调控Toll样受体4-MyD88信号通路干预C反应蛋白诱导肾脏炎症与纤维化中的机制研究

Tubular interstitial fibrosis (TIF) is the final common pathway of chronic kidney disease (CKD) progressing to end-stage renal disease. CKD patients are associated with chronic inflammatory status which may be reflected by the inflammatory biomarker, C-reactive protein (CRP). Our research indicated CRP was also a mediator of the pathogenesis of early renal fibrosis and inflammation. TGF-β/Smad and NF-κB signaling pathways activation and expression of Toll-like receptor 4 (TLR4) were involved but the underlying mechanism remains unclear. Hypothesis: TLR4 activates TGF-β/Smad and NF-κB signaling pathway through TLR4-MyD88 dependent pathway to mediates the acute renal fibrosis and inflammation induced by CRP. In this project, TLR4 shRNA plasma and plasma overexpressing TLR4 gene will be constructed and transfected to HK2 cell with CRP stimulation. We will use shRNA to knockout the renal TLR4 gene expression with ultrasound mediated transfection in CRP transgenic mice after UUO. We will clarify the effect of TLR4 through activation of TLR4-MyD88 pathway, the downstream BAMBI/TGF-β/Smad and NF-κB signaling pathway in acute renal fibrosis and inflammation induced by CRP. Through this study, a better understanding of the machanism of CRP induced renal inflammation and fibrosis will be gained and novel preventive and treatment target for TIF may be identified.

肾小管间质纤维化(TIF)是慢性肾脏疾病(CKD)进展的共同通路，尚缺乏早期有效干预。我们前期研究提示，C-反应蛋白(CRP)直接促进肾脏炎症与纤维化的发生，涉及TGF-β/Smad与NF-κB信号通路激活，但具体机制未明。我们推测TIF发生发展中，以TLR4为核心的TLR4-MyD88信号通路的激活，及对下游BAMBI/TGF-β/Smad纤维化信号通路和NF-κB炎症信号通路的调控，是CRP促进肾脏炎性与纤维化的关键信号通路。本研究拟在前期基础上，采用基因转染、质粒构建，体外差异表达及体内肾脏定向沉默TLR4基因，使用CRP刺激细胞和CRP转基因小鼠UUO模型,从不同水平验证TLR4-MyD88信号通路在CRP促进肾脏炎症与纤维化的关键调控作用，探讨抑制TLR4基因作为TIF早期防治的可行性。

C反应蛋白（CRP）在小鼠单侧输尿管梗阻（UUO）模型中已经显示在肾脏炎症的发生与放大中具有重要作用，本项目拟研究其作用机制。我们推测以TLR4 为核心的TLR4-MyD88 信号通路的激活，是CRP 促进肾脏炎症的关键信号通路之一。本项目在前期基础上，使用CRP刺激HK-2细胞和CRP 转基因小鼠UUO 模型,采用免疫组化、Real-time PCR，Western Blot等方法进行研究。我们发现，伴随CRP转基因小鼠UUO术后更为严重的小管间质损伤与更为明显的炎症反应，其肾内NF-κB与TLR4/MyD88信号通路显著被活化，这一现象提示CRP的促炎作用与NF-κB-TLR4/MyD88信号通路密切相关。我们进一步在HK-2细胞炎症模型中研究其机制。我们发现，HK-2细胞经CRP刺激后1hr后，NF-κB明显上调伴随大量而快速促进炎症介质(IL-1β, TNF-ɑ)与趋化因子（MCP-1）表达，在CRP刺激12hr后TLR4/MyD88表达上调；使用CD32a中和抗体可阻断所有以上变化，这说明CRP发挥作用首先需依赖于CD32a活化。同时，抑制NF-κB信号通路（PDTC）可显著阻断TLR4/MyD88表达、阻断CD32a在12hr的高表达，这一现象说明CRP信号通路以及CRP诱导的TLR4/MyD88表达依赖于NF-κB信号通路的。最后，使用siRNA沉默TLR4后，虽然不能在1hr、但可以在12hr抑制CRP所诱导的CD32a-NF-κB信号通路，这一现象说明CRP可通过CD32a-NF-κB依赖模式在CRP刺激1hr后触发早期炎症，随后激活TLR4/MyD88信号通路并通过TLR4/MyD88-NF-κB正反馈信号模式促进持续的肾脏反应。因此，本项目的研究结论为CRP通过CD32a-NF-κB-TLR4/MyD88环路来调节肾脏炎症。