分子氢调节RIP或（和）线粒体PGAM5/Drp1蛋白抑制缺血再灌注引起的皮肤组织细胞程序性坏死

Skin tissue transplantation such as skin flap transplantation is a routine practice for wound coverage and reconstruction in plastic surgery. Transplanted skin tissue necrosis is a serious problem in clinical practice. Ischemia reperfusion (I/R) injury is the main reason, which induces the skin tissue necrosis and finally causes surgery failure. The recommended mechanism of I/R injury include reactive oxygen species (ROS) accumulation, neutrophil recruitment, NO depletion and mitochondrial alteration. Recent evidences from I/R model of brain, heart and renal indicate that the programmed necrosis (necroptosis) is another contributor to I/R-induced cell death. Thus, strategies which target to prevent I/R injury and reduce the programmed necrosis may protect transplanted skin flap at a certain extent. In 2007, Ohsawa et al reported that inhalation of H2 gas could selectively mitigate oOH, generating an antioxidant effect in a rat model of middle cerebral artery occlusion without affecting the signaling of other ROS. Subsequently, H2 was shown the protective effects on I/R injury in various organs include skin. The mechanism studies about this effect mainly focus on the anti-oxidative and anti-inflammatory property of H2 before. Little data have been published in the view of cell programmed necrosis.In our previous work, we found that H2-saturated saline increased the surviving areas and blood flow perfusion of rat epigastric adipocutaneous flaps and depressed inflammation and cell aptosis in these tissues. However, there are still some issues to be clarified. TNF-α is an inducement factor during the occurrence and development of cell necrosis, and our previous results showed that H2 significantly reduce the TNF-α level in skin flap tissue, indicating that there may be a complex mechanism underlying the protective effect of H2 on the skin flap I/R injury. On the other hand, our recent experiments indicated that receptor interacting protien (RIP) may also involve in I/R skin necrosis and H2 treatment may also interfere its tissue expression. So we deduced that mechanisms which are related with programmed necrosis may be involved in this effect. In this work, we will continue our investigate in the effect of H2 on the necrosis signal regulating pathway. First, we will identify the anti-necrosis ability of H2 in skin flaps by analyzing HMGB1 and Cyclophilin A staining and transmission electron microscope observation. Next, RIP1, RIP3, Phosphoglycerate mutase family member 5(PGAM5), Dynamin-1-like protein (Drp1) and other factors will be studied, which is involoved in necerosis regulating pathway. The primary objective of the present study is to evaluate the anti-necrosis effect of H2 in skin tissue transplantation with abdominal skin flap model, and investigate the alteration in necrosis signal transduction, eventually explore the exact mechanism underlying necrosis protective effect of H2.

皮瓣移植在创面修复和器官再造中应用广泛。在移植过程中，皮瓣往往因缺血再灌注（I/R）损伤发生坏死。近年来，分子氢在防治移植组织器官I/R损伤中的作用开始引起关注，其作用机制也成为研究热点。在既往研究中，我们发现分子氢对I/R损伤引起的皮瓣组织细胞凋亡具有抑制作用，但一些I/R损伤后皮瓣组织却没有细胞凋亡表现，因此，用抗凋亡解释其机制仍不完善。通过进一步研究，我们发现，这部分皮瓣组织的I/R损伤可能和细胞的程序性坏死有关。基于分子氢促进皮瓣成活，抑制皮瓣组织细胞程序性坏死和RIP表达等的前期研究，本项目将进一步从抑制皮肤组织细胞程序性坏死角度探讨分子氢促进皮瓣成活的机制。项目首先通过HE染色和电镜分析等确定分子氢对皮肤组织细胞程序性坏死的抑制作用，然后进一步通过对RIP、线粒体PGAM5/Drp1蛋白等重要程序性坏死调控物质的检测和酶活性分析，阐明分子氢抑制皮肤I/R细胞程序性坏死的机制。

项目背景：.皮瓣移植在创面修复和器官再造中应用广泛。在移植过程中，皮瓣往往因缺血再灌注（I/R）损伤发生坏死。近年来，分子氢在防治移植组织器官I/R损伤中的作用引起越来越大的关注，其作用机制也成为研究热点。在既往研究中，我们发现分子氢对I/R损伤引起的皮瓣组织细胞凋亡具有抑制作用，但一些I/R损伤后皮瓣组织却没有细胞凋亡的表现，因此，用抗凋亡解释其机制仍不完善。通过进一步的深入研究，我们推断细胞的程序性坏死与I/R损伤密切相关。.主要研究内容：.本课题的研究主要分为两大部分：.1.观察了分子氢处理后，SD大鼠腹部缺血再灌注损伤皮瓣细胞程序性坏死发生情况，进一步确认分子氢对细胞程序性坏死通路RIP- MLKL- PGAM5/Drp1。.2.观察了RIP1 抑制剂（Necrostatin-1）对SD大鼠腹部缺血再灌注损伤皮瓣组织细胞坏死的抑制效果及相关调控信号表达。.研究结果：.实验发现，分子氢处理后能够促进缺血再灌注皮瓣的存活，提高皮瓣的平均血流量。分子氢处理组中的皮瓣存活面积比率及平均血流量较对照组有显著提升。.1. 分子氢处理组皮瓣组织的细胞程序性坏死水平明显低于对照组，在细胞程序性坏死的发生发展中起主要调控作用的RIP-1、RIP-3、MLKL、PGAM5、Drp1，经分子氢处理后，与对照组相比，各指标的表达量均有所下调。.2. RIP-1 抑制剂（Necrostatin-1）处理组的细胞程序性坏死比率较对照组有了显著降低，分子氢处理组的RIP-1（1.3529±0.0317）的表达量较对照组有明显的下降，但凋亡蛋白酶Caspase-3（1.0457±0.3646）的活性较对照组无明显变化。.研究意义：.本研究从细胞程序性坏死途径，讨论了经分子氢处理提升皮瓣成活质量的机制。明确了分子氢处理提升大鼠缺血再灌注损伤皮瓣成活质量，与降低细胞程序性坏死水平有直接联系。抑制RIP-1、RIP-3、MLKL、PGAM5、Drp1的表达，减轻缺血再灌注皮肤组织细胞程序性坏死，可能是分子氢改善皮瓣成活状况的关键机制之一。这为分子氢治疗的机理研究提供了更为深入的研究基础。