消退素D2对CVB3诱导的病毒性心肌炎小鼠心肌细胞凋亡的保护作用及机制研究

Protective effect of resolvin D2 on myocardial apoptosis in mice with viral myocarditis and its mechanism

目的探讨消退素D2(RvD2)对柯萨奇B3病毒(CVB3)诱导的病毒性心肌炎心肌细胞凋亡的抑制作用以及可能的机制。方法40只雄性BALB/c小鼠通过随机数字表法分为4组,每组10只,分别为正常对照组、病毒性心肌炎组、RvD2治疗组以及丝氨酸/苏氨酸蛋白激酶(AKT)抑制剂处理组。后3组小鼠腹腔内注射CVB3病毒构建病毒性心肌炎模型;然后RvD2治疗组和AKT抑制剂处理组小鼠连续腹腔注射相应药物7 d。7 d后处死4组小鼠并留取心脏及血清标本。前3组小鼠通过病理染色评估心肌组织炎症程度,ELISA法测定血清肌钙蛋白I(cTnI)、B型脑钠肽(BNP)水平,Western blot法测定心肌组织中凋亡相关蛋白半胱氨酸蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2关联X蛋白(Bax)水平以及AKT蛋白磷酸化水平;最后,通过Western blot法测定AKT抑制剂处理组小鼠心肌组织中AKT蛋白磷酸化水平以及凋亡蛋白水平。结果与对照组相比,病毒性心肌炎组小鼠心肌组织内炎症反应增加,血清cTnI、BNP水平上调,心肌组织内Caspase-3、Bax水平和AKT磷酸化水平上调,而Bcl-2水平下调(均P<0.05)。与病毒性心肌炎组相比,RvD2治疗组小鼠心肌组织内炎症反应降低,血清cTnI、BNP水平下调,心肌组织内Caspase-3、Bax水平下调,而Bcl-2水平和AKT蛋白磷酸化水平上调(均P<0.05)。与RvD2治疗组相比,AKT抑制剂处理组小鼠心肌组织内Caspase-3、Bax水平上调,而Bcl-2水平和AKT蛋白磷酸化水平下调(均P<0.05)。结论RvD2可通过调控AKT信号通路抑制CVB3感染诱导的病毒性心肌炎小鼠心肌细胞凋亡。

Objective To investigate the effect of Resolvin D2 on myocardial apoptosis in mice with viral myocarditis induced and its mechanism.Methods Forty male BALB/c mice were randomly divided into 4 groups with 10 mice in each group:normal control group,viral myocarditis group,RvD2-treated group,and AKT inhibitor-treated group.The viral myocarditis model was induced by intraperitoneal injection of Coxsackievirus B3(CVB3)in BALB/c mice.The RvD2-treated group and AKT inhibitor-treated group were given intraperitoneal injection of RvD2 or AKT inhibitor for 7 days,respectively.After mice were sacrificed,the heart tissues and serum samples were collected.HE staining was used to detect the infiltration of inflammatory cells in the myocardial tissue of the first three groups.And the expression levels of troponin(cTnI)and brain natriuretic peptide(BNP)in the serum were detected by ELISA.The expression levels of apoptosis-related proteins including Caspase 3,Bax and Bcl 2 as well as the phosphorylation levels of AKT protein in the myocardial tissues were detected by Western blot.Results Compared with the normal control group,the degree of inflammatory cell infiltration in the myocardial tissue of the viral myocarditis group was significantly increased,and the expression levels of serum cTnI and BNP as well as the expression levels of Caspase-3 and Bax were significantly up-regulated,while the expression level of Bcl 2 protein was significantly down-regulated(all P<0.05).In the RvD2-treated group,compared with the viral myocarditis group,the degree of inflammatory cell infiltration in the myocardial tissue was significantly reduced and the expression levels of serum cTnI and BNP were significantly down-regulated.The expression levels of Caspase 3 and Bax in the myocardial tissue were significantly down-regulated;while the expression level of Bcl 2 protein and the phosphorylation level of AKT protein were significantly up-regulated(all P<0.05).Compared with the RvD2-treated group,the expression levels of Caspase-3 and Bax in the myocardial tissues were significantly up-regulated in the AKT inhibitor-treated group,while the expression level of Bcl-2 protein and the phosphorylation level of AKT were down-regulated(all P<0.05).Conclusion RvD2 can inhibit myocardial apoptosis in mice with viral myocarditis induced by CVB3 infection by regulating the AKT signaling pathway.