The Effects And Mechanisms Of Endoplasmic Reticulum Stress On Nonalcoholic Fatty Liver Disease

Objective: 1.To explore the effects and mechanisms of endoplasmic reticulum stress on nonalcoholic fatty liver disease(NAFLD)by establishing models of nonalcoholic fatty liver in vivo and in vitro.2.To study the effects of celastrol on the prevention and treatment of NAFLD and its effects on ERS and its possible mechanism by intervening NAFLD cell model with celastrol.Methods: 1.Establishment and identification of NAFLD models in vivo and in vitro: twenty SD male rats were fed with basal diet for one week and then they were randomly divided into control group and NAFLD group.The rats were fed with basal diet in control group while the rats were fed with high fat diet in NAFLD group for five weeks.LO2 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.48 hours later,they were randomly divided into control group and NAFLD group,then cultured in basal medium and high lipid medium(containing 0.2 mmol/L palmitic acid)respectively for 24 hours.To observe the morphological changes of liver tissue by HE staining;and,to observe fat accumulation of liver tissue and LO2 cells by Oil Red O staining.TC and TG kit were used to detect the contents of total cholesterol(TC)and total triglyceride(TG)in liver tissue and LO2 cells respectively.2.The expression of GRP78 protein in liver tissue was observed by immunohistochemistry.PCR and Western-blot were used to detect the expression of ERS-related mRNAand protein signal molecules of ATF6,GRP78,IRE1,SCAP,SREBP-1c and SREBP-2 in NAFLD cell model respectively.3.LO2 cells were divided into control group,NAFLD group,low dose of celastrol group(0.5?g/ml),high dose of celastrol group(1?g/ml)and simvastatin(SIMVA)group.To observe the lipid deposition in LO2 cells,after celastrol intervention by Oil Red O staining.TC and TG kit were used to detect the content of TC and TG in LO2 cells after intervention;PCR and Western-blot were used to detect the mRNA and protein levels of ERS-related signal molecules ATF6,GRP78,IRE1,SCAP,SREBP-1c and SREBP-2 in LO2 cells under celastrol.Results:1.Establishment and identification of NAFLD models in vivo and in vitro: HE staining have showed that the hepatocytes in control group arrange in the central vein and the structure of the hepatic lobules is normal.Nevertheless,the hepatocytes in NAFLD group are arranged disorderly,and a large number of lipid droplets have appeared.Both liver tissue and LO2 cells Oil Red O staining have showed that the control group have little red particles.Moreover,hepatocyte structure is normal and is arranged neatly.LO2 cells in NAFLD group contain a large number of red particles and are accompanied by the occurrence of steatosis.The contents of TC and TG in liver tissue and LO2 cells in control group are lower than those in NAFLD group(P<0.05).2.The expression of GRP78 protein in liver tissue of NAFLD group is higher than that in control group(P<0.05).PCR and Western-blot have showed that the mRNA and protein expression levels of UPR-related signal molecules ATF6,GRP78 and IRE1 in NAFLD group are higher than those in control group(P<0.05).The levels of mRNA and protein expression of sterol-regulated cascade response-related signal molecules SCAP,SREBP-1c and SREBP-2 in LO2 cells in NAFLD group are higher than those in control group(P<0.05).3.Celastrol intervention experiment: Oil Red O staining has showed that LO2 cells contain a lot of red particles in NAFLD group while the Celastrol0.5?g/ml group,Celastrol 1?g/ml group and SIMVA group have different degrees of reduction compared with NAFLD group.The contents of TC and TG in liver tissue in NAFLD group are higher than those in control group(P<0.05).The levels of TC and TG in Celastrol 0.5 ?g/ml group,Celastrol 1?g/ml group and SIMVA group are significantly lower than those in NAFLD group(P<0.05).PCR and Western-blot showed that:(1)the levels of UPR-related signal molecules ATF6,GRP78 and IRE1 mRNA and protein in NAFLD group are higher than those in control group(P<0.05).The levels of ATF6,GRP78 and IRE1 mRNA and protein in Cel 1?g/ml group and SIMVA group are lower than those in NAFLD group(P<0.05).(2)the levels of mRNA and protein expression of sterol-regulated cascade response-related signal molecules SCAP,SREBP-1c and SREBP-2 in LO2 cells in NAFLD group are higher than those in control group(P<0.05).The expression levels of SCAP,SREBP-1c and SREBP-2 mRNA and protein in LO2 cells in Cel 0.5?g/ml group,Cel 1?g/ml group and SIMVA group are lower than those in NAFLD group(P<0.05).The levels of SREBP-1c and SREBP-2 mRNA and protein inthe LO2 cells in Cel 1?g/ml group and SIMVA group are lower than those in Cel 0.5?g/ml group(P<0.05).Conclusion:1.ERS is closely involved in the lipidosis process of NAFLD and plays an major role in the development of NAFLD.2.The UPR and sterol-regulated cascade responsein ERS,and the associated IRE1/XBP-1 pathway and ATF6 pathway participate in the development of NAFLD.3.Celastrol can reduce LO2 cells lipid metabolism disorders and alleviate NAFLD.The mechanism may be linked to the alleviation of ERS in LO2 cells.