下调USP46通过抑制NLRP3去泛素化减轻细胞焦亡改善肾小管上皮细胞缺氧/复氧损伤

Downregulation of USP46 alleviates hypoxia/reoxygenation-induced pyroptosis in renal tubular epithelial cells by inhibiting NLRP3 deubiquitination

    摘要
  • 摘要:
    目的 探讨泛素特异性蛋白酶46（USP46）在缺氧/复氧（H/R）诱导的肾小管上皮细胞焦亡中的作用和机制。
    方法 将肾小管上皮细胞分为阴性对照沉默组（si-CTL组）、沉默USP46组（si-USP46组）、阴性对照沉默+H/R处理组（si-CTL+H/R组）和沉默USP46+H/R处理组（si-USP46+H/R组）。流式细胞术检测各组细胞凋亡情况，实时定量聚合酶链反应检测各组USP46、NOD样受体蛋白3（NLRP3）、消皮素D（GSDMD）、白细胞介素（IL）-18、IL-1β信使RNA（mRNA）表达，蛋白质印迹法检测各组USP46、NLRP3、GSDMD、裂解半胱氨酸天冬氨酸蛋白酶（C-Caspase）-1蛋白表达。检测各组细胞上清中炎症因子和乳酸脱氢酶（LDH）表达情况，检测各组细胞活性氧簇（ROS）和丙二醛（MDA）水平。采用免疫共沉淀验证USP46和NLRP3的蛋白结合情况。
    结果 与si-CTL组比较，si-CTL+H/R组细胞凋亡水平升高，USP46、NLRP3、GSDMD-N、C-Caspase-1蛋白表达水平升高，USP46、NLRP3、GSDMD、IL-18、IL-1β mRNA表达水平升高，IL-18、IL-1β、TNF-α、LDH水平升高，ROS、MDA增多（均为P<0.05）；与si-CTL+H/R组相比，si-USP46+H/R组细胞凋亡水平降低，USP46、NLRP3、GSDMD-N、C-Caspase-1蛋白表达水平降低，USP46、GSDMD、IL-18 mRNA表达水平降低，IL-18、IL-1β、TNF-α、LDH水平降低，ROS、MDA水平降低（均为P<0.05）。免疫共沉淀结果显示USP46能够和NLRP3结合。
    结论 下调USP46能够减轻H/R诱导的肾小管上皮细胞焦亡，其机制可能是通过抑制USP46依赖性的NLRP3蛋白去泛素化水平，促进NLRP3泛素化降解。

     

    Abstract:
    Objective To investigate the role and mechanism of ubiquitin-specific protease 46 (USP46) in hypoxia/reoxygenation (H/R)-induced pyroptosis of renal tubular epithelial cells.
    Methods Renal tubular epithelial cells were divided into negative control siRNA group (si-CTL group), USP46 knockdown group (si-USP46 group), negative control siRNA + H/R treatment group (si-CTL+H/R group), and USP46 knockdown + H/R treatment group (si-USP46+H/R group). Flow cytometry was used to detect cell apoptosis in each group. Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to measure the messenger RNA (mRNA) expression of USP46, NOD-like receptor protein 3 (NLRP3), gasdermin D (GSDMD), interleukin (IL)-18, and IL-1β. Western blotting was used to detect the protein expression of USP46, NLRP3, GSDMD, and cleaved cysteinyl aspartate specific proteinase (C-Caspase)-1. The levels of inflammatory factors and lactate dehydrogenase (LDH) in the cell supernatants were detected, and the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the cells were detected. Co-immunoprecipitation was used to verify the interaction between USP46 and NLRP3.
    Results Compared with the si-CTL group, the si-CTL+H/R group exhibited increased cell apoptosis, elevated protein expression of USP46, NLRP3, GSDMD-N and C-Caspase-1, increased mRNA expression of USP46, NLRP3, GSDMD, IL-18 and IL-1β, higher levels of IL-18, IL-1β, TNF-α and LDH, and increased ROS and MDA levels (all P < 0.05). Compared with the si-CTL+H/R group, the si-USP46+H/R group showed decreased cell apoptosis, reduced protein expression of USP46, NLRP3, GSDMD-N and C-Caspase-1, decreased mRNA expression of USP46, GSDMD and IL-18, lower levels of IL-18, IL-1β, TNF-α and LDH, and decreased ROS and MDA levels (all P < 0.05). Co-immunoprecipitation results indicated that USP46 could bind to NLRP3.
    Conclusions Downregulation of USP46 alleviates H/R-induced pyroptosis in renal tubular epithelial cells, possibly by inhibiting USP46-dependent NLRP3 deubiquitination and promoting NLRP3 ubiquitination and degradation.

     

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