基于AMPK/mTOR自噬通路研究甘草酸二铵对柯萨奇病毒B3病毒性心肌炎小鼠的保护作用

Protective effect of diammonium glycyrrhizinate on mice with coxsackievirus B3 myocarditis by regulating AMPK/mTOR autophagy pathway

目的基于单磷酸腺苷活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)自噬通路探究甘草酸二铵(diammonium glycyrrhizinate,DG)对病毒性心肌炎(viral myocarditis,VMC)小鼠心肌的保护作用及机制。方法50只BALB/c小鼠随机分为对照组、模型组、DG(19.5mg/kg)组、DG(19.5 mg/kg)+Compound C(20 mg/kg)组和DG(19.5 mg/kg)+Compound C(20 mg/kg)+雷帕霉素(2.0 mg/kg)组,每组10只。采用试剂盒检测血清中肌酸激酶同工酶(creatine kinase-MB,CK-MB)活性、心肌钙蛋白Ⅰ(cardiac troponinⅠ,c TnⅠ)水平及心肌组织中白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;采用苏木素-伊红(HE)和TUNEL染色分别检测心肌组织病理形态和细胞凋亡;采用qRT-PCR检测心肌组织柯萨奇病毒3(coxsackievirus B3,CVB3)m RNA表达;采用Western blotting检测心肌组织自噬、凋亡及AMPK/mTOR通路相关蛋白表达。结果与对照组比较,模型组心肌纤维部分断裂,有明显的细胞变形、出血和炎性细胞浸润,心肌细胞凋亡率、血清中CK-MB活性、c TnI水平及心肌组织IL-1β、TNF-α水平均显著升高(P<0.05),心肌组织CVB3 mRNA表达及活化的半胱氨酸天冬氨酸蛋白酶-9(cleaved cystein-asparate protease-9,cleaved Caspase-9)、cleaved Caspase-3、p62、p-mTOR/mTOR蛋白表达均显著升高(P<0.05),心肌组织微管相关蛋白轻链3-Ⅱ/Ⅰ(microtubule-associated protein light chain 3-Ⅱ/Ⅰ,LC3-Ⅱ/Ⅰ)、Beclin-1、p-AMPK/AMPK蛋白表达均显著降低(P<0.05)。与模型组比较,DG明显改善心肌形态,降低心肌细胞凋亡率、血清中CK-MB活性、cTnI水平及心肌组织IL-1β、TNF-α水平(P<0.05),下调心肌组织CVB3 m RNA表达及cleaved Caspase-9、cleaved Caspase-3、p62、p-mTOR/mTOR蛋白表达(P<0.05),上调心肌组织LC3-Ⅱ/Ⅰ、Beclin-1、p-AMPK/AMPK蛋白表达(P<0.05)。AMPK抑制剂Compound C能减弱DG的上述保护作用,且自噬激活剂雷帕霉素能抑制这种减弱作用。结论DG可能通过激活AMPK/mTOR通路诱导自噬,减轻VMC小鼠心肌损伤。

Objective To investigate the protective effect and mechanism of diammonium glycyrrhizinate(DG)on myocardium of mice with viral myocarditis(VMC)based on adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)autophagy pathway.Methods Fifty BALB/c mice were randomly divided into control group,model group,DG(19.5 mg/kg)group,DG(19.5 mg/kg)+Compound C(20 mg/kg)group and DG(19.5 mg/kg)+Compound C(20 mg/kg)+rapamycin(2.0 mg/kg)group,with 10 mice in each group.Kits were used to detect creatine kinase-MB(CK-MB)activity,cardiac troponin I(cTn I)level in serum and levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in myocardial tissue;The pathological morphology and cell apoptosis of myocardial tissue were detected by hematoxylin eosin(HE)and TUNEL staining;qRT-PCR was used to detect coxsackievirus B3(CVB3)mRNA expression in myocardial tissue;Western blotting was used to detect autophagy,apoptosis,and AMPK/mTOR pathway related protein expressions in myocardial tissue.Results Compared with control group,mice in model group showed partial myocardial fiber breakage,significant cell deformation,bleeding and inflammatory cell infiltration,and the myocardial cell apoptosis rate,CK-MB activity,cTnI level in serum and levels of IL-1β,TNF-αin myocardial tissue were significantly increased(P<0.05),CVB3 mRNA and cleaved cystein-asparate protease-9(cleaved Caspase-9),cleaved Caspase-3,p62,p-mTOR/mTOR protein expressions in myocardial tissue were significantly increased(P<0.05),microtubule associated protein light chain 3-II/I(LC3-II/I),Beclin-1 and p-AMPK/AMPK protein expressions in myocardial tissue were significantly reduced(P<0.05).Compared with model group,DG significantly improved myocardial morphology,reduced myocardial cell apoptosis rate,CK-MB activity,cTnI level in serum and IL-1β,TNF-αlevels in myocardial tissue(P<0.05),downregulated CVB3 mRNA and cleaved Caspase-9,cleaved Caspase-3,p62,p-mTOR/mTOR protein expressions in myocardial tissue(P<0.05),upregulated LC3-II/I,Beclin-1,p-AMPK/AMPK protein expressions in myocardial tissue(P<0.05).The AMPK inhibitor Compound C could weaken the aforementioned protective effect of DG,and the autophagy activator rapamycin could inhibit this weakening effect.Conclusion DG may induce autophagy by activating AMPK/mTOR pathway and alleviate myocardial damage in VMC mice.