沙库巴曲/缬沙坦通过PI3K/AKT通路抑制CVB3诱导的病毒性心肌炎小鼠中病毒复制及心肌细胞凋亡  

Sacubitril/valsartan inhibit viral replication and cardiomyocyte apoptosis in mice with CVB3-induced viral myocarditis via PI3K/AKT pathway

作　　者：刘文芹[1] 许竞 刘维克 陈奕琏 邱依旋 林元楠 李岳春[3] Liu Wenqin;Xu Jing;Liu Weike;Chen Yilian;Qiu Yixuan;Lin Yuannan;Li Yuechun(Digital Subtraction Angiography Center,the Second Affiliated Hospital and Yuying Children′s Hospital of Wenzhou Medical University,Wenzhou 325000,China;Cardiovascular Medicine Ward 2,Jinhua Municipal Central Hospital,Jinhua 321000,China;Department of Cardiology,the Second Affiliated Hospital and Yuying Children′s Hospital of Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]温州医科大学附属第二医院,育英儿童医院数字减影血管造影中心,温州325000 [2]金华市中心医院心血管内科二病区,金华321000 [3]温州医科大学附属第二医院,育英儿童医院心内科,温州325000

出　　处：《中华微生物学和免疫学杂志》2023年第7期547-554,共8页Chinese Journal of Microbiology and Immunology

基　　金：浙江省基础公益研究计划项目(LY22H020009);国家自然科学基金(81870281);温州市科技计划项目(Y20210137)。

摘　　要：目的观察沙库巴曲/缬沙坦(LCZ696)对柯萨奇病毒B3(CVB3)诱导的病毒性心肌炎(VMC)小鼠中病毒复制及心肌细胞凋亡的作用,并探究其具体机制。方法40只BALB/c小鼠随机分成4组(n=10):Sham组、Sham+LCZ696组、VMC组和VMC+LCZ696组。向BALB/c小鼠腹腔注射10^(6) TCID_(50)/ml浓度CVB30.1 ml建立VMC模型,Sham组注射等量生理盐水,病毒注射当天被定义为第0天。每天60 mg/kg沙库巴曲/缬沙坦进行灌胃治疗,连续7 d。统计小鼠生存率;小动物超声检测小鼠心功能;ELISA法检测肌酸激酶MB同工酶(CK-MB)水平;Western blot检测小鼠心脏促炎因子(IL-6、TNF-α),凋亡相关蛋白(caspase-3、cleaved-caspase-3、Bax、Bcl-2),CVB3病毒表面蛋白(VP-1)及相关通路蛋白(p-PI3K/PI3K、p-AKT/AKT)水平;PCR检测小鼠心脏中CVB3 mRNA水平;HE染色检测小鼠心脏炎性细胞浸润水平;TUNEL检测小鼠心脏组织中细胞凋亡水平。结果与Sham组相比,VMC组生存率下降,心功能指标(LVEDd、LVEDs、LVEF)减退(P<0.05)。VMC组小鼠血清CK-MB及心脏中IL-6、TNF-α、cleaved-caspase-3/caspase-3、Bax/Bcl-2、VP-1、CVB3 mRNA显著上升(P<0.05),同时AKT表达增多,而其磷酸化水平下降(P<0.05),细胞凋亡增多。LCZ696逆转了以上变化,表现为生存率上升,心功能改善(P<0.05),心脏炎症、凋亡与复制水平下调(P<0.05),p-AKT的水平上升(P<0.05)。沙库巴曲/缬沙坦对正常小鼠生存率、心功能、心肌损伤、心脏炎症、凋亡、病毒复制水平以及PI3K/AKT通路相关蛋白均无明显影响。结论沙库巴曲/缬沙坦可通过调控PI3K/AKT通路,显著抑制VMC小鼠的心肌细胞凋亡水平,并降低CVB3在小鼠心脏中的病毒复制水平,抑制心肌炎症,从而改善小鼠心功能及生存率。Objective To observe the effects of sacubitril/valsartan(LCZ696)on viral replication and cardiomyocyte apoptosis in mice with coxsackievirus B3(CVB3)-induced viral myocarditis(VMC)and to analyze the underlying mechanisms.Methods Forty BALB/c mice were randomly divided into four groups with 10 in each group:Sham,Sham+LCZ696,VMC,and VMC+LCZ696 groups.VMC model was established by intraperitoneal injection of 0.1 ml of CVB3 with a concentration of 10^(6) TCID_(50)/ml into BALB/c mice,while the sham intervention was an equal volume of saline.The day of virus injection was defined as day 0.LCZ696 was administered by gavage at a dose of 60 mg/kg every day for seven consecutive days starting from day 1.Mouse survival rates were calculated.Echocardiography was used to evaluate the cardiac function of mice.The level of creatine kinase-MB(CK-MB)was detected by ELISA.Western blot was used to detect the levels of inflammatory cytokines(IL-6,TNF-α),apoptosis-related proteins(caspase-3,cleaved-caspase-3,Bax,Bcl-2),CVB3 surface protein(VP-1)and p-AKT/AKT in the hearts of mice.CVB3 mRNA in mouse hearts was measured by PCR.Inflammatory cell infiltration and cell apoptosis in mouse hearts were observed by HE staining and TUNEL staining,respectively.Results Compared with the Sham group,the mice in the VMC group had a decreased survival rate and impaired cardiac function(P<0.05).The levels of CK-MB,IL-6,TNF-α,cleaved-caspase-3/caspase-3,Bax/Bcl-2,VP-1,and CVB3 mRNA in the hearts of VMC mice increased significantly(P<0.05),accompanied by increased expression of AKT,decreased phosphorylation of AKT(P<0.05)and increased cell apoptosis.LCZ696 reversed the above changes.It could increase the survival rate,improve the cardiac function(P<0.05),decrease cardiac inflammation,cell apoptosis and viral replication(P<0.05),and increase the phosphorylation of AKT(P<0.05).LCZ696 had no significant effects on the survival rate,cardiac function,myocardial injury,cardiac inflammation,cell apoptosis,viral replication or the expression of PI3K/AKT sig

关 键 词：沙库巴曲/缬沙坦 病毒性心肌炎 PI3K/AKT通路 病毒复制 凋亡 

分 类 号：R542.21[医药卫生—心血管疾病]