远端缺血预处理通过GSK-3β信号调控肝缺血再灌注损伤的作用机制研究

Hepatic ischemia/reperfusion (I/R) injury is a common complication during major hepatic surgeries such as hepatic transplantation and hepatectomy. It greatly slows postoperative recovery and is a burden to medical care. Remote ischemic preconditioning (RIPC) is a novel method where ischemia followed by reperfusion of one organ which is believed to protect remote organs either due to release of biochemical messengers in the circulation or activation of nerve pathways, resulting in release of messengers that have a protective effect. However the details of cycles and phases for effective protection of RIPC remain unknown, especially in hepatic I/R. ..As RIPC is a special kind of IPC, and direct IPC has already been identified to protect organ through multiple signaling pathways during I/R, the mechanism of RIPC in regulating hepatic I/R might be associated with glycogen synthase kinase 3β (GSK3β) which has been shown to be a convergence point for the RISK and SAFE pathways and a target to both potentiate prosurvival cascades and maximize organ protection. We have demonstrated the key role of GSK-3β signaling in mitochondrial metabolism modulation during hepatic I/R (Fu et al. J Hepatol. 2014,61:812). However, the effect of GSK-3β on RIPC-related hepatic I/R protection remains exclusive...Since limb ischemia preconditioning(LIP) has identified to be an easy method in RIPC to provide effective protection in heart, kidney during I/R, we intend to use the in vivo model to further investigate the details of cycles and phases for effective protection of LIP during hepatic I/R. The direct IPC will be used as a positive protective control. Also, by utilizing siRNA technique and using selective signaling inhibitors, we aim to identify the possible signaling pathways and responsible proteins regulated by GSK-3β during RIPC. Furthermore, as direct IPC failed to protect aging liver, we plan to test the protective role of LIP on aging animals during hepatic I/R. Indeed, understanding the mechanism of RIPC in modulating GSK-3β signaling during hepatic I/R is helpful for effective hepatoprotection strategy searching and is of potential clinical significance.

缺血再灌注(I/R)损伤是临床常见病理现象，可严重影响患者预后。远端缺血预处理（RIPC）是一种新的器官保护手段，但其在肝I/R中的作用仍存在争议。以往研究显示RIPC可通过神经、体液途径调控靶器官损伤。我们前期发现下肢RIPC能诱导再灌注早期肝细胞GSK-3β磷酸化，减轻肝损伤，但机制和作用时效仍不清楚。GSK-3β是细胞内重要调控信号，前期我们证实了其在肝I/R中的关键调控作用（J Hepatol. 2014,61:812）。我们推测GSK-3β信号可能同样参与了RIPC的肝I/R保护过程。鉴于此，本课题拟通过动物模型研究下肢RIPC调控GSK-3β的信号机制及肝保护效应。通过与经典的机械缺血预处理（IPC）比较，明确RIPC的最佳方式和时效；研究GSK-3β在其中的上下游调控机制及RIPC的关键效应靶点；最后，在IPC保护缺陷的老年模型中验证RIPC的有效性，为临床肝保护提供新思路。

I/R损伤是肝部分切除及肝移植等常见的临床病理现象，可严重影响患者预后。远端缺血预处理（RIPC）是一种新的器官保护手段。.研究第一阶段课题组利用大鼠70%肝热缺血/再灌注（I/R）损伤模型，筛选最佳下肢RIPC方式，发现缺血前即刻行5min缺血、5min再灌注循环4次的下肢RIPC方式肝保护效果最佳。在对肝I/R损伤机制的研究中，课题组发现：（1）使用Salidroside能减轻大鼠肝损伤，机制与其抗氧化及线粒体保护有关，成果发表于《Eur J Pharmacol》；（2）临床上通过随机对照研究发现苹果酸醋酸林格液有围术期肝缺血保护作用，成果发表中文核心期刊《肝脏》。.第二阶段课题组研究了下肢RIPC对I/R时GSK3β活性和上下游信号及细胞功能影响。使用大鼠肝I/R模型，选择5min缺血、5min再灌注循环4次的最佳下肢RIPC方式处理动物，发现：（1）再灌注不同时段RIPC可激活肝细胞内Akt-GSK3β和cAMP-ERK1/2信号，抑制JAK2-STAT3轴；（2）上游PGE2-EP4信号介导了RIPC调控肝损伤及GSK3β相关线粒体功能过程；（3）直接激动EP4信号能通过cAMP-ERK1/2-GSK3β轴调控线粒体膜通透性转换孔（MPTP）开放，减轻肝细胞坏死和凋亡，成果发表于《Int J Mol Med》。.第三阶段课题组研究了RIPC时外泌体介导下PGE2信号调控EP4及下游GSK-3β等相关信号机制，发现直接强化体液途径，即通过脂肪干细胞外泌体（ADSCs-exo）预处理，同样能起到类似于RIPC时抑炎、抗氧化、激活细胞内cAMP-ERK1/2-GSK3β通路、保护线粒体等作用，减轻肝损伤。 .最后，课题组在老年大鼠肝I/R模型上进一步验证RIPC的保护效果及机制，发现老年肝I/R时RIPC的肝保护作用主要是通过激活ERK1/2-GSK3β信号从而调控MPTP开放，减轻肝损伤。此外，老年大鼠RIPC时血浆外泌体水平激增，后续将进一步验证其作用。.综上，本研究进一步阐明了RIPC调控肝I/R损伤的分子机制，为围术期肝损伤的防治提供了新思路；同时，在当下我国人口老龄化的大趋势下，对老龄器官保护的研究具有应用前景。上述研究成果已于国内学术会议大会报告2次，培养硕士研究生2名，研究内容后期预计还将形成SCI论文1～2篇，并参加国内学术交流1～2次。