AMPK通过调控mTOR/Nix信号通路参与小鼠肝缺血-再灌注损伤

AMPK regulates murine hepatic ischemia-reperfusion injury via mTOR/Nix signaling pathway

目的探讨腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)在肝脏缺血-再灌注损伤(hepatic ischemia-reperfusion injury,HIRI)中的作用机制。方法 (1)分组。将42只小鼠随机分为假手术组(Sham组)、4个时点缺血-再灌注(IR)组(2、6、12及24 h)、Compound C组及Compound C+雷帕霉素(Rapa)组,每组6只小鼠。Compound C组:腹腔注射Compound C(25 mg/kg)1 h后,建模。Compound C+Rapa组:腹腔注射Rapa(1 mg/kg)和Compound C(25 mg/kg)1 h后,建模。4个时点IR组、Compound C组和Compound C+Rapa组小鼠制备HIRI模型。Sham组小鼠仅行开腹、游离第一肝门以及关腹操作。(2)IR最佳时点筛选。检测Sham组和4个时点IR组小鼠血清中的丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)浓度,取肝组织行HE染色观察肝组织的病理学改变,并采用蛋白印迹法检测小鼠肝组织中自噬、凋亡相关蛋白的表达。综合血生化检测结果、HE染色结果和蛋白印迹实验结果确定IR最佳观察时点。(3)AMPK机制研究。取IR-12 h组、Compound C组(建模后12 h)和Compound C+Rapa组(建模后12 h)小鼠肝脏组织,行免疫组化染色观察增殖细胞核抗原(PCNA)的表达,行罗丹明123染色观察线粒体损伤情况,行蛋白印迹实验检测自噬、凋亡相关蛋白的表达,行末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL法)检测肝脏细胞的凋亡情况。结果 (1)IR后12 h为最佳观察时点。与IR-12 h组比较,Sham组,IR-2、6及24 h组的ALT和AST浓度均较低(P<0.05);HE染色结果示IR-12 h组小鼠的肝组织结构破坏最为严重;蛋白印迹实验结果示,与IR-12 h组比较,Sham组,IR-2、6及24 h组的AMPKα、磷酸化型腺苷酸活化蛋白激酶α(p-AMPKα)、Nip3样蛋白X(Nix)及BCL-2同源的水溶性相关蛋白(Bax)的表达水平,以及自噬微管相关蛋白轻链3(LC3)Ⅱ/LC3Ⅰ比值均较低(P<0.05),而磷酸化型哺乳动物Rapa靶蛋白(p-mTOR)的表达水平均较高(P<0.05)。因此IR 12 h为最佳观察时点。(2)与IR-12 h组比较,Compound C组小鼠肝组织中PCNA的表达水平较低(P<0.05),线粒体发光强度较弱,凋亡细胞较多;与Compound C组比较,Compound C+Rapa组小鼠肝组织中PCNA的表达水平较高(P<0.05),线粒体发光强度较强,凋亡细胞较少。(3)与IR-12 h组比较,Compound C组小鼠肝组织中Nix及p-AMPKα的表达水平和LC3Ⅱ/LC3Ⅰ比值均降低(P<0.05),而p-mTOR、含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)及分裂型含半胱氨酸的天冬氨酸蛋白水解酶-3(Cleaved Caspase-3)的表达水平均升高(P<0.05);与Compound C组比较,Compound C+Rapa组小鼠肝组织中p-AMPKα和Nix的表达水平均升高(P<0.05),而p-mTOR、Caspase-3和Cleaved Caspase-3的表达水平均降低(P<0.05)。结论在小鼠HIRI过程中,AMPK通过m-TOR/Nix通路调节线粒体自噬和凋亡。

Objective To investigate the mechanism of AMP-activated protein kinase （AMPK） in hepatic ischemia-reperfusion injury （HIRI）. Methods （1） Grouping. Forty-two mice were randomly divided into Sham group, 4 ischemia reperfusion （IR） group of different times （2, 6, 12, and 24 h）, Compound C group, and Compound C＋repamycin （Rapa） group, each group enrolled in 6 mice. Compound C group： mice were modeled at 1 h after intraperitoneal injection of Compound C （25 mg/kg）. Compound C＋Rapa group： mice were modeled at 1 h after intraperitoneal injection of rapamycin （1 mg/kg） and Compound C （25 mg/kg）. Mice of 4 IR groups, Compound C group, and Compound C＋Rapa group were used to prepare HIRI model. Mice of Sham group were treated only for laparotomy, freeing the first portal hepatis and closing peritoneal. （2） To filter the best IR time. The levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum of mice in Sham group and IR groups of 4 different reperfusion time points were measured. The pathological changes of liver tissues were observed by HE staining, and the expressions of related proteins in liver tissue of mice were detected by Western blot. Considering the results of blood biochemical test, HE staining, and Western blot together to determine the best IR point. （3） The exploration of signal pathway for AMPK. The expressions of proliferating cell nuclear antigen （PCNA） were observed by immunohistochemical staining in the liver tissues of IR-12 h group, Compound C group （12 h after IR） and compound C＋Rapa group （12 h after IR）. The mitochondrial damage was observed by rhodamine 123 staining, and the apoptotic status of liver cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay （TUNEL）. Results （1） The 12 h after IR was the best observation time point. Compared with IR-12 h group, the levels of ALT and AST in Sham group, IR-2, 6, and 24 h groups were lower （P〈0.05）. HE staining showed that liver tissue destruction in IR-12 h group was the most severe. Western blot showed that, expressions of AMPKα, phosphorylated adenylate activated protein kinase a （p-AMPKα）, Nip3-like protein X （Nix）, BCL-2 homologous water-soluble protein （Bax）, as well as ratio of autophagy microtubule- associated protein light chain 3 （LC3） Ⅱ to LC3 Ⅰ of Sham group, IR-2, 6, and 24 h group were all lower than those of IR- 12 h group （P〈0.05）, but the expressions of phosphorylated mammalian target of Rapa （p-mTOR） of Sham group, IR-2, 6, and 24 h group were all higher （P〈0.05）. Therefore, 12 h after IR was the best time to observe. （2） Compared with IR-12 h group, the expression level of PCNA protein in liver tissue of Compound C group was lower （P〈0.05）, the mitochondrial luminescence intensity was weaker and the apoptotic cells were more. Compared with Compound C group, the expression of PCNA protein in the liver tissue of the Compound C＋Rapa group was higher （P〈0.05）, the mitochondrial intensity was stronger and the apoptotic cells were less. （3） Compared with IR-12 h group, the expressions of Nix and p-AMPKα, and ratio of LC3 Ⅱ to LC3 Ⅰ in liver tissue of Compound C group decreased （P〈0.05）, while the expressions of p-roTOR, Caspase-3, and Cleaved Caspase-3 increased （P〈0.05）. Compared with Compound C group, the expressions ofp-AMPKα and Nix in the liver tissue of Compound C＋Rapa group increased （P〈0.05）, while the expressions of p-mTOR, Caspase-3, and Cleaved Caspase-3 decreased （P〈0.05）. Conclusion During the HIRI in mouse, AMPK regulates mitophagy and apoptosis through the mTOR/Nix pathway.