NF-κB蛋白在柯萨奇病毒B3诱导的病毒性心肌炎小鼠心肌细胞焦亡过程中的调控作用

Regulatory role of NF-κB protein in process of coxsackievirus B3-in⁃duced myocardial pyroptosis in mice with viral myocarditis

目的:探讨NF-κB蛋白对柯萨奇病毒B3(CVB3)诱导的病毒性心肌炎小鼠心肌细胞焦亡的调控作用。方法:将编号后的45只雄性BALB/c小鼠根据随机数表法平均分为3组,分别为对照组(normal组)、病毒性心肌炎组(CVB3组)和NF-κB蛋白抑制剂吡咯烷二硫代氨基甲酸(PDTC)处理组(CVB3+PDTC组),每组各15只。7 d后处死各组小鼠,HE染色观察心肌组织内炎症细胞浸润情况;RT-qPCR实验检测心肌组织内CVB3的mRNA表达水平;ELISA实验检测血清中脑钠肽(BNP)和心肌钙蛋白I(cTnI)的表达水平;Western blot实验检测心肌组织内焦亡相关蛋白白细胞介素1β(IL-1β)、gasdermin D(GSDMD)和核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)表达水平,caspase-1/pro-caspase-1比值,以及NF-κB蛋白磷酸化水平。结果:与normal组相比,CVB3组小鼠7 d存活率显著降低,心肌组织内炎症细胞浸润程度显著增加,心肌组织内CVB3的mRNA表达水平显著增加(P<0.05),血清中BNP和cTnI表达水平显著增加(P<0.05),心肌组织内焦亡相关蛋白表达水平、caspase-1/pro-caspase-1比值以及NF-κB蛋白磷酸化水平均显著增加(P<0.05);与CVB3组小鼠相比,CVB3+PDTC组小鼠7 d存活数增加,心肌组织内炎症细胞浸润程度显著降低,心肌组织内CVB3的mRNA表达水平显著降低(P<0.05),血清中BNP和cTnI表达水平显著降低(P<0.05),心肌组织内焦亡相关蛋白表达水平、caspase-1/pro-caspase-1比值以及NF-κB蛋白磷酸化水平均显著降低(P<0.05)。结论:CVB3感染可诱导病毒性心肌炎小鼠心肌组织内焦亡相关蛋白表达水平的上调,且NF-κB信号通路参与心肌组织内细胞焦亡过程的调控。

AIM:To explore the regulatory effect of NF-κB protein on coxsackievirus B3(CVB3)-induced myocardial pyroptosis in mice with viral myocarditis.METHODS:The numbered 45 male BALB/c mice were randomly divided into 3 groups:normal group,viral myocarditis group(CVB3 group)and NF-κB protein inhibitor pyrrolidine dithiocarbamate(PDTC)treatment group(CVB3+PDTC group),with 15 mice in each group.Seven days later,the mice in each group were sacrificed,and HE staining was used to observe the infiltration of inflammatory cells in the myocardial tissues,and RT-qPCR was used to detect the mRNA expression level of CVB3 in the myocardial tissues.The serum levels of brain natriunetic pepide(BNP)and cardiac troponin I(cTnI)were measured by ELISA.Besides,the protein levels of pyroptosis-related molecules,interleukin-1β(IL-1β),gasdermin D(GSDMD)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),as well as the caspase-1/pro-caspase-1 ratio and phosphorylation level of NF-κB in the myocardial tissues were determined by Western blot.RESULTS:Compared with normal group,the 7-day survival rate of the mice in CVB3 group was reduced,while the degree of inflammatory cell infiltration in the myocardial tissue was significantly increased.The mRNA expression level of CVB3 in the myocardial tissues was also significantly increased(P<0.05).The serum levels of BNP and cTnI,the protein levels of pyroptosis-related molecules,the caspase-1/pro-caspase-1 ratio and the phosphorylation level of NF-κB in myocardial tissues were all significantly increased(P<0.05).Compared with CVB3 group,the 7-day survival rate of the mice in CVB3+PDTC group was increased,and the degree of inflammatory cell infiltration in the myocardial tissues was dramatically reduced.Moreover,the mRNA expression level of CVB3,and the serum levels of BNP and cTnI were significantly reduced(P<0.05).The protein levels of pyroptosis-related molecules,as well as the ratio of caspase-1/pro-caspase-1 and the phosphorylation level of NF-κB in the myocardial tissues were all significantly reduced(P<0.05).CONCLUSION:CVB3 infection up-regulates the protein levels of pyroptosisrelated molecules in the myocardial tissues of the mice with viral myocarditis,and NF-κB signaling pathway may participate in regulating the process of pyroptosis in the myocardial tissues.