3-甲基腺嘌呤通过抑制自噬和内质网应激途径保护睡眠紊乱诱导的大鼠肝损伤

3-methyladenine prevents sleep disturbance-induced liver injury by suppressing autophagy and endoplasmic reticulum stress

摘要:

目的 观察与分析3-甲基腺嘌呤（3-methyladenine，3-MA）对睡眠紊乱诱导的大鼠肝损伤的保护作用及其相应的机制。方法 将30只雄性SD大鼠随机分为对照组、模型组和3-MA组，每组10只，对比各组大鼠实验前后体质量变化。处死大鼠后进行以下实验分析：①采用ELISA法检测各组大鼠血清丙氨酸转氨酶（alanine aminotransferase，ALT）与天冬氨酸转氨酶（aspartate aminotransferase，AST）水平变化；②运用HE观察肝脏组织形态学的改变；③使用透射电子显微镜观察各组大鼠肝细胞中自噬体的形成；④采用TUNEL法评估各组肝细胞的凋亡水平，并计算凋亡指数；⑤通过RT-PCR与Western blot检测肝组织自噬相关蛋白微管相关蛋白轻链3（LC3-Ⅱ）、Beclin1、葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）及Caspase-3的mRNA与蛋白表达水平。结果 动物实验结果表明睡眠紊乱会诱导大鼠肝损伤，同时伴随肝脏自噬与内质网应激（endoplasmic reticulum stress， ERS）水平升高；相比对照组，模型组大鼠造模后体质量增量明显减小，而肝细胞形态结构变化明显，同时，血清ALT与AST水平、凋亡指数、自噬体数量及相关标志性蛋白表达水平均明显上升（P<0.05）；相比模型组，肝细胞形态结构、血清ALT与AST水平、凋亡指数、自噬体数量及相关标志性蛋白表达水平均有改善（P<0.05）。结论 睡眠紊乱可诱导SD大鼠肝损伤，进而通过上调LC3-Ⅱ、Beclin1及GRP78蛋白表达和mRNA水平激活自噬与ERS信号通路，3-MA可拮抗自噬与ERS信号通路来保护肝细胞及抗凋亡作用。

Abstract:

Objective To explore the protective effect and corresponding mechanisms of 3-methyladenine（3-MA） on liver injury induced by sleep disturbance.Methods Thirty SD mice were randomly divided into three groups，control group， modeling group，and 3-MA group， with 10 mice in each group. After the end of exposure， the weight changes in mice were measured and compared among the groups before and after the experiments. After the mice were sacrificed， the following experiments were made. ①The level changes of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were detected by ELISA method. ②HE staining was conducted to observe the histomorphological changes of the liver. ③Autophagosomes were analyzed using transmission electron microscope. ④TUNEL method was used to evaluate the apoptosis level of hepatocytes in each group and calculate the apoptosis index. ⑤RT-PCR and Western blot methods were used to detect the mRNA and expression levels of autophagy-related proteins，such as microtubule-associated protein light-chain-3 （LC3-Ⅱ），Beclin1，glucose-regulated protein 78（GRP78），and Caspase-3.Results The animal experiments showed that sleep disturbance could induce the liver damage，and which was accompanied with the increase of autophagy and endoplasmic reticulum stress（ERS）. Compared with the control group，the body weight increment of the model group decreased significantly，while the morphological structure of hepatocytes remarkably changed. Meantime，the levels of ALT and AST，apoptosis index，autophagosomes and the expression of related marker proteins increased respectively（P<0.05）. In contrast with the model group，the morphological structure，serum ALT and AST，apoptosis index，autophagosomes and the expression of related marker proteins were improved（P<0.05）.Conclusion Sleep disturbance exposure can induce liver damage in SD rats，and then further activate autophagy and ERS signaling pathways by up-regulating the protein expression and mRNA level of LC3-Ⅱ，Beclin1 and GRP78. 3-MA can antagonize autophagy and ERS signaling pathways to protect hepatocytes and resist apoptosis.