硒对病毒性心肌炎小鼠心肌细胞凋亡的抑制作用及其作用机制

doi:10.13481/j.1671-587x.20160111

摘要/Abstract

摘要：

目的: 探讨硒对病毒性心肌炎(VMC)小鼠心肌细胞凋亡的抑制作用,阐明其对PI3K-Akt信号传导通路中Akt、磷酸化Akt(p-Akt)及其下游靶基因编码的Bax和Bcl-2蛋白的作用机制。方法: 60只BALB/c小鼠随机分为正常对照组、病毒对照组和硒干预组(n=20)。正常对照组小鼠连续3 d腹腔注射不含病毒的培养液200 μL;病毒对照组小鼠腹腔注射100半数组织培养感染剂量(TCID₅₀)柯萨奇病毒B组病毒(CVB3)液200μL,连续3d;硒干预组小鼠在病毒对照组同样的处理后灌胃给予100 μg·kg^-1亚硒酸钠,每日1次,连续处理14d。接种CVB3的第15天处死小鼠,TUNEL法检测心肌细胞的凋亡情况,光镜下观察心肌病理变化,Western blotting法检测心肌细胞中Akt、p-Akt、Bcl-2、Bax和Cleaved caspase-3蛋白表达水平。结果: 与病毒对照组比较,硒干预组小鼠生存率明显升高(P<0.01)。硒干预组小鼠的心肌病理改变较病毒对照组减轻。硒干预组小鼠心肌细胞凋亡率低于病毒对照组(P<0.01)。与病毒对照组比较,硒干预组小鼠心肌细胞中p-Akt、Bcl-2蛋白表达水平增加,Bax、Cleaved caspase-3蛋白表达水平明显降低,Bax/Bcl-2蛋白比值下降(均P<0.01)。结论: 硒对CVB3感染导致的VMC小鼠心肌细胞有保护作用,其作用机制与硒通过活化心肌细胞的PI3K-Akt信号通路、调控其下游Bcl-2和Bax蛋白表达和抑制心肌细胞凋亡有关。

关键词: 病毒性心肌炎, 硒, 细胞凋亡, Bcl-2, Bax, PI3K-Akt信号通路

Abstract:

Objective: To investigate the inhibitory effect of selenium on the apoptosis of myocardial cells in the mice with viral myocarditis(VMC), and to clarify its influence in the expressions of Akt,p-Akt of PI3K-Akt signaling pathway and their down-stream protein Bax and Bcl-2. Methods: Sixty male BALB/c mice were randomly divided into blank control group,virus control group and selenium intervention group (n=20).The mice in virus control group and selenium intervention group were intraperitoneally injected with 200 μL fluid including 100 TCID₅₀ coxsackievirus B3(CVB3)for 3 d to establish the viral myocarditis models.The mice in blank control group were treated intraperitoneally with 200 μL fluid without virus for 3 d.The rats in selenium intervention group were given 100 μg·kg^-1 sodium selenite by intragastric administration for 14 d continuously after modeling.All the mice were killed on the 15th day after the CVB3 injection.TUNEL analysis was used to detect the apoptosis of myocardial cells,and the pathological changes of myocardial cells were examined by light microscope.The expression levels of Akt,p-Akt,Bax,Bcl-2 and Cleaved caspase-3 were detected by Western blotting method.Results: The survival rate of the mice in selenium intervention group was higher than that in virus control group(P<0.01).The pathological changes of myocardium of the rats in in selenium intervention group were improved compared with virus control group.The apoptotic rate of myocardial cells in selenium intervention group was lower than that in virus control group (P<0.01).Compared with virus control group,the expression levels of Bax and Cleaved caspase-3 in selenium intervention group were decreased and the expression levels of Bcl-2 and p-Akt were increased (P<0.01).Conclusion: Selenium can inhibit the CVB3 virus induced apoptosis and play a protective effect on the myocardium in the mice with VMC through affecting the expressions of Akt,p-Akt of PI3K-Akt signaling pathway and regulating the down-stream Bax and Bcl-2 protein expressions and inhibiting the apoptosis of myocardial cells.

Key words: viral myocarditis, selenium, apoptosis, Bcl-2, Bax, PI3K-Akt signaling pathway

中图分类号:

R-332

黎萍, 郑百红, 陈鹏, 许忠, 张磊, 潘薇. 硒对病毒性心肌炎小鼠心肌细胞凋亡的抑制作用及其作用机制[J]. 吉林大学学报(医学版), 2016, 42(01): 54-58.

