HBx/miR-223通过靶基因调控乙肝相关性肾炎足细胞焦亡的作用及机制研究

Podocyte injury has been identified as a key word resulting in hepatitis B virus-associated glomerulonephritis(HBV-GN), Which contributes to HBV-GN development and progression. Recent studies indicate that pyroptosis leads to the release of pro-inflammatory cytokines. We have recently found podocyte pyroptosis, high expression of NLRP3 inflammasome and downstream inflammatory cytokines in HBV-GN. We also find low expression of miR-223 by miRNAs differential expression chips and real time-PCR. Bioinformatics prediction indicates that miR-223 may regulate the expression of NLRP3. Therefore, we propose that miR-223 is involoved in podocyte pyroptosis in HBV-GN by regulating NLRP3 inflammasome singling pathway. HBx transgenic mice is used to verify this hypothesis, we intend to carry out the project from three aspects. Firstly, we evaluate the relationship between NLRP3 inflammasome singling pathway and podocyte pyroptosis in vivo and vitro. Secondly, we verify NLRP3 as target gene regulated by miR-223. Finally, the influence of miR-223 regulating NLRP3 pathway to podocyte pyroptosis will be further investigated by using gene transfection or RNA interference. Better understanding of podocyte pyroptosis wil not only give insight into disease pathogenesis, but can also lead to development of new therapeutic strategies for treatment of HBV-GN.

足细胞损伤是乙肝相关性肾炎（HBV-GN）发病及进展的重要原因。研究报道细胞焦亡是促炎症介质释放的主要形式，在我们前期研究中发现HBV-GN存在足细胞焦亡，以及NLRP3炎症小体及其下游炎症因子表达水平的增高，通过基因芯片及RT-PCR发现miR-223表达水平下调。进一步生物信息学提示NLRP3是miR-223靶基因。我们推测，miR-223通过调控靶基因NLRP3介导HBV-GN足细胞焦亡。本研究以HBx为诱因，首先通过体内外实验进一步验证NLRP3炎症小体信号通路与足细胞焦亡的关系；其次鉴定miR-223的靶基因NLRP3；最后通过过表达及基因沉默，分别在体外和体内水平观察miR-223对NLRP3炎症小体信号通路及足细胞焦亡的影响，为阐明HBV-GN足细胞损伤机制提供新的理论依据，为临床防治寻找新的干预靶点。

乙型肝炎病毒X蛋白（HBx）的表达可能在HBV-GN的发病机制中起到了至关重要的作用，然而具体机制尚不明确。足细胞焦亡是HBV-GN重要的病理变化之一，线粒体损伤与其密切相关；分泌型磷脂酶A2 IB组（sPLA2-IB）与M型磷脂酶A2受体（M-PLA2R）结合可引起多种细胞效应；MicroRNA（miRNA）与机体多种生理病理过程有关，但其在HBV-GN足细胞焦亡中的作用尚不明确。本研究的主要目的是探讨氧化应激、足细胞膜上M-PLA2R过表达、miRNA在HBx蛋白诱导的 HBV-GN足细胞焦亡中的潜在功能及相关机制。大部分HBV-MN患者肾PLA2R呈阳性，肾组织中PLA2R与蛋白尿相关，肾组织中PLA2R的存在与nt1753的HBx基因突变有关。HBx过表达慢病毒成功感染足细胞后，电镜下观察到细胞发生焦亡相关形态学改变，NLRP3 敲低或 ROS 抑制减弱了 HBx 过表达引起的足细胞焦亡以及相关蛋白表达增加；转染HBx质粒诱导足细胞膜上M型PLA2R表达增加，过表达的PLA2R与其配体结合后导致足细胞焦亡程度增加，加入PLA2R抑制剂或ROS抑制剂后，ROS-NLRP3信号通路焦亡相关蛋白表达减少；与对照组相比，HBx过表达组miR⁃223表达较低，双荧光素酶报告基因实验证明NLRP3是miR⁃223的下游靶点之一。ROS/NLRP3 通路在 HBx过表达引起的足细胞焦亡中起着重要作用。HBx诱导足细胞膜上PLA2R过表达通过显著上调ROS-NLRP3信号通路的水平，从而介导足细胞焦亡。此外，HBx可能通过下调miRNA⁃223靶向NLRP3炎症小体促进HBV⁃GN足细胞焦亡，miR⁃223有望成为治疗HBV⁃GN的潜在靶点。