姜黄素调控miR-21对病毒性心肌炎细胞凋亡、炎症反应及NF-κB信号通路的影响

Effects of Curcumin Regulating miR-21 on Cell Apoptosis,Inflammatory Response,and NF-κB Signaling Pathway in Viral Myocarditis

目的:探讨姜黄素(Cur)是否通过调控微小RNA-21(miR-21)影响病毒性心肌炎(VMC)心肌细胞的凋亡及炎症反应。方法:用柯萨奇病毒B3(CVB3)感染心肌细胞建立VMC心肌细胞模型记为CVB3组,未经任何处理的心肌细胞作为对照组。以不同浓度(0.25,0.50,1.00μmol)的Cur处理心肌细胞进行干预,采用流式细胞术检测细胞凋亡率;以酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、单核细胞趋化因子-1(MCP-1)的含量;采用实时荧光定量聚合酶链反应(RT-qPCR)检测Cur对miR-21表达水平的影响;分别将anti-miR-con、anti-miR-21转染至心肌细胞后使用CVB3处理,分别将miR-con、miR-21 mimics转染至心肌细胞后使用Cur与CVB3处理,采用上述方法检测细胞凋亡率及炎性因子的含量;蛋白免疫印迹法(Western Blot)检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、核因子-κB(NF-κB)信号通路相关蛋白表达。结果:与对照组比较,CVB3组心肌细胞凋亡率[(6.81±0.60)%与(21.38±1.92)%]升高(P<0.05),miR-21[(1.00±0.05)与(1.84±0.12)]、Cleaved Caspase-3表达水平[(0.31±0.01)与(1.21±0.08)]、TNF-α[(105.44±9.15)ng/mL与(623.10±41.06)ng/mL]、IL-6[(89.01±6.11)pg/mL与(453.67±31.32)pg/mL]、MCP-1[(491.16±38.02)pg/mL与(1803.11±82.01)pg/mL]水平升高(P<0.05),Cur处理后能够逆转CVB3对VMC心肌细胞凋亡、炎性因子水平及NF-κB信号通路相关蛋白表达的影响;干扰miR-21表达可降低细胞凋亡率及TNF-α、IL-6、MCP-1水平(P<0.05),抑制Cleaved Caspase-3过表达,miR-21能够部分逆转Cur对VMC心肌细胞凋亡、炎症反应及NF-κB信号通路相关蛋白表达的影响。结论:Cur可通过下调miR-21的表达缓解VMC心肌细胞炎症反应而对心肌细胞发挥保护作用,其作用机制可能与抑制NF-κB信号通路活化及心肌细胞凋亡有关。

Objective:To investigate curcumin(Cur)affects the apoptosis and inflammatory response of viral myocarditis(VMC)cells by regulating microRNA-21(miR-21).Methods:The VMC cardiomyocyte model was established by infecting cardiomyocytes with Coxsackievirus B3(CVB3),which was recorded as the CVB3 group,and the cardiomyocytes without any treatment were used as the control group.Cardiomyocytes were treated with Cur at different concentrations(0.25μmol,0.50μmol,1.00μmol)for intervention,and the apoptosis rate was detected by flow cytometry.Enzyme-linked immunosorbent assay(ELISA)was used to detect the tumor necrosis factor-α(TNF-α),interleukin 6(IL-6),and monocyte chemoattractant-1(MCP-1);quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the effect of Cur on the expression level of miR-21.Cardiomyocytes were treated with CVB3,Cur and CVB3,respectively,after being transfected by anti-miR-con and anti-miR-21,respectively.to detect the apoptosis rate of cells and the content of inflammatory factors by the above method.Western Blot was used to detect the activated cysteine-containing aspartic acid proteolytic enzyme 3(Cleaved Caspase-3)and transcription factor-nuclear factor(NF-κB)signaling pathway-related protein expression.Results:Compared with the control group,the apoptosis rate of cardiomyocytes in the CVB3 group[(6.81±0.60)%vs(21.38±1.92)%]increased(P<0.05);miR-21[(1.00±0.05)vs(1.84±0.12)],Cleaved Caspase-3 expression level[(0.31±0.01)vs(1.21±0.08)],TNF-α[(105.44±9.15)ng/mL vs(623.10±41.06)ng/mL],IL-6[(89.01±6.11)pg/mL vs(453.67±31.32)pg/mL],MCP-1 level[(491.16±38.02)pg/mL vs(1803.11±82.01)pg/mL]increased(P<0.05).Cur treatment could reverse the effects of CVB3 on VMC cardiomyocyte apoptosis,inflammatory factor levels,and NF-κB signaling pathway-related protein expression.Interference with miR-21 expression could reduce cell apoptosis rate,the levels of TNF-α,IL-6,and MCP-1(P<0.05),and inhibit the overexpression of Cleaved Caspase-3.miR-21 can partially reverse the effects of Cur on VMC cardiomyocyte apoptosis,inflammatory response,and NF-κB signaling pathway-related protein expression.Conclusion:Cur could protect cardiomyocytes by down-regulating the expression of miR-21 and alleviating the inflammatory response of VMC cardiomyocytes,and its mechanism might be related to the inhibition of NF-κB signaling pathway activation and cardiomyocyte apoptosis.