NLRP3炎性小体在白念珠菌脓毒症小鼠组织器官中的表达及作用

Expression and Role of NLRP3 Inflammasome in Tissues and Organs of Mouse with Candida albicans Sepsis

目的:研究NLRP3炎性小体信号通路相关分子在白念珠菌脓毒症小鼠重要组织器官中的表达情况,初步探讨NLRP3炎性小体在白念珠菌脓毒症免疫反应中的作用。方法:将C57BL/6小鼠分成对照组、脓毒症组(24 h, 48 h),经尾静脉注射白念珠菌建立白念珠菌脓毒症感染模型,在感染后的24 h、48 h处死小鼠并检测相关指标。取各组血液、肝、肺和肾组织匀浆在血平板上培养后检测其计数,ELISA法检测细胞因子IL-1β以及IL-18含量;qRT-PCR检测各组织匀浆中NLRP3、ASC、caspase-1中mRNA表达情况,Western blot及免疫荧光染色监测各组织NLRP3、ASC、caspase-1蛋白表达情况。结果:白念珠菌菌液(2.5*108 CFU/mL, 1 ml/10g)可导致小鼠发生白念珠菌脓毒症。与健康对照组相比,脓毒症组中的小鼠3种组织匀浆及血清中IL-1β、IL-18表达水平升高;与24 h组相比,3种组织匀浆中NLRP3 mRNA、ASC mRNA、caspase-1 mRNA表达量在感染48 h后显著升高,并且肝脏和肾脏中NLRP3 mRNA、ASC mRNA、caspase-1 mRNA在感染后24 h就显著升高;通过Western blot及免疫组化可见NLRP3、ASC、pro-casepase-1和caspase-1 p20蛋白水平也显著升高,不同组织中的阳性蛋白的表达量随感染时间而增加。与24 h组相比,在48 h组中,NLRP3蛋白在肝、肾、肺组织表达量显著升高(p β和IL-18的释放而发挥促炎作用。

Objective: To investigatethe expression of NLRP3 inflammatory signaling pathway-related molecules in amouse model with Candida albicans sepsis, and explore the immune reaction mechanism of NLRP3 inflammasome in Candida albicans infection. Methods:C57BL/6 mice were injected via caudal vein with Candida albicans and phosphate buffer (PBS) respectively in sepsisgroup (24 h, 48 h) and control group. The animals were sacrificed 24 h and 48 hafter treatment respectively. The corresponding fungi colony-forming units andthe levels of cytokines IL-1β andIL-18 in serum and tissues were measured. qRT-PCR and Western blot analyseswere used to detect NLRP3, ASC, caspase-1mRNA and proteins in tissuehomogenate. The expression of inflammasome was evaluated byimmunohistochemistry. Results: Candidaalbicans sepsis occurred in mice after injection of Candida albicans solution via caudal vein (2.5*108 cfu/ml, 1 ml/10g).Compared with healthy control group, levels of IL-1β and IL-18 increased significantly in all tissue homogenates;compared with 24 h group, the expression levels of NLRP3 mRNA, ASC mRNA andcaspase-1 mRNA in three kinds of tissue homogenates significantly increased 48h after infection, and NLRP3 mRNA, ASC mRNA and caspase-1 mRNA in liver andkidney significantly increased 24 h after infection. The expression of NLRP3,ASC, pro-casepase-1 and caspase-1 p20 protein in different tissues increasedwith the time of infection. Compared with 24 h group, in 48 h group, NLRP3protein expression in liver, kidney and lung tissue significantly increased (pCandida albicans sepsis is highly likelyto be associated with activation of NLRP3 inflammasome, as the expression levelof NLRP3 inflammasome is seen correlated with the severity of Candida albicans infection. Comparedwith lung tissue, there were more positive proteins in liver and kidney, andultimately plays a role in promoting inflammatory response by releasing of IL-1β and IL-18.