Isoflurane Preconditioning Alleviated Murine Liver Ischemia and Reperfusion Injury. by Restoring AMPK/mTOR-Mediated Autophagy
Isoflurane Preconditioning Alleviated Murine Liver Ischemia and Reperfusion Injury. by Restoring AMPK/mTOR-Mediated Autophagy
复制标题
DOI：
10.1213/ane.0000000000002385
复制
发表时间：
2017-10-01
影响因子：
5.7
通讯作者：
Ding, Zhengnian
中科院分区：
文献类型：
--
作者：
Rao, Zhuqing;Pan, Xiongxiong;Ding, Zhengnian
背景：异氟醚对心脏、肾脏和脑的缺血损伤具有药理预适应作用，但异氟醚预适应是否以及如何保护肝脏免受缺血再灌注(IR)损伤尚不清楚。方法：小鼠随机分为异氟醚预适应(ISO)组和对照组，分别给予1.5%异氟烷或载气40min(n=8/组)。采用部分温热肝脏IR模型，评价肝损伤程度。原代肝细胞用1.5%异氟烷预处理2小时，然后用过氧化氢诱导细胞死亡。用乳酸脱氢酶和细胞计数试剂盒-8检测细胞死亡和存活。用免疫印迹法(n=6/组)分析应激肝和肝细胞中的自噬和调节分子。在体内(n=8组)和体外(n=6组)分别给予自噬抑制剂3-甲基腺嘌呤(3-MA)和5‘-腺苷单磷酸活化蛋白激酶(AMPK)抑制剂(n=6)。结果：与对照组相比，ISO组小鼠肝脏损伤明显减轻(丙氨酸氨基转移酶[ALT]水平，对照组比ISO组，8285769vs4896+/-917U/L，P<；.001)。此外，肝脏自噬被ISO恢复，表现为伴随着p62降解增加的Lc3B II蛋白水平升高。原代肝细胞的体外研究还发现，ISO有效地减轻了过氧化氢诱导的肝细胞死亡。此外，3-MA对对照组无明显影响，但取消了ISO对应激肝(ALT水平，磷酸盐缓冲液+ISO与-3-MA+ISO组，5081+/-294vs8663+/-607-U/L，P<；0.001)和肝细胞的保护作用。最后，信号通路分析表明，AMPK被ISO激活。用An进行预处理。AMPK抑制剂也可阻断ISO对肝脏的保护作用(ALT水平，磷酸盐缓冲液+ISO与多索吗啡+ISO组，5081+/-294vs8710+/-500U/L，P<；.001)，而对照小鼠无明显作用。结论：异氟醚预适应可通过AMPK/mTOR介导的肝细胞自噬修复减轻肝脏IR损伤。我们的发现为治疗肝脏IR损伤提供了一种新的潜在的治疗策略。
BACKGROUND: Isoflurane has a pharmacological preconditioning effect against ischemia injury in the heart, kidney, and brain, but whether and how isoflurane preconditioning protects livers against ischemia and reperfusion (IR) injury is unclear.METHODS: Mice were randomly divided into an isoflurane preconditioning (ISO) group and control group, receiving 1.5% isoflurane or carrier gas for 40 minutes, respectively (n = 8/group). A partial warm liver IR model was used, and liver injury was evaluated. Primary hepatocytes were pretreated with 1.5% isoflurane for 2 hours before the induction of cell death by hydrogen peroxide. Cell death and survival were evaluated with the lactate dehydrogenase and cell counting kit-8 assay. Autophagy and regulatory molecules in stressed livers and hepatocytes were analyzed by Western blot (n = 6/group). An autophagy inhibitor (3-methyladenine [3-MA]) and 5' adenosine monophosphate-activated protein kinase (AMPK) inhibitor (dorsomorphin) were administered in vivo (n = 8/group) and in vitro (n = 6/group).RESULTS: Compared to that observed in the control group, mice in the ISO group showed reduced liver injury (alanine aminotransferase [ALT] levels, control versus ISO group, 8285 769 vs 4896 +/- 917 U/L, P < .001) and enhanced hepatocellular antiapoptosis in livers after IR. Furthermore, liver autophagy was restored by ISO as indicated by elevated LC3B II protein levels accompanied with increased p62 degradation. The in vitro study of primary hepatocytes also found that ISO effectively attenuated hepatocyte cell death induced by hydrogen peroxide. In addition, 3-MA pretreatment showed no significant influence in the control group, but abrogated the protective role of ISO both in stressed livers (ALT levels, phosphate-buffered saline + ISO versus-3-MA + ISO group, 5081 +/- 294 vs 8663 +/- 607-U/L, P < .001) and in hepatocytes. Finally, signaling pathway analysis demonstrated that AMPK was activated by ISO. Pretreatment with an. AMPK inhibitor also abrogated liver protection by ISO (ALT levels, phosphate-buffered saline + ISO versus dorsomorphin [DOR] + ISO group, 5081 +/- 294 vs 8710 +/- 500 U/L, P < .001), with no significant effect in control mice.CONCLUSIONS: Our results indicate that isoflurane preconditioning attenuates liver IR injury via AMPK/mTOR-mediated hepatocellular autophagy restoration. Our findings provide a novel potential therapeutic strategy for managing liver IR injury.