循环HMGB1/DNA复合物通过脾免疫细胞RAGE/TLR9受体加剧心肌再灌注损伤的机制研究

Circulating HMGB1/DNA complex plays an important role in immune and inflammatory responses. We have already demonstrated that a HMGB1 (Heart) – RAGE (Spleen) axis is activated following myocardial ischemia and exacerbates infarct size during reperfusion. This activated heart-spleen axis exacerbates inflammatory response in part by enhancing IL-1beta production, a key component of NLRP3 inflammasome. In our preliminary studies, we found that ischemic myocardium released HMGB1/DNA complex during reperfusion, which activated splenic leukocytes. Recently, it is reported that HMGB1 promotes cellular uptake of DNA by activating RAGE. Increased cytosol DNA subsequently binds TLR9 and triggers downstream inflammatory response. One of the downstream pathway following TLR9 activation includes ROS – inflammasome. By using RAGE-/-, TLR9-/- and NLRP3-/- mice as well as techniques of bone marrow transplantation and immune cell adoptive transfer, the current project is to test our hypothesis that ischemic myocardium releases HMGB1/DNA complex into the blood stream, which in turn activates splenic leukocytes via RAGE/TLR9-ROS-NLRP3 inflammasome pathway and ultimately exacerbates infarct size during reperfusion.

循环中HMGB1与DNA以复合物形式存在参与机体免疫调控。申请人发表论文证实HMGB1(心脏 )-RAGE(脾)轴是介导心肌再灌注免疫损伤的核心通路，可显著上调脾免疫细胞IL-1β(NLRP3炎症复合体主要产物)水平。我们预实验提示，缺血心肌释放HMGB1/DNA复合物，激活脾脏介导的免疫炎症反应。文献报道，HMGB1可以通过RAGE受体促进DNA进入免疫细胞作用于胞浆中TLR9受体，上调ROS水平，激活NLRP3炎症复合体。据此，本项目拟在小鼠心肌缺血再灌注模型中，利用RAGE-/-，TLR9KO及NLRP3-/-等基因修饰小鼠，结合骨髓移植/脾细胞重建技术，证实缺血心肌释放HMGB1/DNA复合物，作用于脾免疫细胞RAGE受体促使DNA进入胞内，通过TLR9-ROS通路活化NLRP3炎症复合体，促进全身炎症反应，加剧再灌注心肌损伤。深入探索心肌再灌注免疫损伤机制，寻求新的心肌保护靶点。

近年来心肌缺血再灌注的免疫损伤机制备受关注。申请人发表论文证实HMGB1(心脏 )-RAGE(脾)轴是介导心肌再灌注免疫损伤的核心通路，可显著上调脾免疫细胞IL-1β(NLRP3 炎症复合体主要产物)水平。但心脏脾轴在心肌再灌注免疫损伤中的详细调控机制仍不清楚。本研究计划在小鼠心肌缺血再灌注损伤模型中，探讨在体动物复杂环境下心脏- 脾轴调控心肌缺血再灌注免疫损伤的详细调控机制，验证HMGB1/DNA 复合物在心肌缺血再灌注损伤机制中参与心脏-脾轴免疫调控机制，加剧再灌注心肌损伤。在本次研究中，我们在小鼠心肌缺血再灌注损伤模型中首先证实缺血心肌组织可以释放cfDNA及HMGB1进入血液循环。循环中的cfDNA与HMGB1通过协同作用，通过RAGE/TLR 9受体调控脾免疫细胞介导的全身炎症反应，加剧心肌缺血再灌注损伤的；随后我们进一步探讨了脾单核巨噬细胞NLRP3炎症小体参与心脏(cfDNA/HMGB1)-脾(RAGE/TLR9)轴，促进全身炎症反应，加剧再灌注心肌损伤的机制，首次证实了再灌注早期（0-15分钟）脾NLRP3炎症小体在心肌再灌注免疫损伤机制中具有重要作用；此外在上述研究过程中，我们还发现CD200-CD200R抗炎通路可以通过抑制心脏-脾轴，发挥心肌保护作用并在心肌梗死后心室重塑过程中发挥重要调控作用；使用外源性重组CD200预处理及后处理可以显著下调NLRP3炎症小体及其下游炎症因子水平，抑制全身炎症反应，减轻再灌注心肌损伤。这些研究结果将为完善心肌缺血再灌注免疫损伤理论机制，寻求可能的心肌保护靶点，开发新型有效的心肌保护策略奠定基础。