Page 7 - 南京医科大学自然版

第44卷第5期 南京医科大学学报（自然科学版）
2024年5月 Journal of Nanjing Medical University（Natural Sciences） ·595 ·

·基础研究·

TBK1对NLRC4炎症小体的作用及其机制研究

曾 强，张在奎，孙乃双，陈允梓 *
南京医科大学基础医学院免疫学系，江苏 南京 211166

［摘 要］ 目的：探究TANK结合激酶1（TANK⁃binding kinase 1，TBK1）调控核苷结合寡聚化结构域样受体4（nucleotide⁃binding
oligomerization domain⁃like receptor 4，NLRC4）炎症小体激活的作用及其机制。方法：在鼠伤寒沙门氏菌（Salmonella typhimurium，
S.T）感染的永生化骨髓巨噬细胞（immortalized bone marrow derived macrophage，IBMDM）中，Western blot 检测NLRC4炎症小体
激活及其下游分子半胱天冬蛋白酶1（cysteine aspartic acid specific protease 1，Caspase⁃1）和Gasdermin D（GSDMD）的剪切情况；
乳酸脱氢酶检测试剂盒检测细胞培养基上清中乳酸脱氢酶的含量；蛋白质免疫共沉淀实验确定 TBK1 与 NLRC4的相互作
用及其具体结构域；细胞免疫荧光实验确定TBK1与NLRC4的空间定位；GST pull⁃down实验确定TBK1与NLRC4是否存在直
接相互作用；凋亡相关斑点样蛋白（apoptosis⁃associated speck⁃like protein containing a CARD，ASC）寡聚化检测实验验证NLRC4
炎症小体组装。构建S.T感染C57BL/6小鼠动物模型，观察小鼠生存情况。涂板计数腹腔灌洗液和肺的细菌负荷量；酶联免疫
吸附试验（ELISA）检测腹腔灌洗液及血清中的肿瘤坏死因子（tumor necrosis factor，TNF）⁃α和白细胞介素（interleukin，IL）⁃1β的
含量；流式细胞术检测腹腔灌洗液中的中性粒细胞比例。结果：在S.T感染的IBMDM中，抑制TBK1可减弱NLRC4炎症小体激
活，NLRC4磷酸化水平下降，Caspase⁃1与GSDMD的剪切减少；TBK1与NLRC4存在相互作用，TBK1的N端与NLRC4的NACHT
结构域相互作用；TBK1与NLRC4存在空间上的共定位；TBK1可以磷酸化NLRC4的Ser533位点。S.T动物模型实验显示，抑制
TBK1活性可以显著提高小鼠的生存率；减低小鼠腹腔灌洗液和肺中的细菌负荷量；降低血清以及腹腔灌洗液中的IL⁃1β、TNF⁃α
的表达水平；减少腹腔灌洗液中的中性粒细胞比例。结论：TBK1与NLRC4相互作用，磷酸化NLRC4 Ser533位点，促进NLRC4
炎症小体的激活，为治疗相关疾病提供理论依据和新的潜在靶点。
［关键词］ NLRC4炎症小体；TBK1；鼠伤寒沙门氏菌
［中图分类号］ R393 ［文献标志码］ A ［文章编号］ 1007⁃4368（2024）05⁃595⁃10
doi：10.7655/NYDXBNSN231080

The effects and mechanisms of TBK1 on NLRC4 inflammasome

ZENG Qiang，ZHANG Zaikui，SUN Naishuang，CHEN Yunzi *
Department of Immunology，School of Basic Medicine，Nanjing Medical University，Nanjing 211166，China

［Abstract］ Objective：To investigate the mechanism by which TANK binding kinase 1（TBK1）regulates the activation of nucleotide⁃
binding oligomerization domain⁃like receptor 4（NLRC4）inflammasome. Methods：Western blot was used to detect the activation of
NLRC4 inflammasome and their downstream molecules cysteine aspartic acid ⁃ specific protease 1（Caspase ⁃ 1）and Gasdermin D
（GSDMD）in immortalized bone marrow ⁃ derived macrophages（IBMDM）infected with Salmonella typhimurium（S.T）. A lactate
dehydrogenase detection kit was used to detect the content of lactate dehydrogenase in the supernatant of cell culture medium. The
interaction between TBK1 and NLRC4 and their specific interaction domain was determined through protein co⁃immunoprecipitation
experiments. Cellular immunofluorescence assay was used to determine the spatial localization of TBK1 and NLRC4. The GST pull⁃down
experiment confirmed the direct interaction between TBK1 and NLRC4. The assembly of NLRC4 inflammasome was verified using
apoptosis⁃associated speck⁃like protein containing a CARD（ASC）oligomerization detection experiments. The S.T infected animal
model of C57BL/6 mice was built and the survival of mice was observed. The bacterial load of lung tissues and peritoneal cavity⁃
flushed fluid was analyzed through smear analysis. ELISA was used to detect the content of tunor necrosis factor（TNF）⁃ α and
interleukin（IL）⁃1β in peritoneal cavity⁃flushed fluid and serum. Flow cytometry was used to detect the proportion of neutrophils in
peritoneal cavity ⁃ flushed fluid. Results：In S.T infected IBMDM，inhibiting TBK1 led to a weakened activation of NLRC4

［基金项目］ 国家自然科学基金（82171723）
∗
通信作者（Corresponding author），E⁃mail：chenyunzi@njmu.edu.cn