RIP3在狼疮肾炎进展中的作用机制

Lupus nephritis(LN) is the inflammation of the kidney induced by autoimmunity. We had previously shown that the activation of NLRP3 inflammasome pathway accelerated the progression of LN via inducing Th17/Tregs dysregulation and promoting inflammatory infiltration. Necroptosis is a newly described form of cell death. Recent studies showed that RIP3 mediates inflammation in addition to its involvement in the necroptosis. Our preliminary study showed that RIP3 was activated in LN and RIP3 can activate NLRP3 inflammasome in macrophages. In this study, chemical inhibitor and gene silencing by siRNA will be used to treat MRL/lpr and NZB/W F1 lupus-prone mice in vivo in order to investigate the roles of RIP3 on the activation of NLRP3 inflammasome pathway and its impacts on the progression of LN. The results will be further confirmed by using RIP3 knockout NZM2328 lupus-prone mice. Moreover, macrophages, dendritic cells and glomerular podocytes will be isolated from lupus-prone mice and RIP3 will be knocked out in NZM2328 podocyte cell line to investigate how RIP3 in these cells participates in the inflammation of the kidneys in lupus.

狼疮肾炎(LN)是由自身免疫引起的肾脏炎症。我们报道NLRP3炎性体通路活化促进Th17/Tregs失衡及炎症细胞浸润，加速LN进展。Necroptosis是新发现的细胞死亡形式。最新发现necroptosis通路分子RIP3可直接介导炎症反应。本课题在预实验提示LN中有RIP3活化及RIP3能活化巨噬细胞NLRP3的基础上，应用化学抑制剂和siRNA基因沉默等方法干预MRL/lpr和NZB/W F1自发狼疮小鼠模型，探讨RIP3对NLRP3炎性体通路活化作用及其对LN进展的影响；并用RIP3基因敲除的NZM2328自发狼疮小鼠进一步验证。进而分离小鼠巨噬细胞、树突状细胞和肾小球足细胞，并对NZM2328足细胞株RIP3基因进行敲除，研究RIP3如何介导这些细胞参与狼疮肾脏炎症反应。

研究背景：坏死性凋亡是新发现的细胞死亡形式。最新发现坏死性凋亡通路分子RIP3可直接介导炎症反应，调控NLRP3炎性小体活化。但LN中足细胞NLRP3炎性小体通路是否活化及是否受到坏死性凋亡通路调控仍不明确。.主要研究内容：采用NLRP3抑制剂MCC950及RIP3抑制剂GSK872腹腔注射干预MRL/lpr或NZM2328狼疮小鼠模型，收集小鼠肾脏进行病理染色，观察肾脏组织病理变化、免疫复合物沉积；免疫荧光染色观察肾脏足细胞NLRP3炎性小体活化情况；Western blot观察RIP3/NLRP3炎性小体通路表达情况；分离肾小球制备单细胞悬液进行流式细胞术分析肾脏浸润炎症细胞以及肾脏足细胞NLRP3炎性小体活化变化。采用Protein A+G法去除血清中IgG，进而刺激足细胞，探讨抗dsDNA等自身抗体活化足细胞的机制，观察NLRP3抑制剂及RIP3抑制剂对抗dsDNA等自身抗体诱导的NLRP3炎性小体活化的抑制作用。.关键结果：发病狼疮小鼠肾脏足细胞坏死性凋亡通路及NLRP3炎性小体显著活化；应用NLRP3抑制剂及RIP3抑制剂治疗后狼疮小鼠肾脏足细胞坏死性凋亡通路以及NLRP3炎性小体活化显著降低，肾损害明显减轻、免疫复合物沉积减少，蛋白尿、血自身抗体和血尿素氮水平明显下降，足细胞病变减轻；足细胞内NLRP3炎性小体活化显著降低。.科学意义：RIP3/NLRP3炎性小体通路参与自发狼疮小鼠肾脏损害的发病；靶向RIP3/NLRP3炎性小体通路能抑制足细胞内NLRP3炎性小体活化，减轻狼疮肾炎。