雌二醇正向调控TWEAK表达激活IFN-I信号通路在狼疮性肾炎发病机制中作用的研究

Lupus nephritis (LN) is a major complication of systemic lupus nephritis(SLE) and is associated with high rates of morbidity and mortality. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) superfamily (TNFSF) initially described in 1997. Many studies showed that TWEAK plays an important role in cytokines production,vascular injury and immune regulation in lupus nephritis（LN） through activating multiple downstream signaling pathways.Preliminary study of our group demonstrated that TWEAK involved in kidney injury of SLE through TWEAK-p38 MAPK signaling pathway. Expression of TWEAK increased in peripheral mononuclear cells (PBMC) from patients with LN and in renal tissues of lupus mice, but its upstream regulatory mechanism remains unknown.Recent researchs demonstrated that TWEAK induced several type I IFN pathway genes in C2C12 cells ,It also might be upregulated by estrogen .Type I IFN signaling pathway plays an important role in the immune injury in LN. Thus, the aim of this study is to prove a possible role of upregulated TWEAK by estradiol and to explore the potential role of TWEAK to activate type I IFN signaling pathway in the pathogenesis of LN. We carried out series researchs performed in vivo study with MRL/lpr lupus model and in vitro study with PBMCs from LN . The mice and cells are to be treated with 17?-estradiol，specific estrogen receptor antagonist(including antagonist of ER ICI182780 and ERa MPP) or TWEAK siRNA.Immunofluorescence ,western blot and realtime PCR techniques are to be used, to demonstrate the role of estradiol to upregulate TWEAK expression and TWEAK to active type I IFN signaling pathway,thus proving that estradiol- TWEAK- type I interferon pathway plays an important role in the pathogenesis of lupus nephritis .Target gene silence of TWEAK might be a new therapeutic approach of LN.

肿瘤坏死因子样微弱凋亡诱导剂（TWEAK）是肿瘤坏死因子配体超家族成员之一。近年文献及我们前期研究表明，外周血单一核细胞(PBMC)和肾组织TWEAK表达增高，通过激活下游信号通路（如NF-KB、p38MAPK等）参与了狼疮性肾炎(LN)的发病，但其上游调控机制不明。雌激素是公认的诱导LN发病的致病因子，IFN -I是LN免疫损伤的重要通路之一。文献报道，TWEAK上调成肌细胞I型干扰素(IFN-I)表达。本研究假设TWEAK受雌激素的调控，拟采用去势MRL/lpr小鼠及LN患者PBMC为体、内外模型，予以雌二醇、雌激素受体（ER和ERa）拮抗剂和TWEAK-siRNA干预，运用免疫荧光、real time PCR和western blot等技术，从分子、细胞和整体层面探讨雌二醇上调TWEAK表达，并激活IFN-I信号通路，参与LN免疫损伤的机制，为LN的临床治疗提供新的靶点。

内分泌-免疫系统和细胞因子的“交叉对话”在自身免疫性疾病发病机制中扮演着十分重要的角色，肿瘤坏死因子样微弱凋亡诱导剂（TWEAK）是肿瘤坏死因子配体超家族成员之一。通过激活下游信号通路（如NF-KB、p38MAPK等）参与了狼疮性肾炎(LN)的发病，但其上游调控机制不明。雌激素是公认的诱导LN发病的致病因子，雌二醇是雌激素的主要活性成分， 参与了细胞免疫、 体液免疫和抗原呈递调节。IFN -I是LN免疫损伤的重要通路之一。本研究假设TWEAK受雌激素的调控，采用去势MRL/lpr小鼠及LN患者PBMC为体、内外模型，予以雌二醇、雌激素受体（ER和ERa）拮抗剂和TWEAK-shRNA干预，运用免疫荧光、real time PCR和western blot等技术，从分子、细胞和整体层面探讨雌二醇上调TWEAK表达，并激活IFN-I信号通路，参与LN免疫损伤的机制。 研究结果表明TWEAK参与了狼疮小鼠肾脏损伤，雌二醇通过ERа上调LN患者PBMC中TWEAK表达，增加MRL/lpr小鼠肾组织 TWEAK mRNA及蛋白表达，采用雌激素受体拮抗剂及雌激素受体共培养则TWEAK mRNA及蛋白表达下调。LV-TWEAK-shRNA干预后小鼠肾脏HE染色，PAS染色及Masson染色积分明显下调，MRL/lpr 狼疮肾炎小鼠肾组织IFN-a及LY6E蛋白表达降低。该研究阐明雌二醇正向调控TWEAK表达，激活IFN –I通路参与狼疮肾炎免疫损伤机制，阐明 LN时TWEAK靶基因调控的实际意义。