OBJECTIVETo establish an in vitro cell model for investigating hepatic steatosis in non-alcoholic fatty liver disease.

METHODSL-02 cells cultured in 1640 containing 10% fetal bovine serum were divided into control group and model group. At 70%-80% confluency, L-02 cells in the model group were exposed to a long-chain mixture of free fatty acids (FFA, oleate and palmitate ) for 24 h, and cells in control group were treated with fresh medium. Lipid droplets in the cells were observed and total lipid content was determined with Oil Red O staining. The morphology of lipid droplets, trilyceride level, malonaldehyde content and cell apoptosis rate were evaluated to verify the cell model, and the effect of Huganqingzhi tablet on the lipid droplets was observed.

RESULTSA large number of lipid droplets were found in the cell model, which showed markedly increased level of triglyceride without significant changes of malonadehyde content or cell apoptosis rate. Intervention with two doses of Huganqingzhi tablet significantly decreased the number of lipid droplets and trilyceride content in the cell model.

CONCLUSIONhepatic steatosis L-02 cell model can be established by long-chain mixture of free fatty acids (oleate:spalmitate=2:1) for therapeutic drug studies.

Apoptosis ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Fatty Acids, Nonesterified ; chemistry ; Fatty Liver ; Humans ; Malondialdehyde ; analysis ; Non-alcoholic Fatty Liver Disease ; Triglycerides ; analysis

OBJECTIVE: To investigate the effects of sera from rats fed with tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism. METHODS: FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. RESULTS: Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT ( < 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels ( < 0.05), reduced accumulation of the lipid droplets ( < 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ ( < 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis ( < 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 ( < 0.001) and P-PERK ( < 0.01) in FFAs-induced cells with PKC-δ knockdown. CONCLUSIONS HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.
Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Endoplasmic Reticulum ; ultrastructure ; Endoplasmic Reticulum Stress ; drug effects ; Fatty Acids, Nonesterified ; Fenofibrate ; administration & dosage ; Hep G2 Cells ; Humans ; Hypolipidemic Agents ; administration & dosage ; Microscopy, Electron, Transmission ; Non-alcoholic Fatty Liver Disease ; blood ; etiology ; prevention & control ; RNA, Messenger ; blood ; Rats ; Serum ; Tablets ; Triglycerides ; blood