γ射线照射导致心肌细胞坏死性凋亡和线粒体损伤

Effect of γ-ray irradiation on cardiomyocyte necroptosis and mitochondrial injury

目的探讨γ射线照射引起的心肌细胞死亡方式及其线粒体损伤效应。方法用;Coγ射线单次照射大鼠H9C2心肌细胞,按照射后时间分为照射后24,48和72 h组,按照射剂量分为细胞对照组及5,10和20 Gy组。用CCK-8试剂盒检测细胞存活率;APC-AnnexinⅤ/7AAD凋亡试剂盒检测细胞凋亡;Western印迹法检测细胞凋亡标志物——活化的胱天蛋白酶3和胱天蛋白酶原3及坏死性凋亡标志物——受体相互作用蛋白激酶1(RIPK1)、RIPK3、磷酸化混合系列蛋白激酶样结构域(p-MLKL)和线粒体酪氨酸磷酸化酶1(PTPMT1)蛋白表达水平;通过荧光探针H2DCF-DA标记,用流式细胞术检测细胞产生活性氧(ROS)水平;通过JC-1探针标记,分别用激光共聚焦显微镜和流式细胞术定性和定量检测细胞线粒体膜电位;通过钙黄绿素-AM探针标记,分别用激光共聚焦显微镜和流式细胞术定性和定量检测细胞线粒体膜通道孔(mPTP)开放状态。结果与细胞对照组相比,20 Gy照射各时间组及5和10 Gy照射48和72 h组H9C2细胞存活率降低(P<0.05);20 Gy照射各时间组H9C2细胞凋亡率显著增加(P<0.01),活化的胱天蛋白酶3水平升高(P<0.05);10 Gy照射48和72 h组H9C2细胞凋亡率增加(P<0.05),48 h组活化的胱天蛋白酶3水平升高(P<0.05)。20 Gy照射各时间组RIPK1,RIPK3和P-MLKL表达水平显著升高(P<0.05,P<0.01),ROS增加(P<0.01),线粒体膜电位下降(P<0.01),mPTP开放增加(P<0.01);10 Gy照射48 h组RIPK1,RIPK3和p-MLKL表达水平显著升高(P<0.05,P<0.01),各时间组ROS产生增加(P<0.05,P<0.01)且线粒体膜电位下降(P<0.05,P<0.01),48 h组mPTP开放增加(P<0.01)。5 Gy照射各时间组RIPK3表达水平显著升高(P<0.01),24 h组ROS产生增加(P<0.05),48 h组线粒体膜电位下降(P<0.05)且mPTP开放增加(P<0.05)。各照射剂量各时间组PTPMT1表达水平均显著降低(P<0.05,P<0.01)。结论γ射线照射可导致大鼠心肌细胞H9C2发生坏死性凋亡,并导致线粒体损伤及氧化应激水平升高,PTPMT1表达降低可能与该过程有关。

OBJECTIVE To investigate the way in which cardiomyocytes die and mitochondrial damage caused by γ-ray irradiation. METHODS H9 C2 cells(rat cardiomyocytes) that had been irradiated with a single dose of;Co γ-ray were divided into 24, 48 and 72 h groups according to the duration of irradiation before being re-divided into the cell control group and 5,10 and 20 Gy groups according to the irradiation dose. CCK8 kit was used to detect the cell survival rate. The apoptosis rate was detected by APC-AnnexinⅤ/7 AAD apoptosis kit. Western blotting was used to detect the protein expression levels of apoptosis marker—cleaved-caspase 3, pro-caspase 3 and necroptosis marker—receptor protein-interacting kinase 1(RIPK1), RIPK3, phosphorylated mixed series protein kinase-like domain(p-MLKL) and protein tyrosine phosphatase, mitochondrial 1(PTPMT1). The level of reactive oxygen species(ROS)was detected by flow cytometry with fluorescent probe H2 DCF-DA labeling, and the changes of mitochondrial membrane potential(ΔΨm) were detected by flow cytometry or laser confocal fluorescence microscopy with JC-1 probe labeling. The open state of the mitochondrial permeability transition pore(mPTP) was detected by flow cytometry or the laser confocal fluorescence microscope with CalceinAM probe labeling. RESULTS Compared with the cell control group, the survival rate of H9 C2 cells at each time point of 20 Gy group decreased(P<0.05), so did the survival rate of 5 Gy and 10 Gy groups at 48 and 72 h time point. The apoptosis rates of H9 C2 cells were significantly increased at each time point of 20 Gy group(P<0.01), and levels of cleaved-caspase 3 were increased(P<0.05). The apoptosis rates of H9 C2 cells of 10 Gy group were increased at 48 and 72 h time points(P<0.05), so did the level of cleaved-caspase 3 of 10 Gy group at 48 h time point(P<0.05). The levels of RIPK1, RIPK3 and p-MLKL were significantly increased at each time point of 20 Gy group(P<0.05, P<0.01), so did ROS production(P<0.01), but mitochondrial membrane potential was decreased(P<0.01), and opening of mPTP was increased(P<0.01). The levels of RIPK1, RIPK3 and p-MLKL of 10 Gy group at 48 h time point were significantly increased(P<0.05, P<0.01). In addition, at each time point of 10 Gy group, ROS production was increased(P<0.05, P<0.01), but mitochondrial membrane potential was decreased(P<0.05, P<0.01).Furthermore, the opening of mPTP of 10 Gy group was increased at 48 h time point(P<0.01). In 5 Gy group, the levels of RIPK3 were significantly increased at each time point(P<0.01), ROS production was increased at 24 h time point(P<0.05), mitochondrial membrane potential was decreased at 48 h time point(P<0.05), and opening of mPTP was increased at 48 h time point(P<0.05). What′s more,the levels of PTPMT1 were significantly decreased at each dose and each time point(P<0.05, P<0.01).CONCLUSION γ-Ray irradiation can induce H9 C2 cell apoptosis, necroptosis, mitochondrial damage and oxidative stress. The decrease of PTPMT1 expression may be related to this process.