芳基烃受体AhR对CVB3诱导病毒性心肌炎小鼠心肌细胞损伤和焦亡的影响

Effects of Aryl Hydrocarbon Receptor AhR on Myocardial Cell Injury and Pyroptosis in CVB3-Induced Viral Myocarditis Mice

目的:探究抑制芳基烃受体(aryl hydrocarbon receptor,AhR)对柯萨奇B3病毒(coxsackievirus 3,CVB3)诱导的小鼠病毒性心肌炎(viral myocarditis,VMC)心肌损伤及细胞焦亡的影响。方法:将60只BALB/c小鼠随机分为对照组(Control)、AhR抑制剂组(AhR inhibitor)、模型组(VMC)、VMC+AhR抑制剂组(VMC+AhR inhibitor),每组15只。VMC组和VMC+AhR抑制剂组小鼠腹腔注射CVB3建立心肌炎模型后,AhR抑制剂组和VMC+AhR抑制剂组小鼠腹腔注射AhR抑制剂CH223191(10mg/kg)。10d后,通过高分辨率Vevo2100小动物超声成像系统检测各组小鼠心脏超声指标,ELISA法检测血清心肌肌钙蛋白l(cardiac troponin l,cTnl)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和IL-18的含量,苏木精-伊红(HE)染色观察心肌组织病理学变化,TUNEL染色检测心肌组织细胞的凋亡情况,免疫组织化学染色测定心肌组织NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor pyrin domain containing 3,NLRP3)与消皮素D(Gasdermin-D,GSDMD)的阳性表达水平,实时荧光定量PCR和Western blot检测心肌组织焦亡相关蛋白含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase-1)、细胞凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing CARD,ASC)、NLRP3及GSDMD在mRNA和蛋白上的表达水平。结果:与对照组比较,VMC组小鼠FS和EF下降,LVIDd和LVAWd增加,血清中cTnl、TNF-α、IL-1β以及IL-18的含量均上升,心肌组织内细胞排列紊乱,伴随大量炎性细胞浸润,心肌组织内TUNEL阳性率升高,NLRP3阳性表达率和GSDMD阳性表达率也升高,caspase-1、ASC、NLRP3及GSDMD的mRNA相对表达量和蛋白相对表达量均上调,差异均具有统计学意义(P<0.05);与VMC组比较,VMC+AhR抑制剂组小鼠FS和EF升高,LVIDd和LVAWd减小,血清中cTnl、TNF-α、IL-1β以及IL-18的含量均降低,心肌组织病理损伤明显减轻,心肌组织内TUNEL阳性率下降,同时,NLRP3阳性表达率和GSDMD阳性表达率均降低,caspase-1、ASC、NLRP3及GSDMD的mRNA相对表达量和蛋白相对表达量均下调,差异均具有统计学意义(P<0.05)。结论:抑制AhR对CVB3诱导的小鼠病毒性心肌炎起保护作用,能够减轻心肌组织损伤,并抑制心肌细胞焦亡。

Objective:To explore the effect of inhibiting aryl hydrocarbon receptor(AhR)on myocardial injury and cell pyroptosis in mice with coxsackievirus 3(CVB3)-induced viral myocarditis(VMC).Methods:Totally 60 BALB/c mice were randomly divided into control group(Control),AhR inhibitor group(AhR inhibitor),model group(VMC),and VMC+AhR inhibitor group(VMC+AhR inhibitor),15 mice in each group.Mice in VMC group and VMC+AhR inhibitor group were intraperitoneally injected with CVB3 to establish a myocarditis model.Mice in AhR inhibitor group and VMC+AhR inhibitor group were intraperitoneally injected with AhR inhibitor CH223191(10 mg/kg).After 10 days,the cardiac ultrasound indexes of each group were detected by the high-resolution Vevo2100 small animal ultrasound imaging system;ELISA method was used to detect serum cardiac troponin l(cTnl),tumor necrosis factor-α(tumor necrosis factor-α,TNF-α),interleukin-1β(interleukin-1β,IL-1β)and IL-18;Hematoxylin-Eosin(HE)staining was used to observe the pathological changes of myocardial tissue;TUNEL staining to detect the apoptosis of myocardial tissue cells;immunohistochemical staining to determine the positive expression levels of NOD-like receptor pyrin domain containing 3(NLRP3)and Gasdermin-D(GSDMD)in myocardial tissue;real-time fluorescent quantitative PCR and Western blot to detect expression levels on mRNA and protein of cysteinyl aspartate specific proteinase(caspase-1),apoptosis-associated speck-like protein containing CARD(ASC),NLRP3 and GSDMD in myocardial tissue.Results:Compared with the control group,FS and EF decreased,LVIDd and LVAWd increased,the levels of cTnl,TNF-α,IL-1βand IL-18 in serum increased,the cell arrangement in myocardial tissue was disordered,accompanied by a large number of inflammatory cell infiltration,the increase of TUNEL positive rate in myocardial tissue,the NLRP3 positive expression rate and GSDMD positive expression rate.The relative expression of caspase-1,ASC,NLRP3 and GSDMD mRNA and protein were all upregulated,and the differences were statistically significant(P<0.05);Compared with the VMC group,FS and EF were elevated,LVIDd and LVAWd were reduced,serum levels of cTnl,TNF-α,IL-1βand IL-18 were decreased,myocardial histopathological damage was significantly reduced,TUNEL positive rate in myocardial tissue was decreased,meanwhile,NLRP3 positive expression rate and GSDMD positive expression rate.The relevant expression of caspase-1,ASC,NLRP3 and GSDMD mRNA and protein were downregulated,and the differences were statistically significant(P<0.05).Conclusion:Inhibition of AhR played a protective role against CVB3-induced viral myocarditis in mice anda was able to reduce myocardial tissue damage and inhibit cardiomyocyte scorching.