NLRP3炎症小体和NF-κB信号通路参与化脓隐秘杆菌感染小鼠肝脏炎症反应的研究

NLRP3 inflammasome and NF-κB signaling pathways are involved in the inflammatory response of mice liver infected with Trueperella pyogenes

为探究化脓隐秘杆菌(Trueperella pyogenes)在肝脏中诱导炎症小体活化的机制,本研究以2×106 cfu/只腹腔注射化脓隐秘杆菌的小鼠为感染模型,分别于感染后不同时间采集小鼠肝脏组织,分别提取肝细胞RNA和蛋白质,将RNA反转录成cDNA作为模板,利用荧光定量PCR检测小鼠肝脏组织中NLRP-3、ASC、IL-18、IL-1β和TNF-αm RNA的转录水平,同时通过western blot检测小鼠肝细胞中炎症小体信号通路蛋白NLRP-3、AIM2、ASC、Caspase-1、NLRP-1和细胞焦亡信号通路相关蛋白GSDMD-C、IL-18和IL-1β的表达。结果显示,与对照组相比,感染化脓隐秘杆菌后1 d、2 d和7 d的小鼠肝组织内炎症小体信号通路蛋白基因及促炎细胞因子基因的转录水平均显著升高(P<0.05);western blot检测感染后1 d、2 d、4 d和7 d的小鼠肝细胞蛋白,结果显示,与对照组相比,小鼠肝细胞内除NLRP-1的表达无差异外,其他炎症小体信号通路相关蛋白及促炎细胞因子表达量均显著或极显著升高(P<0.05,P<0.01)。上述结果证实化脓隐秘杆菌可诱导肝细胞中NLRP-3/ASC和AIM2/ASC炎症小体复合物的激活,继而促进Caspase-1的成熟与GSDMD的切割,引起肝细胞焦亡,导致大量促炎细胞因子释放。本研究进一步通过western blot检测化脓隐秘杆菌感染后1 d、2 d、4 d和7 d的小鼠肝细胞中NF-κB信号通路中p65和磷酸化p65蛋白(p-p65)的表达水平,结果显示,与对照组相比,p-p65蛋白的表达量显著升高(P<0.05),表明NF-κB信号通路被激活。同时,本研究也对化脓隐秘杆菌感染小鼠后1 d、2 d、4 d和7 d采集的肝脏组织进行病理学观察,结果显示,与对照组织相比,肝细胞均发生变性及坏死。本研究证实在化脓隐秘杆菌感染的小鼠肝细胞内炎症小体和NF-κB信号通路参与了感染期间的炎症反应,为化脓隐秘杆菌引起肝脏炎症反应发生的机制研究提供参考依据。

To investigate the mechanism of Trueperella pyogenes(T.pyogenes)inducing inflammasome activation in the liver,the mice were used as the infection model with intraperitoneally challenged with T.pyogenes at a dose of 2×106 cfu/mouse.By collecting mouse liver tissue and extracting RNA and protein from hepatocytes,the RNA was reverse transcribed into cDNA as a template,the mRNA transcriptional levels of NLRP-3,ASC,IL-18,IL-1βand TNF-αwere detected by qRT-PCR,and the inflammasome signaling pathway proteins NLRP-3,AIM-2,ASC and Caspase-1 and the pyroptosis signaling pathway proteins GSDMD-C,IL-18 and IL-1β,and the NF-κB signaling pathway proteins p65and p-p65 were detected by western blot.The results showed that the mRNA transcriptional levels of inflammasome signaling pathway and pro-inflammatory cytokines in mice liver infected with T.pyogenes at days 1,2 and 7 were significantly increased(P<0.05)compared to the control group.Western blot was used to detect the protein in liver tissue of mice on day 1,2,4 and 7 of infection,and it showed that compared with the control group,there was no significant difference in the expression of NLRP-1,while the expression levels of other inflammasome signaling pathway related proteins and pro-inflammatory cytokines significantly or extremely significantly increased(P<0.05,P<0.01).The results confirmed that T.pyogenes induced the activation of NLRP-3/ASC and AIM2/ASC inflammasomes in hepatocytes,which promoted the maturation of Caspase-1 and the cleavage of GSDMD,causing pyroptosis of hepatocytes,resulting in the release of a large number of pro-inflammatory cytokines.Further detection of expression levels of p65 and p-p65 in NF-κB signaling pathway in mouse hepatocytes at day 1,2,4,and 7 after infection with T.pyogenes was performed using western blot,and it showed a significant increase in p-p65 protein expression compared to the control group(P<0.05),indicating the activation of NF-κB signaling pathway.Meanwhile,histopathology examinations on the liver tissues of mice infected with T.pyogenes at day 1,2,4 and 7 were also conducted,and the results showed that the hepatocytes all underwent degeneration and necrosis compared with the control tissue.In conclusion,this study confirmed that inflammasome and NF-κB signaling pathways in hepatocytes of mice infected with T.pyogenes are involved in the inflammatory response during infection,providing a theoretical basis for the research about the mechanism of liver inflammation caused by T.pyogenes.