雷帕霉素对氨基半乳糖／脂多糖诱导急性肝损伤小鼠保护作用及其机制

Protective mechanism of rapamycin on D - amino galactoose/lipopolysaccharide - induced acute liver injury in mice

目的探讨雷帕霉素对氨基半乳糖（Gal-N）/脂多糖（LPS）诱导急性肝损伤小鼠保护作用及其机制。方法45只C57/b6小鼠经腹腔注射Gal-N（700 mg/kg）和LPS（10 μg/kg）建立急性肝损伤模型，并随机分为模型组、低剂量组、中剂量组和高剂量组。同时选取15只小鼠作为对照组。低剂量组、中剂量和高剂量组在建模20 min后分别经腹腔注射5、10、20 mg/kg。对照组和模型组经腹腔注射等体积的磷酸盐缓冲液（PBS）。两组在治疗4 h后眼球取血检测天冬氨酸氨基转移酶（AST）和丙氨酸氨基转移酶（ALT）的水平；采用酶联免疫吸附试验（ELISA）试剂盒检测肿瘤坏死因子-α（TNF-α）的水平；采用苏木素-伊红（HE）染色检测各组小鼠肝组织病理学变化；采用Western blot分析小鼠肝脏组织中自噬底物微管相关蛋白1轻链3（LC3）和p62的水平。结果与对照组AST和ALT[（30.41±5.23） U/L和（110.23±12.02）]比较，模型组、低、中和高剂量组小鼠血清AST和ALT水平显著升高[（489.39±35.65） U/L和（310.24±41.25） U/L；（385.21±30.59） U/L和（278.96±35.68） U/L；（279.94±35.14） U/L和（214.37±34.12）；（159.24±30.12） U/L和（168.63±28.32） U/L]，差异有统计学意义[（t＝8.120，t＝6.384）；（t＝7.013，t＝5.132）；（t＝5.198，t＝4.318）；（t＝3.957，t＝3.048），P均＝0.000]。与模型组比较，低、中和高剂量组小鼠血清AST和ALT水平显著下调，差异有统计学意义[（t＝3.012，t＝3.419）；（t＝3.901，t＝4.012）；（t＝4.820，t＝5.018）, P均＝0.000]。与对照组TNF-α水平[（150.44±23.19） ng/L]比较，模型组、低、中和高剂量组小鼠TNF-α水平显著升高[（509.44±49.32）、（401.43±38.09）、（298.44±29.41）、（218.29±24.21） ng/L]，差异有统计学意义（t＝6.109，t＝5.184，t＝4.109，t＝3.921，P均＝0.000）。与模型组比较，低、中和高剂量组小鼠TNF-α水平显著下调，差异有统计学意义（t＝2.194，t＝3.331，t＝5.016，P均＝0.000）。HE染色显示模型组小鼠肝脏严重受损，肝脏红细胞弥散，肝细胞间隙增加，空泡变形，肝索排列紊乱。随着雷帕霉素剂量的增加，小鼠肝脏病理性损伤程度呈剂量依赖性缓解。与对照组比较，模型组自噬被抑制，经雷帕霉素处理后细胞自噬水平明显增强。结论雷帕霉素通过抑制哺乳动物雷帕霉素靶蛋白激酶活性，诱导自噬发生，降低TNF-α，达到了保护肝脏的目的。

Objective To investigate the protective mechanism of rapamycin （Rapa） on D-amino galactoose （Gal-N）/lipopolysaccharide （LPS）-induced acute liver injury in mice.Methods The acute liver injury model was established by intraperitoneal injection of Gal-N （700 mg/kg） and LPS （10 g/kg） in 45 C57/B6 mice and the animals were randomly divided into model group, low dose Rapa group, middle dose Rapa group and high dose Rapa group. At the same time, 15 mice were selected as control group. The mice in low dose Rapa group, medium dose Rapa group and high dose Rapa group were injected with 5, 10 and 20 mg/kg Rapa at 20 min after modeling respectively. The mice in control group and model group were intraperitoneally injected with equal volume of phosphate buffer （PBS）. Aspartate aminotransferase （AST） and alanine aminotransferase （ALT） levels of serum were determined. The tumor necrosis factor-α （TNF-α） levels were analyzed by enzyme linked immunosorbent assay （ELISA） Kit. The pathological changes in liver tissues of mice were observed by hematoxylin and eosin （HE） staining. Autophagy substrates microtubule-associated protein 1 light chain 3 （LC3） and p62 were analyzed by Western blotting.Results Compared with AST and ALT of control group [（30.41±5.23） U/L and （110.23±12.02）], serum AST and ALT levels in model group, low, middle and high dose group [（（489.39±35.65） U/L and （310.24±41.25） U/L）; （385.21±30.59） U/L and （278.96±35.68） U/L; （279.94±35.14） U/L and （214.37±34.12）; （159.24±30.12） U/L and （168.63±28.32） U/L]significantly increased [ （t=8.120, t=6.384）; （t=7.013, t=5.132）; （t=5.198, t=4.318）; （t=3.957, t=3.048）; P=0.000]. Compared with the model group, the serum AST and ALT levels in the low, middle and high dose groups significantly reduced [ （t=3.012, t=3.419）; （t=3.901, t=4.012）; （t=4.820, t=5.018）, P=0.000]. Compared with TNF-alpha in the control group [（150.44±23.19） ng/L], the level of TNF- α in the model group, low, middle and high dose group [（509.44±49.32）, （401.43±38.09）, （298.44±29.41）, （218.29±24.21） ng/L] significantly increased （t=6.109, t=5.184, t=4.109, t=3.921, P=0.000）. Compared with the model group, the TNF-α levels in the low, middle and high dose groups significantly reduced （t=2.194, t=3.331, t=5.016, P=0.000）. HE staining showed that the liver of model group mice was seriously damaged, the liver red blood cells diffused, the intercellular space of liver cells increased, the vacuoles deformed and the hepatic cords arranged irregularly. With the increase of rapamycin dose, the pathological damage degree of liver in mice was dose-dependent. Compared with the control group, autophagy in the model group was inhibited, and the autophagy level obviously enhanced after rapamycin treatment.Conclusion Rapa can induce autophagy and decrease TNF-α by inhibiting the activity of mammalian target of Rapa kinase, thus protecting the liver.