二甲基甲酰胺致H9c2心肌细胞炎性损伤研究

Research on the inflammatory injury induced by N,N-dimethylformamide in H9c2 cardiomyocytes

目的探讨二甲基甲酰胺(DMF)对H9c2心肌细胞炎性损伤的影响及其可能的机制。方法取离体培养H9c2心肌细胞,设对照组和50、100、200 mmol/L组,分别予浓度为0、50、100、200 mmol/L DMF处理12 h;设对照组和2、4、6、8、12 h组,分别予浓度为200 mmol/L DMF处理0、2、4、6、8、12 h。以比色法检测乳酸脱氢酶(LDH)活力,紫外分光光度法检测肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)活力,酶联免疫吸附法检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、IL-8水平,荧光探针法检测活性氧水平,免疫荧光细胞化学(IFC)法检测核转录因子-κB(NF-κB)p65蛋白定位。结果与对照组比较,50、100、200 mmol/L组细胞LDH、CK和CK-MB活力均升高(P<0.05),均呈剂量-效应关系(P<0.01);6、8、12 h组细胞LDH、CK和CK-MB活力均升高(P<0.01),呈时间-效应关系(P<0.01)。与对照组比较,200 mmol/L组细胞TNF-α、IL-1β、IL-6和IL-8水平均升高(P<0.05),4和12 h组细胞TNF-α水平均升高(P<0.05),2、4、6、8和12 h组细胞IL-1β水平均升高(P<0.05),2和4 h组细胞IL-6水平均升高(P<0.05),2 h组细胞IL-8水平升高(P<0.05);且TNF-α、IL-1β和IL-6水平在4 h组达到高峰,IL-8水平在2 h组达到高峰。2、4和6 h组细胞内活性氧水平均高于对照组(P<0.05);活性氧水平在2 h组达到高峰。IFC法对细胞内NF-κB p65蛋白定位显示,染毒后2、4 h组细胞核内p65蛋白荧光强度较对照组增强。结论 DMF对H9c2心肌细胞造成炎性损伤,且活性氧和NF-κB可能参与炎性损伤过程。

Objective To explore the effect of N,N-dimethylformamide（ DMF）-induced inflammatory injury in H9c2 cardiomyocytes and its mechanism. Methods H9c2 cardiomyocytes were cultured in vitro and randomly divided into 4different groups： control group,50 mmol / L-group,100 mmol / L-group,200 mmol / L-group. These 4 groups of cells were treated with different DMF concentrations（ 0,50,100,200 mmol / L） for 12 hours. The cells were also divided into 6groups and treated with 200 mmol / L DMF at different time points（ 0,2,4,6,8,12 h） ： control group,2 h-group,4 hgroup,6 h-group,8 h-group and 12 h-group. The level of lactate dehydrogenase（ LDH） was detected by colorimetry. The levels of creatine kinase（ CK） and isoenzyme of creatine kinase（ CK-MB） were detected by ultraviolet spectrometry. The levels of tumor necrosis factor-α（ TNF-α）,interleukin（ IL）-1β,IL-6,and IL-8 were detected by enzyme linked immunosorbent assay. The level of reactive oxygen species（ ROS） was detected by fluorescence probe. The location of nuclear factor-kappa B（ NF-κB） p65 protein was detected by immunofluorescence cytochemistry（ IFC） staining. Results The levels of LDH,CK and CK-MB in the 50 mmol / L-group,100 mmol / L-group and 200 mmol / L-group were higher than that of the control group（ P〈0. 05） and showed a significant dose-effect（ P〈0. 05）. The levels of LDH,CK and CK-MB in the 6 h-group,8 h-group and 12 h-group were higher than that of the control group（ P〈0. 01） and showed a significant time-effect（ P〈0. 01）. The levels of TNF-α,IL-1β,IL-6 and IL-8 of the 200 mmol / L-group were higher than the control group（ P〈0. 05）. Compared with the control group,the levels of TNF-α of the 4 h-group,12 h-group were higher（ P〈0. 05）,the levels of IL-1β of the 2 h-group,4 h-group,6 h-group,8 h-group and 12 h-group were higher（ P〈0. 05）,the levels of IL-6 of the 2 h-group and 4 h-group were higher（ P〈0. 05）,the level of IL-8 of the 2 h-group was higher（ P〈0. 05）. In addition,the levels of TNF-α,IL-1β and IL-6 reached a peak at 4 h-group and the level of IL-8 reached a peak at 2 h-group. The ROS levels of the 2 h-group,4 h-group and 6 h-group were higher than the control group（ P〈0. 01）,and the level of ROS reached a peak at 2 h-group. Furthermore,IFC staining showed that the fluorescence intensity of NF-κB p65 protein in nucleus of the 2h-group and 4 h-group increased after treatment with DMF,comparing with the control group. Conclusion DMF leads to inflammatory injury in H9c2 cardiomyocytes. ROS and NF-κB might be involved in the process.