ERK信号通路对病毒性心肌炎心肌细胞CAR表达的影响

目的:观察细胞外信号调节激酶(Extracellular Regulated Protein Kinases,ERK1/2)信号通路对病毒性心肌炎(Viral Myocarditis,VMC)心肌细胞柯萨奇病毒-腺病毒受体(Coxsackie-adenovirus Receptor,CAR)表达的影响。方法:新生SD大鼠心肌细胞体外培养48 h后随机分为3组,除对照组外均体外接种柯萨奇B3m病毒(Coxsackievirus B,CVB),建立VMC细胞模型。C组:DMEM对照组;V组:CVB3m感染组;U+V组:接种病毒前30 min,给予ERK1/2通路抑制剂U0126(10μmol/L)。各组分别于种毒后12 h、24 h、36 h取心肌细胞,用Western blot法测定ERK1/2活化水平及CAR表达量,并按上述时间点观察各组心肌细胞形态、搏动情况、细胞损伤程度,取培养液测定乳酸脱氢酶(LDH)水平。结果:在接种病毒后12 h,V组与C组相比,P-ERK1/2表达增高(3.25±0.61 vs 0.59±0.09,P0.05),CAR表达增高(1.03±0.17 vs 0.78±0.11,P0.05),逐步出现细胞病变,细胞搏动停止,培养液中LDH水平明显增高(1016.67±67.75 vs 336.34±28.67,P0.05),心肌酶学的升高与镜下心肌细胞损伤程度平行;U+V组与V组相比,P-ERK1/2表达降低(1.66±0.28 vs 3.25±0.61,P0.05),CAR表达明显增高(1.73±0.27 vs 1.03±0.17,P0.05),但细胞损伤却明显减轻,LDH水平明显降低(410.06±13.62 vs 1016.67±67.75,P0.05)。动态观察24 h、36 h,同样出现上述变化趋势。结论:ERK1/2信号转导通路参与心肌细胞感染CVB发生急性损伤的过程,并参与调控CAR的表达。在病毒感染后36 h内,阻断ERK1/2信号通路,CAR表达上调,并未加重心肌细胞损伤。

Roles of ERK Signal Transduction Pathway to Coxsackie-adenovirus Receptor (CAR) in Cardiocytes during Viral Myocarditis

Objective:To investigate the role of extracellular signal-regulated kinase (ERK) signal transduction pathway to coxsackie-adenovirus receptor (CAR) in cardiocytes during viral myocarditis (VMC).Methods:Primary cultured new born SD rat cardiocytes were infected by CVB3m to establish a model of viral myocarditis. Forty-eight hours after culturing, cardiomyocytes were divided into 3 groups. Group C: cardiomyocytes were incubated with DMEM as controls; group V: cardiomyocytes were incubated with CVB solutions; group U+V: cardiomyocytes were incubated with U0126 (10 umol/L) for 30 min initially, and then incubated with CVB solutions. After processing, the level of P-ERK1/2 and CAR were detected by Western blot. CVB mediated cytopathic effects were observed after co-culturing for further 12, 24, 36 hours respectively, lesions of the cardiocytes were examined by LDH assay.Results:Twelve hous after infected by CVB, comparing to group C, the level of P-ERK1/2 in group V increased (3.25± 0.61 vs 0.59± 0.09, P<0. 05), the exrpession of CAR increased (1.03± 0.17 vs 0.78± 0.11, P>0.05), the beating rate decreased gradually and then stopped, the level of cardiac enzymes increased (1016.67± 67.75 vs 336.34± 28.67, P<0.05). In group U+V, comparing to group V, the level of P-ERK1/2 decreased (1.66± 0.28 vs 3.25± 0.61, P<0.05), the exrpession of CAR increased significantly (1.73± 0.27 vs 1.03± 0.17, P<0. 05), the injury to cardiocyte extenuates, so was the level of cardiac enzymes (410.06± 13.62 vs 1016.67± 67.75, P<0.05). During the following 24 and 36 hours, we can also observe the same change.Conclusion:When cardiocytes were infected by CVB3m in acute phase, ERK1/2 signal transduction pathway was involved in it. It also participated in the regulation of CAR. Inhibition of this pathway did not aggravate the injury of cardiac cells.