VDR-NFκB-NLRP3轴在狼疮性肾炎发病中的作用机制研究

Inflammation plays an important role in the pathegenesis and progression of lupus nephropathy(LN), and NF-κB induced NLRP3 activation is associated with LN inflammation. Although recent evidence suggests that vitamin D receptor (VDR) is widely recognized as a key reno-protective factor through its complex anti-inflammatory effects, the specific mechanism remains to be recognized deeply. Our previous studies demonstrated that nuclear VDR expression was significant down regulated in LN patients, which is negatively associated with the expressions of pro-inflammatory factor NF-κB. Our preliminary in vitro studies have further demonstrated that VDR and NF-κB need to bind with importin-4 for nuclear transportation to exert their biological effect. In addition, the expression of NLRP3、IL-1β and IL-18 of VDR null mice is higher than that of wild type mice. Therefore, we can speculate that VDR could competitively inhibit importin-4 binding of NF-κB for its subsequent nuclear transportion, and then affect the activation of NF-κB-NLRP3 axis to exert anti-inflammatory effect. To explore these processes, in this project we will focus on verifing the effect of VDR on the NF-κB-NLRP3 axis which participate in the inflammation of LN, as detection methods by cell molecular biology. Then establish podocyte line, transiently transfect with epitope-tagged full-length, N-terminal, C-terminal constructs or siRNAs of VDR, NF-κB or importin-4, and investigate with CoIP whether VDR could competitively inhibit importin-4 binding of NF-κB and its subsequent nuclear transportion. We will further explore novel molecular mechanisms of VDR-NF-κB-NLRP3 axis involved in the pathogenesis of LN inflammatory actions with lupus mice and VDR-/- mice based on our in vitro findings in order to provide new clues and evidence basis for the prevention and treatment of LN.

炎症反应在狼疮性肾炎(LN)的发生发展中起重要作用，NF-κB诱导炎性小体NLRP3活化参与LN炎症过程，VDR可通过抗炎机制发挥肾脏保护作用，但具体机制尚不明确。我们前期研究发现LN患者存在核内VDR表达下调，且与NF-κB负相关，VDR与NF-κB入核转录均需importin-4介导；此外，VDR敲除小鼠肾脏NLRP3、IL-1β等表达增加。由此我们提出假说：VDR通过与importin-4结合竞争性抑制NF-κB入核，进而影响NF-κB-NLRP3轴的活化以拮抗LN的炎症反应。本项目拟采用细胞分子生物学技术观察VDR在LN状态下对NF-κB-NLRP3轴表达及功能的影响；通过过表达及基因沉默技术，证实VDR可竞争性抑制NF-κB与importin-4结合及入核转运过程；拟在基因敲除动物模型上证实VDR-NF-κB-NLRP3轴参与LN发病过程，以期为LN早期防治提供新线索和实验支持。

研究背景：炎症反应在狼疮肾炎(LN)发生发展中起重要作用，NF-κB诱导NLRP3活化参与LN的炎症过程，维生素D受体（VDR）可通过抗炎保护肾脏，具体机制尚未完全阐明。据报道核转录因子VDR需通过核转运蛋白importin-4介导入核，前期发现NF-κB与importin-4也可结合，且LN患者核内VDR表达下调，与NF-κB负相关。本研究旨在探究VDR与NF-κB入核转运时相互影响的可能机制，及VDR对NF-κB-NLRP3轴的影响，以期为LN早期防治提供新线索。主要研究内容：MRL/lpr狼疮小鼠给予帕立骨化醇（pari）干预，检测小鼠VDR、NF-κB、NLRP3、Casepase-1、IL-1β、IL18、Bax及Bcl-2表达。MRL/lpr小鼠血清刺激鼠肾小管上皮细胞（mRTEC细胞），给予pari、转染NF-κB及NLRP3 siRNA干预，检测上述炎症及增殖、凋亡相关指标表达；流式检测细胞凋亡；过表达VDR后CO-IP检测VDR、NF-κB与importin-4的结合。重要结果：1、MRL/lpr小鼠肾组织NLRP3、Caspase-1，血清IL-1β、IL-18表达均增高，Pari干预可显著减低NF-κB-NLRP3通路因子的表达。2、MRL/lpr小鼠肾组织VDR表达减低，NF-κB表达增高，pari干预增加VDR表达的同时减低NF-κB的表达。3、MRL/lpr小鼠血清干预可减低mRTEC细胞VDR总蛋白及核蛋白的表达，增加NF-κB总蛋白及核蛋白的表达，给予pari后，VDR表达增加、NF-κB表达减少，过表达VDR后NF-κB与importing-4结合明显减低。4、MRL/lpr小鼠血清干预mRTEC细胞可增加NLRP3、Caspase-1、IL-1β及IL-18的表达，Pari可减低上述指标的表达。5、NF-κB siRNA转染后，NLRP3通路因子表达减低更明显，沉默NLRP3后下游炎症因子表达更低。6、小鼠血清干预后细胞Bax增加，Bcl-2减低，同时凋亡更明显；Pari可减低Bax表达、增加Bcl-2表达并减轻凋亡；沉默NF-κB和NLRP3后，上述过程更为明显。科学意义：揭示VDR可能通过与NF-κB竞争性结合importin-4入核转运，阻抑NLRP3炎症通路活化，减轻细胞凋亡，进而抗炎保护肾脏，为LN早期防治提供新思路。