内质网应激在高脂饲养小鼠脂肪肝形成中的作用

The role of endoplasmic reticulum stress in the formation of fatty liver in high-fat diet fed mice

目的探讨内质网应激在高脂饲养小鼠脂肪肝形成中的作用。方法将8周雄性C57BL／6J小鼠随机分成两组：高脂饲养组（给予含60％卡路里的高饱和脂肪酸饲养）和正常对照组，饲养16周；使用高脂肪酸体外孵育原代肝细胞，模拟高脂状态；通过HE染色、苏丹Ⅳ染色反映肝组织脂质沉积情况；通过RT．PCR和Western blottling检测固醇调节元素结合蛋白（SREBP-1）和脂肪酸合成酶（FAS）基因和蛋白表达以反映肝细胞内脂质合成，检测葡萄糖调节蛋白78（GRP78）、蛋白激酶R样内质网激酶（PERK）、磷酸化真核细胞翻译启始因子2仅（eIF2ct）以及增强子结合蛋白同源蛋白（CHOP）表达以反映肝细胞内质网应激。结果肝组织HE染色可见高脂组肝细胞胞浆内充满大量脂肪空泡，苏丹Ⅳ染色可见肝细胞内存在大量大小不一的红色脂滴；过氧化物酶体增殖激活受体OL（PPARα）的mRNA水平明显高于正常对照组。与正常对照组比较，高脂组肝脏内质网应激的标志蛋白GRP78、PERK、磷酸化eIF2α和CHOP等表达也明显上调。另外，用脂肪酸如油酸孵育原代培养的正常肝细胞24-72h，亦呈时间依赖性地上调GRP78、PERK、磷酸化eIF2α和CHOP等蛋白表达，并伴有SREBP-1和FAS蛋白表达增加。结论长期高脂饲养小鼠可导致肝脏脂肪合成与沉积增加，其机制可能与肝脏内质网应激有关。

Objective To explore the influence of endoplasmic reticulum stress on fatty liver in mice feeding with high-fat diet. Methods The 8-week-old male C57BL/6J mice were randomly divided in- to two groups： high-fat diet group （with 60% calories by high saturated fatty acid） and control group （with chow diet） , both groups had been fed for 16 weeks. H＆E-staining and Sudan 1V-staining reflected lipid deposition in liver. The levels of 78-kDa glucose-regulated protein （ GRP78 ） , protein kinase R-like endo- plasmic reticulum kinase （PERK）, phosphorylated ct subunit of eukaryotic initiation factor 2 （p-eIFla）, C/EBP homologous protein （ CHOP）, steroid regulated element binding proteins 1 （ SREBP-1 ）, and fatty acid synthetase （FAS） protein were determined by Western blot to reflect the endoplasmic reticulum stress and lipid synthesis. Results In liver of high fat diet （HFD） group, H＆E staining showed that the cyto- plasm of hepatocytes were filled with vacuoles, Sudan IV staining also displayed that many different sizes of red lipid drops exist in hepatocytes. Compared to the liver of control group, high-fat diet induced endoplas- mie reticulum stress and elevated lipid synthesis, as evidenced by increases in the level of peroxisome prolif-erator-activated receptor alpha （PPARct） mRNA expression, and the protein levels of GRP78, PERK, phosphorylated eIF2α, CHOP were also significantly increased. In primary normal hepatocytes incubated with exogenous oleic acid intervention for 24 - 72 hours, the expression of GRP78, PERK, phosphorylated eIF2α, CHOP protein levels, and the expression of SREBP-1 and FAS protein were significantly increased in dose-dependent manner. Conclusions Feeding with high-fat diet led to accumulation of lipid deposition in liver and fatty liver, the underlying mechanisms might be related to induction of endoplasmic reticulum stress.