益气活血方调控TLR2/TLR4介导缺血再灌注肾损伤作用及机制研究

Renal ischemia reperfusion injury（RIRI）often lead to acute kidney injury（AKI）,recently reports showed that toll-like receptors (TLR)-mediated inflammation is a key link in the pathogenic process.In our study, TLR2, TLR4 and MyD88 knockout mice are used to establish RIKI-AKI model,Laser capture microdissoction in situ hybridization and immunofluorescence double-labeled confocal laser technology are also used to research the mechanism of TLR2 and TLR4-mediated renal damage ,to study the regulate effect of Yiqi Huoxue Fang to TLR2 and TLR4 in vivo. And as for in vitro，the primary cultured renal tubular epithelial cells which is separated by microdissected are applied to explore the signaling pathway of MyD88 dependent or MyD88 independent TLR2/4expression, by knocking out TLRs or blocking the signaling pathway，so as to study the regulate effect of Yiqi Huoxue Fang to TLR2 and TLR4 pathway for the first time. Our research will clarify the mechanism of TLR2/4-mediated RIRI-AKI, and will prove the traditional Chinese medicine can regulate TLR2 / 4 mediated the RIKI-AKI, and further, we will explore the molecular mechanism of TLR2/4 mediated RIKI-AKI, to provide the clinical and theoretical basis for the best time of interference and therapeutic targets.

缺血再灌注（RIKI）常引发急性肾损伤（RIKI -AKI），新近报道Toll样受体（TLR）介导的炎症反应是关键致病环节。本研究用TLR2、TLR4和MyD88基因敲除鼠建立RIKI -AKI模型，采用激光切割微分离技术、原位杂交、免疫荧光双标激光共聚焦技术等在体研究TLR2和TLR4介导肾损害的机制，研究益气活血方对TLR2和TLR4的调控；体外应用微分离原代培养的肾小管上皮细胞，通过TLRs基因敲除及特异信号分子阻断方法，研究TLR2与TLR4表达依赖MyD88或非MyD88（TRIF）信号途径,首次研究益气活血方对TLR2与TLR4信号通路的作用。研究结果将解析TLR2和TLR4介导RIKI -AKI的机制，创新性证实中药复方可以调控TLR2/4介导的的RIKI -AKI，阐明中药复方调控TLR2/4介导RIKI -AKI的分子机制，为临床干预靶点及最佳时机提供实验依据。

肾缺血再灌注损伤（IRI）是急性肾损伤最重要机制之一。新近报道IRI与TLRs受体激活炎症相关。本研究建立IRI模型，观察不同时间肾损伤及修复、Toll样受体2 (TLR2)和Toll样受体4 (TLR4)及相关炎症因子；Toll样受体信号传导通路中髓样细胞分化因子(MyD88)表达变化，研究益气活血方（复方肾华片，简称SHT）对IRI模型的保护效应及作用机制。将动物分为5组：假手术组(Sham)，模型组(Model)、黄芪甲苷组(Astragaloside, 150mg / kg∙d )、肾华低剂量组(SHT-L, 1.5g / kg∙d SHT)、肾华高剂量组(SHT-H, 3.0g / kg∙d SHT)。IRI成模，观察干预后1d、3d、5d、7d血肌酐、尿素氮、肾脏病理损害；TlR2/TLR4、MyD88的mRNA及蛋白表达； IL-6、 IL-12 mRNA及蛋白水平；IL-8和IFN-γ蛋白变化。结果：与Sham相比，1d后Model组肾损伤严重（Scr：189.42±21.50，P <0.05），病理受损重（4.50±0.55，P <0.05）；TLR2，TLR4，MyD88；TNF-α及IL-6都高于其它组（P <0.05）；IL-8和TNF-γ蛋白表达最高；TLR2\TLR4在1d 至sd 上升趋势最显。3d时 IL-6、IL-12表达峰值。5d时MyD88蛋白及mRNA表达峰值,但与肾脏受损不平行。与Model比，1d、3d、5d、7d，Astragaloside组、SHT-L组和SHT-H组TlR2/TLR4、MyD88蛋白和IL-6、IL-12的mRNA及蛋白表达均降低(P<0.05)，血肌酐及肾脏病理积分下降(P<0.05)。较Astragaloside组和SHT-L组，SHT-H组MyD88蛋白及mRNA表达，及IL-8和IFN-γ蛋白表达均下降(P<0.05)，肾功能及肾脏病理积分改善(P<0.05)。结论：益气活血方肾华片（SHT）可能通过调控MyD88依赖的TLRs信号传导通路，抑制多种炎症因子释放，减轻和修复IRI大鼠的急性肾损伤，且疗效呈现良好的剂量效应。本研究提供了SHT防治急性肾损伤可能的TLRs信号传导通路机制，为临床干预靶点及最佳时机提供了实验依据。共发表论文15篇，其中SCI9篇，统计源6篇；培养博士和硕士各1名。