NLRP3炎症小体介导单核细胞增生李斯特菌感染小鼠和巨噬细胞的细胞焦亡

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摘要目的: 探讨单核细胞增生李斯特菌（Listeria monocytogenes，Lm）感染引起小鼠和巨噬细胞的细胞焦亡作用机制。方法：体内实验，C57B/6小鼠分为Lm组和阴性对照组（NC组），分别经小鼠尾静脉注射Lm菌液5×104 CFU/鼠和无菌PBS，24 h后取脾脏、肝脏、脑组织，蛋白质印迹法检测焦亡及核苷酸结合寡聚化结构域样受体蛋白3（nucleotidebinding oligomerization domainlike receptor protein 3，NLRP3）炎症小体相关蛋白的表达。体外实验，分Lm组、NC组、LPS+ATP组、Lm+MCC950组和LPS+ATP+MCC950组。以感染复数（multiplicity of infection，MOI）=10 Lm感染THP-1源巨噬细胞，ELISA检测细胞上清液IL-1β，实时荧光定量PCR和蛋白质印迹法分别检测细胞焦亡及NLRP3炎症小体mRNA和蛋白的表达，免疫荧光检测Lm感染后凋亡相关斑点样蛋白（apoptosisassociated specklike protein containing a CARD，ASC）斑点及与NLRP3的共定位。结果： Lm感染小鼠和巨噬细胞后，消化道皮肤素D（gasdermin D，GSDMD）蛋白表达水平均明显升高（P<0.05），且出现GSDMD蛋白氨基端裂解片段（gasdermin D N-terminal，GSDMD-NT）。Lm感染巨噬细胞1 h、3 h、6 h，NLRP3、caspase-1、IL-1β、IL-18 mRNA水平均明显升高（P<0.05），且3 h升高最明显。Lm感染小鼠的脾、肝、脑组织NLRP3、pro-caspase-1、pro-IL-1β及裂解片段caspase-1 p20、IL-1β-17蛋白表达量均明显升高（P<0.05）。Lm感染巨噬细胞后IL-1β分泌水平升高，细胞上清液中检测到裂解片段caspase-1 p20、IL1β17的分泌，细胞裂解液中NLRP3、procaspase1、proIL1β蛋白表达升高（P<0.05），荧光显微镜观察到ASC斑点形成且与NLRP3蛋白存在共定位。加入NLRP3特异性抑制剂MCC950预处理后，巨噬细胞GSDMDNT明显减少（P<0.05）。结论： Lm感染导致的细胞焦亡主要与NLRP3炎症小体的激活有关。

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	袁琳
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	陈盛霞

关键词 ：单核细胞增生李斯特菌, 细胞焦亡, NLRP3炎症小体, 白细胞介素-1&beta, 凋亡相关斑点样蛋白

Abstract：Objective: To investigate the potential mechanism of Listeria monocytogenes （Lm）infectionon pyroptosis in mice and macrophages.Methods: In vivo experiments, C57B/6 mice were divided into Lm group and negative control group（NC group）, then the mice were injected with Lm 5×104 CFU/mouse and sterile PBS via tail vein, respectively. The spleen, liver and brain tissues of mice were collected 24 hours later. The pyroptosis and NLRP3 inflammasome related protein expression were detected by Western blotting. In vitro experiments were divided into Lm group, NC group, LPS+ATP group, Lm+MCC950 group and LPS+ATP+MCC950 group. Multiplicity of infection （MOI）= 10 Lm was used to infect THP-1 derived macrophages, ELISA was used to detect IL-1β from cell supernatant, quantitative real-time PCR （qRT-RCR）and Western blotting were used to detect expression of mRNA and protein about pyroptosis and NLRP3 inflammation. Immunofluorescence （IF）was used to detect ASC-speck and co-localization with NLRP3. Results: After Lm infection of mice and macrophages, the expression level of gasdermin D （GSDMD）protein was significantly increased, and gasdermin D N-terminal （GSDMD-NT）was appeared. The levels of NLRP3, caspase-1, IL-1β, and IL-18 mRNA were significantly increased after Lm infected macrophages at 1 h, 3 h, and 6 h （P<0.05）, and the increase was the most obvious at 3 h. The protein expression of NLRP3, pro-caspase-1, pro-IL-1β and cleavage fragment caspase-1 p20, IL-1β-17 in Lminfected mouse spleen, liver, brain tissue were all significantly increased （P<0.05）.In macrophages by Lm infection, the secretion of IL-1β was increased, the secretion of caspase-1 p20 and IL-1β-17 was detected in the cell supernatant, and the expression of NLRP3, pro-caspase-1, pro-IL-1β protein in the cell lysate was increased （P<0.05）, then ASC-speck was formed and co-localized with NLRP3 protein was observed by fluorescence microscope. After pretreatment with specific NLRP3 inflammasome inhibitor MCC950, GSDMD-NT of macrophages was significantly reduced （P<0.05）. Conclusion: Pyroptosis caused by Lm infection is mainly related to the activation of NLRP3 inflammasome in mice and macrophages.

收稿日期: 2020-10-25

基金资助:
科技部基础平台资助项目（TDRC-2019-194-30）

通讯作者: 陈盛霞（通讯作者），教授，博士生导师，E-mail：chensxia@ujs.edu.cn

作者简介: 袁琳（1996—），女，硕士研究生

引用本文:

袁琳，朱昱蓉，林琳，黄霜，姜旭淦，陈盛霞. NLRP3炎症小体介导单核细胞增生李斯特菌感染小鼠和巨噬细胞的细胞焦亡[J]. 江苏大学学报:医学版, 2021, 31(04): 307-313.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2021/V31/I04/307

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