ERS Influence Lipidoses In Hepatocytes Through SERBP-1c-FAS

Background and ObjectiveNonalcoholic fatty liver disease (NAFLD) is a chronic metabolicdisorder induced by excessive lipids accumulated in hepatocytes. As itsincidence trends to raise year by year in the world, it is necessary for us toclarify its pathogenesis. Recent researches suggest the relations betweenNAFLD and endoplasmic reticulum stress (ERS). SREBP-1c and LXRsparticipate in denovo synthesis of fatty acid in hepatocytes by regulatingFAS on downstream target genes,and the expression control of SREBP-1ccontains transcription and posttranscription, that is precursor protein andactive protein respectively.We establish the molde of ERS in hepatic cellswith thapsigargin to study the effects on lipids deposition under ERS.Moreover, we will observe the expressive changes of FAS, SREBP-1c andLXR through Q-PCR and Western-blot, and discuss the possiblemechanism of thapsigargin inducing ERS to formfatty deposition inhepatocytes.Methods1. Cells culture: L02cell and HepG2cell was respectively cultured in RPMI1640and DMEM containing10%fetal bovine serum,0.0625g/Lpenicillin,0.1g/L streptomycin and HepG2cell in37℃,5%CO2saturatedhumidity incubator.2. Group division: there were control group(with no addition) andexperimental group (treated with1the optimal concentrated thapsigargin).3. CCK-8was used for determination of cell proliferation and WesternBlot for test of the expression of GRP78(glucose regulated protein78).Lipids depositon in the hepatocytes were observed by biochemical assay(TG kits) and oil red O staining.4. Real-time PCR and Western Bloting were used to test theexpression of LXRα, SREBP-1c and FAS mRNA and protein changes.ResultsPart one1. We test L02cell proliferation inhibition rate induced by differentconcentration thapsigargin after24h,48h,72h through CCK-8. Becausehepatocytes are excessively inhibited by thapsigargin after72h(theinhibition rate is20%), we choose24h and48h to estabolish the model.2. The expression of GRP78: Western blotting analysis show thatGRP78expression in the experimental group was remarkably higher thanthe control group （F=29.39、34.56，p=0.0008、0.0005for L02cell,HepG2cell respectively). 3. After L02cells were exposed to TG for24h, there is no statisticsignificant difference between experimental group and control group, butafter48h, fatty deposition increase in experimental group（p＜0.01).Therefore,48h would be the induction time in consequence researches.After HepG2cell were exposed to TG for48h, experimentalgroup(276.98±6.68μg) reveals more fatty deposition than controlgroup(121.52±4.73μg)（P＜0.01）.4. Oil red O staining: Compared with the control group, morphologychanges emerge in L02cell and HepG2cell and obvious fatty deposition inthe expremental groupPart two1. The expression of LXRα mRNA, SREBP-1c mRNA in hepatocytes:after the hepatocytes were exposed to1μmol/L thapsigargin for48h, theexpression of SREBP-1c mRNA in experimental group boost（t=7.4334、10.3923，P=0.0017、0.0005，＜0.05. Meanwhile, There was no statisticalsignificant difference in terms of the expression of LXRα mRNA betweenexperimental and control group （t=0.5774、0.9448，P=0.5946、0.3983，＞0.05).2. After the test of precursor protein and active protein of SREBP-1c,SREBP-1c remains as protein, there is increase in experimental group ofprecursor protein（t=5.4153、6.2429，P=0.0056、0.0034，＜0.05), and activeprotein tends to be same（t=8.2914、9.9675，P=0.0012、0.0006，＜0.05). The expression of FAS is the same as SREBP-1c, as the hepatocytes inexperimental group increase compared to the control group（t=9.4185、6.1189，P=0.0007、0.0036，＜0.05). However, there was no statisticalsignificant difference in terms of the expression of LXRα betweenexperimental and control group（t=0.3884、1.2546，P=0.7175、0.2779，＞0.05）.Conclusion1.Thapsigargin may induce ERS in hepatocytes to form fattydeposition.2.During this course, the expressions of SREBP-1c and FAS increase,showing that SREBP-1c-FAS is the important mechanism to induce ERSto fatty deposition in hepatocytes.3.During this course, LXRα mRNA and protein level reveal nosignificant changes, suggesting few correlation of LXRs-SERBP-1c andLXRs-FAS with the process of fatty deposition.