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  • Title: [Mechanism of podocyte pyroptosis aggravated by up-regulation of phospholipase A2 receptor by hepatitis B virus X protein].
    Author: Feng MX, An Q, Yu YN, Chen YQ, Yang XQ, Li BS, Jiang W.
    Journal: Zhonghua Yi Xue Za Zhi; 2023 Jun 13; 103(22):1714-1723. PubMed ID: 37302862.
    Abstract:
    Objective: To explore the effect and underlying mechanism of increased expression of M-type phospholipase A2 receptor (PLA2R) on podocyte membrane induced by hepatitis B virus X protein (HBx) on podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis (HBV-GN). Methods: Transfection of the HBx gene into human kidney podocytes was used to mimic the HBV-GN pathogenesis process. Subsequently, podocytes were divided into the following eight groups: normal control plus secretory phospholipase A2-ⅠB (sPLA2-ⅠB) group, empty plasmid plus sPLA2-ⅠB group, HBx group, HBx plus sPLA2-ⅠB group, HBx plus sPLA2-ⅠB plus PLA2R control siRNA group, HBx plus sPLA2-ⅠB plus PLA2R-siRNA group, HBx plus sPLA2-ⅠB plus ROS control siRNA group, and HBx plus sPLA2-ⅠB plus ROS-siRNA group. Podocyte morphology was observed under a transmission electron microscope, and PLA2R expression was detected under a fluorescence microscope. Podocyte pyroptosis and reactive oxygen species (ROS) expression were analyzed by flow cytometry, and the mRNA and protein expression of PLA2R, nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, interleukin (IL)-1β and IL-18 were determined by real-time fluorescence quantitative PCR and Western blot. Results: Compared with the control group, the expression of PLA2R on podocyte membrane significantly increased after transfection with HBx plasmid in vitro (4.07±0.41 vs 1.01±0.17, P<0.001). Transmission electron microscope and fluorochrome-labeled inhibitor of caspases/propidium iodide (FLICA/PI) double staining suggested that overexpressed PLA2R combined with sPLA2-ⅠB caused aggravated podocyte injury and increased pyroptosis (20.22%±0.36% vs 7.86%±0.28%, P<0.001). Moreover, the expression levels of ROS (4 324 515±222 764 vs 12 920±46, P<0.001), NLRP3 (48.30±2.73 vs 1.00±0.11, P<0.001), ASC (4.02±0.84 vs 1.01±0.15, P<0.001), caspase-1 (3.99±0.42 vs 1.00±0.11, P<0.001), IL-1β (9.08±0.75 vs 1.00±0.09, P<0.001) and IL-18 (19.20±0.70 vs 1.00±0.02, P<0.001) increased when PLA2R was overexpressed. In contrast, with the addition of PLA2R-siRNA or ROS-siRNA to knockdown the expression of related substances, podocyte injury was alleviated and the degree of pyroptosis decreased, and the expressions of genes related to the downstream signaling pathway (NLRP3, ASC, caspase-1, IL-1β and IL-18) decreased (all P<0.01). Conclusion: HBx may promote podocyte pyroptosis in HBV-GN by targeting the ROS-NLRP3 signaling pathway via the upregulation of PLA2R. 目的： 探讨乙型肝炎病毒X蛋白（HBx）诱导足细胞膜上M型磷脂酶A2受体（PLA2R）表达增加对乙型肝炎病毒相关性肾炎（HBV-GN）中足细胞焦亡的影响及其作用机制。 方法： 采用转染HBx基因至人肾脏足细胞内的方法模拟HBV-GN发病过程，并将足细胞分为以下8组：正常对照+分泌型磷脂酶A2-ⅠB（sPLA2-ⅠB）组、空白质粒+sPLA2-ⅠB组、HBx组、HBx+sPLA2-ⅠB组、HBx+sPLA2-ⅠB+PLA2R对照siRNA组、HBx+sPLA2-ⅠB+PLA2R-siRNA组、HBx+sPLA2-ⅠB+ROS对照siRNA组及HBx+sPLA2-ⅠB+ROS-siRNA组。电镜下观察足细胞形态，荧光显微镜下利用免疫荧光染色观察足细胞膜上PLA2R的表达，流式细胞术分析足细胞焦亡率及活性氧（ROS）表达，实时荧光定量PCR及Western 印迹测定PLA2R、核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）、凋亡相关斑点样蛋白（ASC）、胱天蛋白酶1（caspase-1）、白细胞介素（IL）-1β及IL-18 mRNA及蛋白的表达情况。 结果： 与正常对照组相比，体外转染HBx质粒后足细胞膜上M型PLA2R表达增加（4.07±0.41比1.01±0.17，P<0.001），电镜及胱天蛋白酶荧光染料抑制物/碘化丙啶（FLICA/PI）双染细胞焦亡检测提示过表达的PLA2R与其配体sPLA2-ⅠB结合后足细胞损伤加重，焦亡程度增加（20.22%±0.36%比7.86%±0.28%，P<0.001），且PLA2R过表达时ROS（4 324 515±222 764比12 920±46，P<0.001）、NLRP3（48.30±2.73比1.00±0.11，P<0.001）、ASC（4.02±0.84比1.01±0.15，P<0.001）、caspase-1（3.99±0.42比1.00±0.11，P<0.001）、IL-1β（9.08±0.75比1.00±0.09，P<0.001）及IL-18（19.20±0.70比1.00±0.02，P<0.001）表达水平增加。相反，加入PLA2R-siRNA或ROS-siRNA敲低相关物质表达后，足细胞损伤减轻，焦亡程度下降，下游信号通路相关基因NLRP3、ASC、caspase-1、IL-1β及IL-18基因表达均减少（均P<0.01）。 结论： HBx可能通过上调PLA2R靶向ROS-NLRP3信号通路促进HBV-GN中足细胞焦亡。.
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