ROS/PARP-1/AIF信号通路在糖皮质激素诱导的MC3T3-E1细胞凋亡过程中的作用研究

The role of ROS/PARP-1/AIF signaling pathway in glucocorticoid induced apoptosis in MC3T3-E1 cells

目的通过用地塞米松诱导小鼠胚胎成骨细胞前体细胞(MC3T3-E1)的凋亡,建立激素性股骨头坏死的细胞模型,探讨抗氧化剂(N-乙酰-L-半胱氨酸,NAC)和钙蛋白酶的抑制剂(PD151746)对MC3T3-E1细胞抑制凋亡的作用和可能机制的研究。方法 CCK-8检测NAC和PD151746不同药物浓度对MC3T3-E1细胞增殖的影响,选择合适的药物浓度。细胞分组:正常组(10%FBS),激素组(地塞米松1×10^(-6) mol/L),NAC组(NAC1 mmol/L+地塞米松1×10^(-6) mol/L)和PD151746组(简称PD组,PD 10 mol/L+地塞米松1×10-6 mol/L),流式细胞仪检测细胞凋亡率和ROS含量,激光扫描共聚焦显微镜检测各组ROS荧光强度和钙离子荧光强度,Western blot测定细胞蛋白AIF、PARP-1和Cyt-c的表达水平。结果 CCK-8结果显示,NAC和PD151746的实验浓度分别取1 mmol/L和10 mol/L。流式细胞仪检测凋亡率结果显示,激素组、NAC组和PD组的凋亡率与正常组比较,差异有统计学意义(<0.05),激素组与NAC组、PD组比较差异有统计学意义(<0.05)。流式细胞仪和激光扫描共聚焦显微镜检测各组ROS含量,结果显示激素组荧光强度最高,与NAC组和PD组比较明显增高(<0.05),其他三组与正常组比较差异也具有统计学意义(<0.05)。激光扫描共聚焦显微镜检测钙离子荧光强度,与NAC组和PD组比较明显增高(<0.05),其他三组与正常组比较差异也具有统计学意义(<0.05)。Western blot检测各组AIF、PARP-1和Cyt-c的表达水平,激素组、NAC组和PD组的凋亡率与正常组比较,差异有统计学意义(<0.05),激素组与NAC组、PD组比较差异有统计学意义(<0.05)。结论 NAC能抑制地塞米松诱导的ROS产生,PD151746能有效抑制钙蛋白酶的活性,抑制PAPR-1/AIF的释放,抑制成骨细胞凋亡,ROS/PARP-1/AIF通路参与了地塞米松诱导MC3T3-E1细胞的凋亡过程。

Objective A cell model was established of steroid necrosis of the femoral head by using dexamethasone induced apoptosis of mouse embryonic osteoblast precursor cells(MC3 T3-E1). To investigate the effects of antioxidants(Nacetyl-L-cysteine) and calpain inhibitor(PD151746) inhibit apoptosis in MC3 T3-E1 cells and study on the possible mechanism. Methods CCK-8 detect the effects of different drug concentrations of NAC and PD151746 on the proliferation of MC3 T3-E1 cells and select the appropriate drug concentration. The osteoblasts were divided into the following groups:normal group(10% FBS), hormone group(1×10^-6 mol/L Dex), NAC group(NAC 1 mmol/L+1×10^-6 mol/L Dex)and PD151746 group referred to as PD group(PD 10 mol/L+ 1×10^-6 mol/L Dex). Flow cytometry was used to detect apoptotic rate and ROS content, laser scanning confocal microscopy was used to detect ROS fluorescence intensity and calcium dissociation in each group. The expression levels of AIF, PARP-1 and Cyt-c in each group were detected by Western blot. Results CCK-8 results showed that the experimental concentrations of NAC and PD151746 were 1 mmol/L and 10 mol/L, respectively. Flow cytometry showed that the apoptotic rate of hormone group, NAC group and PD group was significantly higher than that of normal group( <0.05), and there was significant difference between hormone group and NAC group and PD group( <0.05). Flow cytometry and laser scanning confocal microscopy were used to detect the ROS content in each group. The results showed that the fluorescence intensity of hormone group was the highest,which was significantly higher than that of NAC group and PD group( <0.05), and the difference between other three groups and normal group was also significant( <0.05). The fluorescence intensity of calcium ions detected by laser scanning confocal microscopy was significantlyhigher than that of NAC group and PD group( <0.05), and there was a significant difference between the other three groups and the normal group( <0.05). Western blot was used to detect the expression of AIF, PARP-1 and Cyt-c in each group. The apoptotic rate of hormone group, NAC group and PD group was significantly higher than that of normal group( <0.05). There was significant difference between hormone group and NAC group and PD group( <0.05).Conclusion NAC can inhibit the production of ROS induced by dexamethasone, PD151746 can effectively inhibit the activity of calpain, inhibition of the release of PAPR-1/AIF, inhibiting apoptosis of osteoblasts. ROS/PARP-1/AIF pathway is involved in apoptotic process of dexamethasone induced MC3 T3-E1 cells.