LI Ping, ZHENG Baihong, CHEN Peng, XU Zhong, ZHANG Lei, PAN Wei. Inhibitory effect of selenium on apoptosis of myocardial cells in mice with viral myocarditis and its mechanism[J]. Journal of Jilin University Medicine Edition, 2016, 42(01): 54-58.

参考文献

[1] Pankuweit S,Klingel K.Viral myocarditis: from experimental models to molecular diagnosis in patients[J].Heart Fail Rev,2013,18(6):683-702.

[2] Fairweather D,Stafford KA,Sung YK.Update on coxsackievirus B3 myocarditis[J].Curr Opin Rheumatol,2012,24(4):401-407.

[3] Chen Z,Yang L,Liu Y,et al.LY294002 and Rapamycin promote coxsackievirus-induced cytopathic effect and apoptosis via inhibition of PI3K/Akt/mTOR signaling pathway[J].Mol Cell Biochem,2014,385(1/2):169-177.

[4] 郑百红,毓明涛,郑百波,等.微量元素硒对急性病毒性心肌炎患儿脂质过氧化损伤的影响[J].吉林大学学报:医学版,2004,30(3):437-440.

[5] 李小华,王爱民,刘莉莉,等.硒对4-羟基壬烯酸和β淀粉样蛋白诱导的神经细胞凋亡抗氧化保护作用[J].中华老年心脑血管病杂志,2014,16(4):428-430.

[6] Musik I,Kocot J,Lewandowska A,et al.The investigation of the possible protective influence of selenium on antioxidant barrier in heart of rats exposed to lithium[J].Life Sci,2015,132(1):1-5.

[7] Benstoem C,Goetzenich A,Kraemer S,et al.Selenium and its supplementation in cardiovascular disease-what do we know?[J].Nutrients,2015,7(5):3094-3118.

[8] Yao H,Han X,Han X.The cardioprotection of the insulin-mediated PI3K/Akt/mTOR signaling pathway[J].Am J Cardiovasc Drugs,2014,14(6):433-442.

[9] Ursu ON,Sauter M,Ettischer N,et al.Heme oxygenase-1 mediates oxidative stress and apoptosis in coxsackievirus B3-induced myocarditis[J].Cell Physiol Biochem,2014,33(1):52-66.

[10] 邓旺,赵燕,何婧,等,胰岛素通过PI3K信号通路对肺泡上皮钠通道β亚基表达的调控[J].吉林大学学报:医学版,2014,40(2):233-237.

[11] Diehl N,Schaal H.Make yourself at home: viral hijacking of the PI3K/Akt signaling pathway[J].Viruses,2013,5(12):3192-3212.

[12] Liang K,Ye Y,Wang Y,et al.Formononetin mediates neuroprotection against cerebral ischemia/reperfusion in rats via downregulation of the Bax/Bcl-2 ratio and upregulation PI3K/Akt signaling pathway[J].J Neurol Sci,2014,344(1/2):100-104.

[13] Umoh NA,Walker RK,Al-Rubaiee M,et al.Acute alcohol modulates cardiac function as PI3K/Akt regulates oxidative stress[J].Alcohol Clin Exp Res,2014,38(7):1847-1864.

[14] 张静,马翠丽,王志国.黄芪甲苷对大鼠心肌局部缺血再灌注损伤的改善作用及其对PI3K/Akt/mTOR信号通路的影响[J].吉林大学学报:医学版,2014,40(5):991-996,1130.

[15] Chau DH,Yuan J,Zhang H,et al.Coxsackievirus B3 proteases 2A and 3C induce apoptotic cell death through mitochondrial injury and cleavage of eIF4GI but not DAP5/p97/NAT1[J].Apoptosis,2007,12(3):513-524.

[16] Saraste A,Arola A,Vuorinen T,et al.,Cardiomyocyte apoptosis in experimental coxsackievirus B3 myocarditis[J].Cardiovasc Pathol,2003,12(5):255-262.

[17] Zhang T,Saghatelian A.Emerging roles of lipids in BCL-2 family-regulated apoptosis[J].Biochim Biophys Acta,2013,1831(10):1542-1554.

[18] 潘晓波,孙景辉,高齐.儿童病毒性心肌炎诊疗进展[J].中国实验诊断学,2014,18(5):863-867.

[19] 黎萍,郑百红,潘薇,等.硒对病毒性心肌炎小鼠心肌的保护作用及其机制研究[J].中国实验诊断学,2015,19(3):352-354.