摘要
目的 在细胞水平上探讨亚砷酸钠(NaAsO2)诱导小鼠海马神经元细胞(HT22细胞)凋亡的潜在机制,为解决神经退行性疾病提供参考.方法 采用CCK-8法检测NaAsO2对HT22细胞活性的影响.不同剂量(0、2、4、6 μmol/L)NaAsO2处理HT22细胞24 h,采用Annexin V-FITC/PI流式细胞术检测细胞凋亡率;采用JC-1探针结合荧光显微镜检测HT22细胞线粒体膜电位的水平变化.采用蛋白质印迹法(Western blot)检测细胞中凋亡相关蛋白Bcl-2、Bax、Cleaved-caspase 3的表达情况.采用实时荧光定量PCR及Western blot检测FTO/DRP1 mRNA及蛋白的表达情况.结果 与对照组比较,不同剂量NaAsO2处理组HT22细胞活性明显降低(F=716.700,P＜0.01),线粒体膜电位明显下降(F=2 028.000,P＜0.01),细胞凋亡率明显升高(F=56.030,P＜0.01).Bcl-2的表达明显下调(F=63.250,P＜0.01),Bax和Cleaved-caspase 3 的表达明显上调(F=39.580、57.480,P＜0.01),Bax/Bcl-2表达也明显上调(F=51.250,P＜0.01).此外,FTO表达水平升高(F=62.680,P＜0.01),DRP1和p-DRP1(Ser616)表达水平明显升高(F=155.000、78.300,P＜0.01),P-DRP1(Ser616)/DRP 1 的表达水平也明显升高(F=18.050,P＜0.01).结论 砷可能是通过激活FTO/DRP1信号通路,激活线粒体介导的内在HT22细胞凋亡途径导致细胞凋亡.
Abstract
Objective To explore the potential mechanism of apoptosis of mouse hippocampal neurons(HT22 cells)induced by sodium arsenite(NaAsO2)at the cellular level,and to provide reference for solving neurodegenerative diseases.Methods The effect of NaAsO2 on the viability of HT22 cells was detected by CCK-8 method.HT22 cells were treated with different doses of NaAsO2(0,2,4,6 μmol/L)for 24 hours,the apoptosis rate of HT22 cells was detected by Annexin V-FITC/PI flow cytometry,and the changes of mitochondrial membrane potential of HT22 cells were detected by JC-1 probe and fluorescence microscope.Western blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bax and Cleaved-caspase-3.Real-time fluorescence quantitative PCR and Western blot were used to detect the expression of FTO/DRP1 mRNA and protein.Results Compared with the control group,the viability of HT22 cells treated with different doses of NaAsO2 decreased significantly(F=716.700,P＜0.01),the mitochondrial membrane potential decreased significantly(F=2 028.000,P＜0.01),and the apoptosis rate increased significantly(F=56.030,P＜0.01).The expression of Bcl-2 was down-regulated(F=63.250,P＜0.01),the expression of Bax and Cleaved-caspase-3 was up-regulated(F=39.580,57.480,P＜0.01),and the expression of Bax/Bcl-2 was also up-regulated(F=51.250,P＜0.01).In addition,the expression of FTO,DRP1,p-DRP1(Ser616)and P-DRP1(Ser616)/DRP1 increased significantly(F=62.680,155.000,78.300,18.050,P＜0.01).Conclusion Arsenic may induce apoptosis by activating FTO/DRP1 signal pathway and mitochondrial-mediated apoptosis pathway in HT22 cells.
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