内质网应激在脂肪酸致肝细胞损伤中的作用

Investigation on the role of endoplasmic reticulum stress in the fatty acids-induced hepatocyte injury

目的通过建立不同的肝细胞脂肪变模型探讨脂肪酸对肝细胞内质网应激的作用，同时检测牛磺熊去氧胆酸（TUDCA）对脂肪变肝细胞内质网应激相关基因表达的影响。方法以正常成人肝细胞株HL-7702为研究对象，以软脂酸或混合脂肪酸建立肝细胞脂肪变模型，以细胞计数试剂盒细胞增殖法测定和计算细胞活力，观察细胞形态和脂肪变情况，用三酰甘油试剂盒测定细胞内三酰甘油含量。予TUDCA干预，实时PCR方法检测肝细胞内质网应激前后葡萄糖调节蛋白78（GRP78）和CCAAT／增强子结合蛋白同源蛋白（CHOP）的mRNA相对表达量。结果混合脂肪酸浓度达0．5mmol／L或软脂酸浓度达0．125mmol／L即可影响肝细胞活力。单纯软脂酸对肝细胞的损伤作用较混合脂肪酸更强。混合脂肪酸组肝细胞内三酰甘油含量逐渐升高，软脂酸组肝细胞内三酰甘油含量仅于作用12h时显著增加，其后则无显著改变。混合脂肪酸组不同浓度、不同作用时间下GRP78mRNA和CHOPmRNA相对表达量与对照组相比差异均无统计学意义（P值均〉0．05）。0．5mmol／L软脂酸作用后肝细胞内CHOPmRNA相对表达量显著升高，但其对GRP78mRNA相对表达量无明显影响。1．0mmol／L软脂酸作用后肝细胞内GRP78mRNA和CHOPmRNA相对表达量均显著升高。TUDCA干预低剂量软脂酸组肝细胞内质网应激前后GRP78mRNA和CHOPmRNA相对表达量差异无统计学意义（P值均〉0．05）。TuDCA干预高剂量软脂酸组肝细胞内质网应激前后CHOPmRNA表达量差异有统计学意义（12h时为8．6400比5．1032，24h时为13．7948比6．4928，P值分别=0．042和0．017），但GRP78mRNA表达量差异无统计学意义（P值均〉0．05）。结论相同的脂肪酸浓度下，软脂酸的比例越高对肝细胞的损伤作用越大。软脂酸呈时间一剂量依赖性地调控肝细胞内质网应激。TUDCA可在一定程度上改善软脂酸引起的内质网应激。

Objective To explore the role of fatty acid on endoplasmic reticulum （ER） stress and to detect the influence of tauroursodeoxycholic acid （TUDCA） on the expression of ER related genes in steatosis hepatoeytes by establishing different models of hepatic steatosis. Methods Healthy adult hepatocyte cell line HL-7702 was taken as research object. The model of hepatic steatosis was established with palmitic acid alone or mixed fatty acids. The cell viability was measured and calculatedthrough CCK-8 positive cell proliferation assay. The cell morphology and steatosis was observed. The intracellular triglyceride content was tested with the triglyceride determination kit. With TUDCA intervention, the relative expression of Glucose-regulated protein 78 （GRP78） and CCAAT/Enhancer binding protein homologous protein （CHOP） at mRNA level was determined by real-time PCR. Results The viability of hepatocyte was influenced once the concentration of mixed fatty acid reached 0.5 mmol/L or palmitic acid was 0. 125 mmol/L. The effect of palmitic acid alone was stronger than that of mixed fatty acid on hepatocyte injury. The content of intra-hepatocyte triglyceride gradually increased in mixed fatty acid group, which only significantly increased after treated for 12 hours in palmitic acid alone group and then there was no significant change. There was no significant difference in relative expression of GRP78 and CHOP at mRNA level in various concentrations and treated time of mixed fatty acid group. After treated with palmitic acid at 0.5 mmol/L, intra-hepatocyte relative expression of CHOP at mRNA level increased obviously, however there was no effect on GRP78 mRNA expression. After treated with palmitie acid at 1.0 mmol/L, both intra-hepatocyte GRP78 and CHOP mRNA relative expression increased. There was no significant difference in GRP78 and CHOP mRNA relative expression before and after ER stress in TUDCA intervented low dose palmitie acid group. There was significant difference in CHOP mRNA relative expression before and after ER stress in TUDCA intervented high dose palmitie acid group （at 12hrs： 8. 6400 to 5. 1032; at 24hrs： 13. 7948 to 6. 4928, P= 0. 042 and 0. 017）, while no significant difference in GRP78 mRNA expression. Conclusion At same fatty acid concentration, the larger propotion palmitic acid has, the more severe injury hepatocyte has. The regulation of palmitic acid on intra-hepatic ER stress is a time and dose dependent manner. TUDCA may improve palmitic acid induced ER stress to some degree.