阻断MyD88探索TLR信号通路对小鼠肾缺血再灌注损伤的长期影响及其机理

Ischemia-reperfusion injury (IRI) was considered not only the cause of graft dysfunction and increased the rejection reaction short time after operation, but also the affect of increased chronic graft injury and long-term survival. Although there are many experimental studies of drug showed certain anti-IRI effects, but the results were not effective because of most drugs are blocking the intermediate links of the immune response. So these drugs can not be applicated on the clinical area. In recent years, the innate immune system was found as the start site of the immune response, played an important role in IRI. Inhibition TLR/MyD88 signaling pathway of the innate immune blocked the excesses immune response after IRI from the very beginning, which showed good effect short time after IRI. It has not been reported for long-term effects after IRI by blocking this signaling pathway. TJ-M2010, Our own created MyD88 inhibitor, was confirmed to have excellent effect short time after IRI. This project intends to use MyD88 inhibitor and gene knockout techniques to explore the long-term effects of TLR/MyD88 on renal IRI. To study the MyD88 inhibitor on the long-term effects of IRI and its internal mechanisms not only for establishing the role of innate immunity in IRI, but also for the theoretical foundation to develop MyD88 inhibitors.

缺血再灌注损伤（IRI）不仅是术后近期移植物功能障碍和排斥反应增加的原因，也能够影响移植物的慢性损伤和长期存活。虽然有许多实验研究药物有一定抗IRI作用，但大多数只是阻断免疫反应的中间环节，效果不理想，更无任何临床应用。近年发现固有免疫系统作为免疫反应的启动部位，在IRI中发挥重要作用。阻断固有免疫的主要信号通路TLR/ MyD88，可以从源头上阻断IRI后过激的免疫反应，对于IRI后近期的疗效优良，但尚未见对于IRI后长期的损伤及修复研究的报道。我们自主创制的MyD88抑制剂TJ-M2010已证实对IRI近期损伤有极好的防治疗效，本课题拟利用MyD88抑制剂以及基因敲除等技术手段，进一步探讨TLR/MyD88对肾IRI长期损伤的影响，探索MyD88抑制剂对IRI长期损伤的防治作用及其内在机制，为确立固有免疫在IRI损伤中的作用、也为MyD88抑制剂的研发奠定理论基础。

缺血再灌注损伤现象在我们的临床工作中经常遇到并且难以避免，如器官移植血供恢复，休克、DIC微循环再通，冠脉解痉等。我们前期实验研究发现，创新性的MyD88抑制剂TJ-M2010可以明显提高小鼠致死性肾缺血再灌注损伤后的生存率。而在缺血再灌注损伤后的长期效果并未涉及。本项目通过应用TJ-M2010 以及MyD88-/-小鼠来研究 TLR/MyD88 信号通路对于小鼠肾缺血再灌注损伤后的长期影响，包括对成纤维细胞的聚集、胶原蛋白的沉积，结合手术后近期的炎性反应的指标变化、纤维化信号通路的激活等探讨其作用的机制，用体外实验辅助验证。实验结果表明，TJ-M2010-2治疗或联合使用MR1抗体相对于IRI组在近期表现出显著抑制IL1β，IL6和TNF-α水平，增加IL10水平，并且减少ROS产生，显著降低肾MPO活性；抑制Caspase-3以及增加Bcl-2的表达，减轻IRI诱导的细胞凋亡。在长期效果中表现出降低血清TGF-β1水平，减少Fibronectin，Collagen IV及α-SMA沉积。在体外试验中TJ-M2010-2能反转由TGF-β1诱导的EMT过程。这为临床上研发缺血再灌注损伤药物提供更可靠的选择，TJ-M2010-2很有潜力成为有效IRI保护药物。