A Study Of Endoplasmic Reticulum Stress Mediates Hepatoctes Injury In Patients With Non-Alcoholic Fatty Liver Disease And Possible Mechanisms

Part One Relationship of serum ALT,AST,GGT level and lipid peroxidation in patients with NAFLDObjectives:NAFLD is a spectrum of liver diseases associated with obesity and IR.Lipid peroxidation plays a ciritcal role in NAFLD progressing.For better understanding the relations of obesity,hyperglycemia, hyperlipidemia,lipid peroxidation and aminotansferase increasing in patients with NAFLD,the serum changes of ALT,AST and GGT were analysed with GSH,GSSG and MDA.The aims were to evaluate lipid peroxidation in hepatocytes injury in vivo and provide some theoretic clues for in vitro study and therapeutic strategies in clinic practice.Methods:89 NAFLD patients and 30 healthy controls were randomly enrolled in the study.The NAFLD was diagnosed by the NAFLD diagnostic standard published by Chinese Medical Association.The height, weight,waistline,hip circumference,BMI,WHR and blood pressure were measured.Biochemical assay was used to determine serum level of FBG, ALT,GGT,TG,HDL-C,MDA,GSH,GSSG.The relationships between serum ALT,AST,GGT and other clinical indexes or biochemical parameters were analyzed using multiple linear regression analysis.Result:Except age and height,the systolic blood pressure,diastolic blood pressure,BMI,weight,waistline,hip circumference and WHR of patients with NAFLD are all significantly higher than healthy controls (131.0899±16.06mmHg vs 111.7667±9.6622mmHg, 80.7865±10.7508mmHg vs 72.1333±5.5939mmHg,27.4738±2.7044Kg/m~2 vs 22.348±1.4957Kg/m~2,73.6067±10.1148Kg vs 60.6667±6.1214 Kg, 0.9888±0.0675m vs 0.7497±0.0494m,1.3933±0.0606m vs 0.88±0.0585m, 0.985±0.0364 vs 0.853±0.0197,P＜0.05).Biochemical parameters show that NAFLD patients have elevated serum FBG(6.9204±2.3619mmol/L vs 4.703±0.5628mmol/L),TG(2.1498±1.1454mmol/L vs 1.1369±0.1712mmol/L),TChol(4.9415±0.9635 mmol/L vs 4.2047±0.3735mmoil/L),LDL(2.7342±0.7426 mmol/L vs 2.1693±0.2936mmol/L),FA(0.6481±0.1497mmol/L vs 0.5193±0.1095mol/L) level,whereas HDL is significantly lower than healthy controls(1.0488±0.1953 mmol/L vs 1.374±0.0878mmol/L,P＜0.05). Serum ALT,AST,GGT level of NAFLD patients are relatively significantly higher than control.(55.8202±31.4638u/L vs 21.7667±6.447u/L,40.3258±25.947u/L vs 17.7333±3.805u/L,and 60.3933±50.9723u/L vs 20.3±6.984u/L,P＜0.05).The result of lipid peroxidation analysis implied that GSSG and MDA serum concentration of NAFLD patients are also higher than healthy controls (44.5996±8.4965μmol/L vs 26.4127±2.04μmol/L,3.4401±0.6452μmol/L vs 2.2683±0.1016μmol/L,P＜0.05),while serum GSH is significantly lower than healthy controls(254.2137±35.9436μmol/L vs 320.3693±36.5549, P＜0.05).Multiple linear regression analysis revealed that though parameters which related to obesity and metabolic syndrome including BMI,weight,hyperlipidemia,hyperglycemia correlate with the liver disfunction,the lipid peroxidation indexes including MDA,GSSG,GSH and GSH/GSSG exert greatest effect on serum ALT,AST level.On GGT, MDA and GSSG exert greatest effect but not GSH and GSH/GSSG.Conclusions:NAFLD patients possess the nature of higher hip circumference,BMI,WHR,blood pressure,serum level of FBG,LDL,TG, TChol,FA and concomitant decreased level of HDL,GSH and elevated serum level of ALT,AST,GGT.Hyperglycemia,hyperlipidemia and obesity participate in the pathogenesis of NAFLD,but the lipid peroxidation is the one which plays the key role. Part Two Endoplasmic reticulum stress induce by palmitate sodium in L02 hepatocytes and cell apoptosis signaling pathwaysBackgrouds:Endoplasmic