硒对一氧化氮介导心肌细胞凋亡保护性作用的研究

Protective effects of selenium on nitric oxide mediated myocardial apoptosis

目的探讨硒对一氧化氮(NO)介导心肌细胞凋亡的保护性作用。方法将体外培养的AC16心肌细胞分为对照组、硒处理组、硝普钠(sodium nitroprusside,SNP)处理组、硒+SNP处理组,SNP为外源性NO供体。对照组无任何干预,加入与各处理组等体积的培养液,硒处理组加入的硒剂量为100 μg/L,SNP处理组加入的SNP剂量为1.0 mmol/L,硒+SNP组在100 μg/L硒预处理4 h后加入1.0 mmol/L SNP;培养24 h后收集细胞或上清液。采用Griess法检测上清液NO含量,流式细胞仪检测细胞活性氧水平,荧光倒置显微镜下观察细胞膜电位变化情况及细胞凋亡状况,实时荧光定量PCR和蛋白免疫印迹法检测细胞凋亡相关基因B淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax)、Bcl-2 mRNA和蛋白表达水平。结果对照组、硒处理组、SNP处理组和硒+SNP处理组NO含量分别为(10.3 ± 1.8)、(9.2 ± 2.1)、(15.2 ± 3.5)、(14.3 ± 2.6)μmmol/L,SNP对NO含量存在主效应(F=23.33,P < 0.05);细胞活性氧水平分别为31.63 ± 1.40、29.52 ± 2.86、60.62 ± 4.83、50.08 ± 2.41,硒、SNP对活性氧水平存在主效应(F=12.19、187.20,P均< 0.05),二者也存在交互效应(F=5.42,P < 0.05);细胞线粒体膜电位水平分别为0.42 ± 0.11、0.37 ± 0.07、7.25 ± 1.91、5.21 ± 1.59,硒、SNP对细胞线粒体膜电位水平存在主效应(F=14.21、440.01,P均< 0.05),二者也存在交互效应(F=12.89,P < 0.05)。硒对细胞核固缩比例、Bax蛋白水平、Bcl-2 mRNA水平存在主效应(F=9.52、10.84、22.17,P均< 0.05);SNP对细胞核固缩比例,Bax、Bcl-2 mRNA水平,Bcl-2蛋白水平存在主效应(F=192.86、21.90、16.09、18.39,P均< 0.05);硒与SNP对Bax、Bcl-2 mRNA及蛋白水平存在交互效应(F=20.51、7.59、15.38、11.97,P均< 0.05)。结论SNP能够引起AC16心肌细胞发生凋亡,硒和SNP对AC16心肌细胞存在交互效应,硒对NO介导的心肌细胞凋亡有保护作用。

Objective To investigate the protective effects of selenium on nitric oxide(NO)-mediated myocardial apoptosis. Methods The AC16 cardiomyocyte cultured in vitro were divided into control group, selenium treatment group, sodium nitroprusside (SNP) treatment group and selenium+SNP treatment group, SNP was the exogenous NO donor. There was no intervention in the control group, and an equal volume of the culture solution was added to the treatment groups. The selenium treatment group added a dose of 100 μg/L of selenium, the SNP treatment group added a dose of 1.0 mmol/L of SNP, and the selenium+SNP treatment group was pretreated by 100 μg/L selenium for 4 h followed by 1.0 mmol/L SNP;the cells or supernatants were collected after 24 h of culture. The content of NO was detected by Griess method in supernatants. The level of cell reactive oxygen species was detected by flow cytometry. The changes of cell mitochondrial membrane potential and apoptosis were observed under fluorescence microscope. The real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of apoptosis-related genes B-cell lymphoma-2 (Bcl-2) associated X protein (Bax) and Bcl-2, respectively. Results The NO content in the control group, selenium treatment group, SNP treatment group and selenium+SNP treatment group were (10.3 ± 1.8),(9.2 ± 2.1),(15.2 ± 3.5),(14.3 ± 2.6)μmmol/L, respectively;SNP had a main effect on NO content (F=23.33, P < 0.05). The cell reactive oxygen species were 31.63 ± 1.40, 29.52 ± 2.86, 60.62 ± 4.83, 50.08 ± 2.41, respectively;selenium and SNP had main effects on reactive oxygen species(F=12.19, 187.20, P < 0.05), selenium combined with SNP had an interactive effect on reactive oxygen species (F=5.42, P < 0.05). The cell mitochondrial membrane potential levels were 0.42 ± 0.11, 0.37 ± 0.07, 7.25 ± 1.91, and 5.21 ± 1.59, respectively;selenium and SNP had main effects on cell mitochondrial membrane potential levels (F=14.21, 440.01, P < 0.05), selenium combined with SNP had an interactive effect on cell mitochondrial membrane potential levels (F=12.89, P < 0.05). Selenium had main effects on nuclear pyknosis ratio, Bcl-2 mRNA and Bax protein expressions (F=9.52, 10.84, 22.17, P < 0.05);SNP had main effects on nuclear pyknosis ratio, Bax and Bcl-2 mRNA expressions, and Bcl-2 protein expression (F=192.86, 21.90, 16.09, 18.39, P < 0.05);selenium combined with SNP had an interactive effect on Bax, Bcl-2 mRNA and protein expressions (F=20.51, 7.59, 15.38, 11.97, P < 0.05). Conclusion The SNP can induce apoptosis of AC16 cardiomyocyte;selenium combined with SNP has an interactive effect on AC16 cardiomyocyte, indicating that selenium has protective effect on NO modiated myocardial apoptosis.