动态PH研究缺血预调适和后调适对骨骼肌缺血再灌注损伤的影响及其应用

Ischemic preconditioning(IPC) and postconditioning(PostC) can elicit powerful endogenous protection, which greatly decrease ischemia reperfusion injury(IRI) to involved tissues and organs. Since the two phenomena were first reported in succession, a number of experiments on various tissues and species have been carried out by different investigators to verify its effects and to explore its mechanisms which are not yet completely elucidated to date. A number of studies revealed that IPC and PostC both evoked the protection via triggering and activating the reperfusion injury salvage kinase(RISK) pathway. Recently, several studies showed that a burst of reactive oxygen species and the abrupt change of metabolic acidosis state played key roles in the IRI at onset of reperfusion, which precipitated the opening of mitochondrial permeability transition pore and therefore led to cells death. Our hypothesis is that IPC and PostC decrease IRI via impeding the abrupt PH change，and thus activate key kinases in RISK pathway. This project is to investigate the effects of IPC and PostC on PH change in skeletal muscle during ischemia reperfusion, and its protective effects against IRI. Our aim is to determine whether IPC and PostC induce its protection against IRI via regulating PH change on reperfusion meanwhile activating the key kinases in RISK pathway by methods of dynamically monitoring PH change and detecting expression and activation of key kinases in RISK pathway. Finally, we formulate analogous perfusates basing on the dynamic PH curves and test its protective effect against IRI. Our study of this project may form experimental basis for future clinical application of formulated perfusates in surgical situations of IRI.

缺血预调适和后调适能引发机体强大内源性保护，大大降低组织器官的缺血再灌注损伤。此现象被发现以来得到不少实验证实但其相关机制尚未完全阐明。既往研究显示，缺血预调适和后调适都可通过再灌注损伤存活激酶（RISK）通路发挥其诱导保护作用；而缺血再灌注后大量活性氧族的产生以及酸性微环境的突发改变是再灌注损伤的重要因素，可促使线粒体通透转换孔开放引起细胞死亡。我们假设缺血预调适和后调适可能影响这一环节而降低缺血再灌注损伤。本项目探讨缺血预调适和后调适对骨骼肌缺血再灌注PH值的影响及其在缺血再灌注损伤中的保护作用，通过动态检测缺血预调适和后调适PH值的变化,同时检测RISK通路的关键酶及其与PH变化的相关性，从组织病理学、生物化学和分子生物学等多方面分析了解缺血预调适和后调适是否通过调节PH变化而诱发对缺血再灌注损伤的保护作用，并根据PH值动态曲线模拟配制灌注液验证其保护作用，为临床实践提供理论依据。

缺血预调适(IPC)和后调适(PoC)能引发强大内源性保护对抗组织器官缺血再灌注损伤(IRI)，其相关机制目前尚未完全阐明。既往研究显示，缺血再灌注后大量氧自由基的产生以及酸性微环境的突发改变是再灌注损伤的重要因素，可促使线粒体通透转换孔(mPTP)开放引起细胞死亡。研究认为，再灌注损伤存活激酶（RISK）信号通路的激活是IPC和PoC引发保护作用的重要机制，最终作用于mPTP抑制其开放。本项目研究首先验证IPC和PoC对大鼠肢体骨骼肌IRI的保护效果并筛选最优方案。然后动态检测优选IPC和PoC引发的PH改变并探究其在骨骼肌IRI保护中的作用。同时，检测RISK信号通路关键酶的表达及活化以及mPTP开放状态。结果显示，优选PoC组骨骼肌组织PH动态测量曲线相较于缺血再灌注对照组，其于再灌注初期出现一个酸性延滞平台，pH为6.81±0.133，时长为2min 40sec；而优选IPC组再灌注初期PH检测未见酸性延滞平台。与缺血再灌注对照组相比，优选PoC组RISK通路关键酶总Akt、ERK1/2、以及eNOS表达变化均不明显(P >0.05)，但磷酸化的Akt、ERK1/2、eNOS-S1177表达均明显增加(P < 0.05)，而磷酸化的eNOS-Thr495表达显著降低(P < 0.05)。Ca2+诱导mPTP开放实验结果显示优选PoC限制其开放。这些结果表明RISK通路机制参与PoC对大鼠骨骼肌IRI保护作用。最后模拟优选PoC 组PH动态曲线配制酸性灌注液输入大鼠骨骼肌IRI模型，测试其保护作用。结果发现，模拟酸性灌注液输注同样具有对抗IRI的保护作用，再经RISK信号通路关键酶的表达及活化检测发现，其结果类似于PoC作用的结果。推测认为，PoC引起的再灌注初期PH改变可能参与RISK信号通路的激活,但其相关性及机理仍需深入研究。总之，IPC和PoC均具有对抗大鼠肢体骨骼肌IRI的保护效果但有方案差异性。模拟优选PoC方案再灌注初期PH曲线输注酸性灌注液同样具有对抗大鼠骨骼肌IRI的保护效果。本项目研究结果将对骨科临床上大量出现的与IRI密切相关的骨骼肌坏死和功能障碍于再灌注初期应用配制酸性灌注液进行防治有重要启迪意义。