白术内酯Ⅰ对病毒性心肌炎小鼠Toll样受体4的调控作用

Regulation of Toll-like receptor 4 in mice with viral myocarditis by atractylenolideⅠ

目的:探讨白术内酯Ⅰ(ATR-Ⅰ)对病毒性心肌炎(VM)小鼠的治疗作用及其对Toll样受体4(TLR4)的影响。方法:通过对C57BL/6小鼠腹腔注射柯萨奇病毒B3(CVB3)诱导VM小鼠模型,并随机分为5组:Control组、VM组、VM+L-ATR-Ⅰ组、VM+M-ATR-Ⅰ组和VM+H-ATR-Ⅰ组,每组12只。Control组小鼠为正常饲养的小鼠,其他组小鼠为VM建模小鼠。VM+L-ATR-Ⅰ组、VM+M-ATR-Ⅰ组和VM+H-ATR-Ⅰ组小鼠分别灌胃0.5 ml 60、120和240 mg/kg的ATR-Ⅰ溶液;Control组和VM组小鼠分别灌胃0.5 ml 0.5%羧甲基纤维素钠溶液。各组小鼠均灌胃7 d。使用超声仪器检测各组小鼠的舒张期内径(LVIDd)、左心室收缩期内径(LVIDs)、左心室短轴缩短率(FS)和左心室射血分数(LVEF)。ELISA和qRT-PCR检测血清和心肌组织中IL-1β、IL-6及TNF-α水平。然后对小鼠心肌组织进行HE染色和Masson三色染色。qRT-PCR或Western blot检测心肌组织中TLR4、NF-κB p65及p-NF-κB p65的mRNA或蛋白水平。结果:与VM组相比,VM+M-ATR-Ⅰ组和VM+H-ATR-Ⅰ组中LVIDd和LVIDs降低,FS和LVEF升高(P<0.05);与VM组相比,VM+L-ATR-Ⅰ组、VM+M-ATR-Ⅰ组和VM+H-ATR-Ⅰ组血清和心肌组织IL-1β、IL-6及TNF-α水平降低(P<0.05)。心肌损伤程度均呈剂量依赖性减轻。各组小鼠心肌组织中NF-κB p65蛋白水平差异无统计学意义(F=0.219,P=0.927)。与VM组相比,VM+L-ATR-Ⅰ组、VM+M-ATR-Ⅰ组和VM+H-ATR-Ⅰ组心肌组织TLR4mRNA和蛋白水平及p-NF-κB p65蛋白水平降低(P<0.05)。结论:在CVB3诱导的VM小鼠模型中,ATR-Ⅰ可剂量依赖性地抑制心肌炎症,减轻心肌损伤并改善心功能,其机制可能与TLR4/NF-κB信号通路有关。

Objective:To investigate the therapeutic effect of atractylenolideⅠ(ATR-Ⅰ)on viral myocarditis(VM)mice and its effect on Toll-like receptor 4(TLR4).Methods:C57BL/6 mice were intraperitoneally injected with Coxsackie virus B3(CVB3)to induce VM,and randomly divided into 5 groups:Control group,VM group,VM+L-ATR-Ⅰgroup,VM+M-ATR-Ⅰgroup and VM+H-ATR-Ⅰgroup,with 12 mice in each group.Mice in Control group were fed normally.Other groups of mice modeled for VM mice.Mice in VM+L-ATR-Ⅰgroup,VM+M-ATR-Ⅰgroup and VM+H-ATR-Ⅰgroup were intragastrically given 0.5 ml 0.5%of ATRⅠsolution of 60,120 and 240 mg/kg,respectively;mice in Control group and VM group were given 0.5 ml sodium carboxymethyl cellulose solution,respectively.All groups of mice were given gavage for 7 days.Diastolic inner diameter(LVIDd),left ventricular systolic inner diameter(LVIDs),left ventricular fractional shortening(FS)and left ventricular ejection fraction(LVEF)of each group of mice were measured by ultrasonic instrument.Levels of IL-1β,IL-6 and TNF-αin serum and myocardial tissue were determined by ELISA and qRT-PCR.Then myocardial tissue of mice was stained with HE and Masson trichrome.mRNA or protein levels of TLR4,NF-κB p65 and p-NF-κB p65 in myocardial tissue were detected by qRT-PCR or Western blot.Results:Compared with VM group,LVIDd and LVIDs in VM+M-ATR-Ⅰgroup and VM+H-ATR-Ⅰgroup were decreased,while FS and LVEF were increased(P<0.05),compared with VM group,levels of IL-1β,IL-6 and TNF-αin serum and myocardial tissue of VM+L-ATR-Ⅰgroup,VM+MATR-Ⅰgroup and VM+H-ATR-Ⅰgroup were decreased(P<0.05);degree of myocardial injury was significantly reduced in a dosedependent manner.There was no significant difference in NF-κB p65 protein level in myocardial tissue among all groups(F=0.219,P=0.927).Compared with VM group,TLR4 mRNA and protein level,and p-NF-κB p65 protein level of myocardial tissue in VM+L-ATR-Ⅰgroup,VM+M-ATR-Ⅰgroup and VM+H-ATR-Ⅰgroup were decreased(P<0.05).Conclusion:In CVB3-induced VM mouse model,ATR-Ⅰcan dose-dependently inhibit myocardial inflammation,reduce myocardial injury and improve cardiac function,and the mechanism may be related to TLR4/NF-κB signaling pathway.