南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (10): 1560-1565.doi: 10.12122/j.issn.1673-4254.2022.10.17

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NLRC4在具核梭杆菌诱导巨噬细胞焦亡中发挥调控作用

江伟艺，邓子龙，赵望泓   

  1. 南方医科大学南方医院口腔科，广东 广州 510515
  • 出版日期:2022-10-20 发布日期:2022-10-31

NLRC4 plays a regulatory role in F. nucleatum-induced pyroptosis in macrophages

JIANG Weiyi, DENG Zilong, ZHAO Wanghong   

  1. Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
  • Online:2022-10-20 Published:2022-10-31

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摘要： 目的 探讨具核梭杆菌感染引起巨噬细胞焦亡的作用及机制。方法 巨噬细胞RAW264.7与具核梭杆菌共同培养，设置未感染组和具核梭杆菌感染组（以MOI和感染时间设置梯度）。乳酸脱氢酶和荧光双染检测细胞坏死率；Western blot检测焦亡相关蛋白caspase-1、GSDMD和IL-1β的活化情况；qRT-PCR分析NLRP3、NLRC4、AIM2、NLRP1炎症小体激活情况，采用siRNA敲低可能参与调控的关键分子，分为siRNA敲低组与阴性对照组，比较两组细胞在具核梭杆菌感染时的焦亡和坏死率，验证其调控作用。结果 具核梭杆菌感染组的细胞肿胀破碎、胞内出现空泡，乳酸脱氢酶释放增加，PI阳性细胞数增多，坏死率升高（P<0.05）；Western blot结果显示caspase-1、GSDMD和IL-1β被活化且呈MOI和时间依赖性升高（P<0.05）；qRT-PCR筛选出NLRC4可能参与调控，siRNA敲低NLRC4可抑制具核梭杆菌引起的caspase-1/GSDMD通路的激活，减少细胞坏死率和炎症因子IL-1β的表达（P<0.05）。结论 具核梭杆菌感染能够诱导巨噬细胞RAW264.7经caspase-1/GSDMD信号通路发生焦亡并释放炎症因子，且NLRC4炎症小体发挥关键的调控作用。

关键词: 具核梭杆菌；焦亡；巨噬细胞；NLRC4炎症小体

Abstract: Objective To explore the mechanism of F. nucleatum-induced pyroptosis in macrophages and the regulatory role of inflammasomes. Methods Lactate dehydrogenase (LDH) cytotoxicity assay and Hoechst 33342/PI double fluorescence staining were used to analyze cytolysis in F. nucleatum- infected macrophage RAW264.7 cells. The expressions of pyroptosis-related proteins caspase-1, GSDMD and IL-1β were determined using Western blotting. Inflammasome activation in the cells was analyzed by detecting the mRNA expressions of NLRP3, NLRC4, AIM2, and NLRP1 with qRT-PCR. RNA interference technique was used to knock down the key molecules involved in pyroptosis regulation in the macrophages, and the pyroptosis and necrosis rates of the cells following F. nucleatum infection were examined. Results The results of LDH cytotoxicity assay and double-fluorescence staining showed that F. nucleatum infection caused swelling and lytic cell death in RAW264.7 cells. F. nucleatum infection resulted in the activation of caspase-1 and GSDMD and upregulated IL-1β expression in a multiplicity of infection (MOI)- and time-dependent manner (P<0.05). qRT-PCR revealed significantly increased expression of NLRC4 mRNA in the macrophages after F. nucleatum infection (P<0.05). NLRC4 silencing by siRNA strongly inhibited the activation of caspase-1/GSDMD pathway and reduced cell death (P<0.05) and IL-1β expression in F. nucleatum-infected cells. Conclusion NLRC4 inflammasome drives caspase-1/GSDMD-mediated pyroptosis and inflammatory signaling in F. nucleatuminfected macrophages, suggesting the potential of NLRC4 inflammasome as a therapeutic target for F. nucleatum infections.

Key words: Fusobacterium nucleatum; macrophages; pyroptosis; NLRC4 inflammasome