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

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。

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.