基于中医“祛瘀生新”理论研究消肿止痛合剂治疗皮瓣缺血再灌注损伤中p38MAPK-PPARγ/NF-κB信号通路作用机制及全基因表达谱分析

Ischemic reperfusion injury of skin flap is a common problem in the field of trauma repair. How to improve the survival rate of skin flap is one of the focus of research. In recent years, based on the theory of "removing blood stasis and new", traditional Chinese medicine has made some progress in the treatment of ischemia and reperfusion injury of skin flap. Earlier studies have found that the mixture of relieving swelling and relieving pain can reduce TNF- - alpha and IL-6 in soft tissue and prevent the release of inflammatory mediators after trauma. The p38MAPK-PPAR - gamma /NF- kappa B signaling pathway is closely related to the skin flap ischemia reperfusion injury, and is an important target for controlling oxidative stress and inflammatory response. Therefore, we hypothesized: the swelling swelling pain relieving mixture can make the p38MAPK-PPAR gamma /NF- kappa B signaling pathway express exactly in the process of oxidative stress or inflammatory reaction after skin flap transplantation, thus reducing ischemia reperfusion injury. This study uses model and cell model of rats with Xiaozhongzhitong mixture intervention, using gene chip detection results show the chip using the RT-PCR method, and analyze the changes of p38MAPK-PPAR gamma /NF- B pathway function rule and the key genes, transcription factors, through the analysis of the differences between chip genes and signaling pathways, revealing Xiaozhongzhitong mixture treatment of skin flap ischemia reperfusion injury in the mechanism of micro environment.

皮瓣缺血再灌注损伤是创伤修复领域的常见问题，如何提高皮瓣成活率始终是研究的焦点之一。近年来，基于“祛瘀生新”理论，中医药在皮瓣缺血再灌注损伤治疗方面取得了一定的研究进展。前期研究发现，消肿止痛合剂能降低软组织TNF-α和IL-6, 阻止外伤后炎症介质的释放。而p38MAPK-PPARγ/NF-κB信号通路与皮瓣缺血再灌注损伤密切关系，是控制氧化应激和炎性反应的重要靶点。因此我们假设：消肿止痛合剂能够使p38MAPK-PPARγ/NF-κB信号通路在皮瓣移植后氧化应激或炎症反应过程中精确表达，从而降低缺血再灌注损伤。本研究借助大鼠模型及细胞模型，以消肿止痛合剂干预，采用全基因芯片检测，使用RT-PCR方法验证芯片结果，并分析p38MAPK-PPARγ/NF-κB信号通路作用规律及关键基因、转录因子的变化，通过对芯片差异基因筛选及信号通路分析，揭示消肿止痛合剂治疗皮瓣缺血再灌注损伤微环境中的作

目的：探讨消肿止痛合剂对皮瓣缺血再灌注损伤的影响和基因表达谱的分析，完善消肿止痛合剂治疗皮瓣缺血再灌注损伤的作用机理。方法：SPF级SD大鼠60只随机分为六组（n=10）:假手术组、模型对照组、消肿止痛合剂组、p38MAPK抑制剂组、PPARγ抑制剂组，NF-κB抑制剂组。剃除大鼠背部术区毛发，在除假手术组外大鼠背部构建随意皮瓣（8cm×2cm），术后用3-0号缝合线原位缝合皮瓣。对大鼠形态学观察。western blot检测皮瓣组织p38MAPK、PPARγ、IκBα、NF-κB蛋白表达。用HE染色、TUNEL染色及qRT-PCR法检测大鼠皮瓣血管内皮细胞中炎性因子的浸润程度、细胞核的破坏程度以及p38MAPK、PPARγ、NF-κ B的分布特点及其mRNA表达水平。采用全基因芯片检测观察消肿止痛合剂对皮瓣缺血再灌注损伤基因表达谱的影响。采用红外热谱检测大鼠皮瓣的最高温度和最低温度；苏木精-伊红染色观察皮瓣水肿和白细胞浸润。结果：术后7d与模型组相比p38MAPK抑制剂组和NF-κB抑制剂组大鼠存活面积最大；消肿止痛合剂组、p38MAPK抑制剂组、PPARγ抑制剂组，NF-κB抑制剂组TNF-α、IL-6、ICAM-1均减少，消肿止痛合剂组大鼠皮瓣组织中PPARγ、IκBα表达升高、p38MAPK、NF-κB的表达减少。筛选差异基因生物功能包括白细胞迁移、炎症反应、免疫系统过程的正调控，免疫系统过程、免疫反应、中心粒细胞迁移等；参与的信号通路包括趋化因子和细胞因子信号通路介导的炎症、白细胞介素信号通路等。术后假手术组皮瓣存活面积最大，模型对照组与PPARγ抑制剂组皮瓣存活面积最小；HE染色与TUNEL染色结果模型对照组与PPARγ抑制剂组大鼠皮瓣组织细胞破坏严重，可见明显凋亡细胞，模型组大鼠皮瓣组织细胞呈单层排列，细胞核完整，清晰；qRT-PCR实验结果：与模型组相比，消肿止痛合剂组大鼠皮瓣组织中p38MAPK、NF-κB的表达被抑制（P<0.05），而PPARγ的表达有所升高（P<0.05），当加入阻断剂后，皮瓣组织中p38MAPK、NF-κB及PPARγ的表达进一步被抑制。结论：消肿止痛合剂明显减少皮瓣缺血再灌注损伤的炎症反应，有效的减少了TNF-α、IL-6、ICAM-1和p38MAPK、NF-κB的表达。