游离脂肪酸混合物对肝细胞脂毒性及脂代谢相关基因表达的影响

目的:研究游离脂肪酸(FFA)混合物对肝细胞L-02的脂毒性及脂代谢相关基因表达的影响。方法:正常肝细胞L-02分别用正常培养基和0.5、1、2 mmol/LFFA混合物(油酸和软脂酸的比例为2:1)培养24 h后,尼罗红染液室温避光染色,激光共聚焦显微镜及流式细胞仪确定细胞内脂质堆积情况。组织细胞酶法测定试剂盒测定细胞内甘油三酯含量。MTT法分析细胞存活率,Annexin V-PI凋亡检测试剂盒分析肝细胞的凋亡情况,丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)测定试剂盒分别检测培养液中ALT和AST活性。实时定量PCR技术检测脂代谢相关的脂肪分化相关蛋白(ADRP)和固醇调节元件结合蛋白1(SREBP-1)的mRNA表达情况。结果:各浓度FFAs混合物均可剂量依赖性增加肝细胞脂肪堆积和肝细胞甘油三酯含量,且1 mmol/LFFA混合物可增高肝细胞的甘油三酯含量2.6倍,与非酒精性脂肪肝病人的变化基本相同。2 mmol/LFFA混合物可降低肝细胞L-02细胞存活率并诱导细胞凋亡,而0.5 mmol/L和1 mmol/LFFA混合物对细胞无明显影响。与对照组相比,各浓度FFA混合物对细胞上清中ALT和AST活性无明显影响。1 mmol/LFFA混合物作用后可分别上调肝细胞的ADRP和SREBP-1 mRNA的表达2.660和2.758倍。结论:FFA混合物可诱导肝细胞L-02脂肪变性且2mmol/LFFA混合物可造成轻度细胞损伤。脂代谢相关基因ADRP和SREBP-1表达上调与FFA混合物诱导的脂肪变性相关。

Lipotoxicity of free fatty acid mixture and effect of free fatty acid mixture on lipid metabolism-related genes in L-02 cells

AIM：To investigate the lipotoxicity of free fatty acid (FFA) mixture and the effect of the FFA mixture on lipid metabolism-related genes in L-02 cells. METHODS：A normal human hepatocytes-derived cell line L-02 was treated with 0.5, 1 and 2 mmol/L FFA mixture (oleate and palmitate, 2∶1) for 24 h. The cellular total lipid accumulation was determined after Nile red staining by confocal laser scanning microscopy and flow cytometry. Intracellular triglyceride (TG) content was measured using an enzymatic kit. The viability of L-02 cells was determined by MTT assay and the apoptosis-inducing effect of FFA mixture was evaluated by annexin V/PI staining. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the culture medium were detected by ALT and AST kits. The mRNA levels of lipid metabolism-related genes, adipose differentiation-related protein (ADRP) and sterol regulatory element-binding protein 1 (SREBP-1), were examined by quantitative real-time PCR. RESULTS：All the different concentrations of FFA mixture increased intracellular lipid accumulation and TG content in a dose-dependent manner. FFA mixture at concentration of 1 mmol/L increased intracellular TG by 2.6 folds, which matched with the change in non-alcoholic fatty liver disease patients. Treatment for 24 h with 0.5 mmol/L and 1 mmol/L FFA mixture did not trigger apparent cell death and apoptosis, while treatment with 2 mmol/L FFA mixture resulted in a marked decrease in cell viability and induced early and late stages of apoptosis in L-02 cells. The levels of ALT and AST in the culture supernatant had no significant difference between control group and FFA treatment group. Treatment with 1 mmol/L FFA mixture up-regulated the expression of ADRP and SREBP-1 by 2.660 and 2.758 folds, respectively. CONCLUSION：FFA mixture induces the hepatic steatosis and 2 mmol/L FFA mixture causes mild cells damage in L-02 cells. The up-regulation of ADRP and SREBP-1 may be involved in FFA-induced hepatic steatosis.