reticulum stress(ER Stress) can be triggered by hypoxia,nutrients deficiency,oxidative stress and accumulation of mis-or un-folded proteins.Once being promoted,ER Stress singaling pathways are activated to suppress protein synthesis and hydrolyse misfolded protein. For those cells which can not be recovered from ER stress,ER stress related apoptosis is induced to maintain internal environment balance. Previous studies had proved that prolonged or overwhelming ER stress is a strong damage factor via energy depletion,vast cell apoptosis and calcium imbalance.Lipid peroxidation is an inducing factor of ER stress and saturated fatty acid is a strong apoptosis inducer of hepatocytes.Excessive ER stress induced by palmitate sodium may be the underline mechanisms. Our aim was to investigate the role of endoplasmic reticulum stress(ER Stress) in the saturated fatty acid induced hepatocytes apoptosis.Methods:The hepatocytes steatosis was established by L02 hepatocytes and palmitate sodium co-culturing.Cell viability and apoptosis were analysed by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and Annexin V/PI flow cytometry.MDA(malonaldehyde) and GSSG measurement were used to analyze lipid peroxidation.ER stress and apoptosis signaling were estimate by p-PERK western-blotting,ATF4 western-blotting and mRNA quantitative analysis and CHOP western-blotting.Caspase-4 activity was measured by cleavage of the Ac-LEVD-pNA substrate to pNA.Reduced glutathione was applied as antioxidants to explore the role of lipid peroxidation in palmitate sodium induced apoptosis.Results:1.Hepatocytes steatosis was successfully established and verified by oil "o" staining with lipid droplets accumulation inside the L02 hepatocytes cytoplasms.The lipid accumulation became prominent as palmitate sodium treatment prolonged.2.Compared with control,elevated ALT,AST,MDA and GSSG level of L02 hepatocytes treated with palmitate sodium could be verified.A significantly drop of these biochemical parameters could be noticed by GSH application.3.Cell viability and apoptosis assay revealed that palmitate sodium induces hepatocytes apoptosis in a dose-and time-depend manner.A significant cell protective effect can be detected after GSH was applied(P＜0.05).4. Western-blotting detected elevated GRP78 protein level after palmitate sodium treatement and decreased GRP78 expression after GSH application. p-PERK western blotting analysis showed increase of p-PERK expression after 12 or 24 hours treatment with palmitate sodium and dramatic decrease after treatment in combination of palmitate sodium and GSH.ATF4 mRNA began to increase after 2 hours co-culturing and reached its peak after 12 hours at a level of 2.7 folds more than baseline.When reduced glutathione was applied,a dramatic reduction of both ATF4 mRNA and protein can be found.Significant changes of CHOP were failed to detect(P＞0.05). Caspase-4 was dramatically activated by palmitate sodium treatment and significantly supreesed by GSH applicatrion.Conclusions:Excessive cytoplasm lipid accumulation and following ER stress are the key events which mediate the palmitate sodium induced hepatocytes apoptosis.Lipid peroxidation is the main factor which provokes the ER stress and the subsequent consequences.Among those cell signaling pathways of ER stress,the PERK-eIF-2alpha-ATF4 was apparently activated by lipid peroxidation.The unchangeableness of CHOP implied that apoptosis signal transduction is not mainly mediated by ATF4 activation and CHOP expression.Caspase-4 activation is the major pathway which mediated ER stress-induced L02 hepatocytes apoptosis.