraperitoneal injection.Blood samples from abdominal aortic were collected and the livers were immediately removed.Liver morphological changes and pathological changes were observed by naked eye and light microscope,and the liver cells steatosis degree was graded.The contents of TG,TC,LDL-C,HDL-C,FFA,FBG,AST,ALT in the serum,and as well as TC,TG,MDA,SOD in the liver homogenate were detected by corresponding kits.The expression level of CYP2E1 m RNA in hepatic tissue was analyzed by reverse transcription-polymerase chain reaction(RT-PCR).Rseults: Experiment 1 Effect of ARP on steatotic hepatocytes model 50% FBS-induced steatosis in human liver LO2 cell was used to evaluate the functions of ARP in regulation of lipid metabolism and inhibition of oxidative stress.After oil red O staining,compared with control group,great deals of orange red and red lipid droplets were found in the luminal side of the cells,and the level of TG was significantly increased in model group(P<0.01).Compared with model group,ARP could inhibite the growth of steatosis LO2 cells,reducing the accumulation of hepatic lipid through lowering the level of TG(P<0.01),and suppressing the leakage volume of enzymes(AST and ALT)(P<0.01)and also could enhance the activities of SOD and GSH-Px(P<0.01).Experiment 2 Effect of ARP on NAFLD rats Compared with control group,model group significantly increased serum TC,TG,LDL-C,FFA,FBG,ALT,AST(P<0.05 or 0.01),liver index and the hepatic levels of TC,TG,MDA(P<0.01),NAFLD activity score(NAS)and the expression of CYP2E1 m RNA(P<0.01),and reduced serum HDL-C and hepatic SOD levels(P<0.05 or 0.01).Furthermore,the morphological evaluation also revealed that model group disorganized hepatocyte,hepatic lobule boundary is not clear,liver cell volume is increased and swollen,and diffuse bullous fatty steatosis.Compared with model group,the administration of ARP for eight weeks significantly reduced the TC,TG,LDL-C and FFA levels(P<0.05 or 0.01),inhibited the incremental changes in ALT and AST(P<0.01),increased the HDL-C level and the hepatic activity of antioxidant enzyme(SOD)(P<0.05 or 0.01),and decreased the hepatic levels of TC,TG and the hepatic lipid peroxides(MDA)content(P<0.05 or 0.01)in NAFLD rats.Furthermore,the AFP-treated groups also down-regulated the m RNA of CYP2E1(P<0.05)in the hepatic tissue.Conclusion: 1.50% FBS and HFE were able to successfully induce steatosis in human liver LO2 cell and NAFLD rats,both of which are consistent with the human NAFLD disease course.2.ARP significantly reduced the steatosis intercellular content of TG and the serum levels of TC,TG,LDL-C and FFA,increased the serum level of HDL-C,decreased the disorder of lipid metabolism,lowered the hepatic lipids levels of TC,TG,and ameliorated liver lipid metabolism and lipid accumulation in steatotic hepatocytes.3.ARP can effectively reduce leakage volume of AST,ALT in 50% FBS-induced cellular steatosis and repair liver cell injury by inhibiting the incremental changes of ALT,AST in NAFLD rats,conditioned the hepatocytes,and maintained hepatic structural integrity.4.ARP can down-regulate the expression of CYP2E1 m RNA in liver tissue of NAFLD rats and alleviate the degree of lipid peroxidation and oxidative stress in vivo and in vitro.The possible mechanisms include,reducing the intake of hepatocyte FFA,the synthesis of hepatic triglyceride,the toxic effects of excess FFA,and liver fat accumulation,thereby improve mitochondrial ?-oxidation,effectively remove ROS and free radicals,improve liver function and hepatic steatosis,protect hepatocytes from lipid peroxidation and oxidative stress,and effectively reverse NAFLD.