硒对肿瘤坏死因子-α诱导的AC16心肌细胞炎性损伤的影响

Selenium attenuates the inflammatory injury induced by tumor necrosis factor-α in AC16 cardiomyocytes

目的探讨硒对肿瘤坏死因子-α(TNF-α)诱导的AC16心肌细胞炎性损伤的影响。方法将体外培养的AC16心肌细胞分为对照组、硒预处理组(100μg/L亚硒酸钠预处理4 h)、50μg/L TNF-α组、硒预处理+50μg/L TNF-α组、100μg/L TNF-α组、硒预处理+100μg/L TNF-α组。各组分别培养24 h后,收集细胞培养液和细胞进行检测。Griess法检测细胞培养液一氧化氮(NO)含量;荧光倒置显微镜观察细胞Hoechst 33342核染色,分析核固缩比例;实时荧光定量PCR检测B淋巴细胞瘤-2基因(Bcl-2)、B淋巴细胞瘤-2相关X蛋白(Bax)及诱导型一氧化氮合酶(iNOS)mRNA表达水平,蛋白质印迹法检测核因子-kappa B(NF-κB)p65、NF-κB抑制蛋白-α(IκB-α)蛋白表达水平。结果对照组、硒预处理组、50μg/L TNF-α组、硒预处理+50μg/L TNF-α组、100μg/L TNF-α组和硒预处理+100μg/L TNF-α组细胞培养液NO含量分别为(10.58±2.32)、(9.07±0.73)、(15.53±3.97)、(12.05±1.11)、(30.65±4.16)、(19.02±3.72)μmol/L。硒、TNF-α对细胞培养液NO含量均存在主效应(F=37.71、99.07,均P<0.001),且二者间存在交互效应(F=11.80,P<0.001)。硒、TNF-α对核固缩比例,Bcl-2、Bax mRNA表达水平均存在主效应(F=56.37、462.81,18.04、32.85,18.38、170.77,均P<0.05),且硒、TNF-α对核固缩比例,Bax mRNA表达水平均存在交互效应(F=21.48、19.96,均P<0.001)。硒、TNF-α对iNOS mRNA表达水平均存在主效应(F=129.98、1051.76,均P<0.001),且二者间存在交互效应(F=53.28,P<0.001)。硒、TNF-α对NF-κB p65、IκB-α蛋白表达水平均存在主效应(F=73.65、145.49,710.20、105.66,均P<0.001),且二者间均存在交互效应(F=26.73、197.59,均P<0.001)。结论硒可能通过NF-κB信号系统抑制iNOS表达,保护心肌细胞免受TNF-α诱导的炎性损伤。

Objective To exploring the effect of selenium on tumor necrosis factor-α(TNF-α)induced inflammatory injury of AC16 cardiomyocytes.Methods AC16 cardiomyocytes cultured in vitro were divided into control group,selenium pretreatment group(100μg/L sodium selenite pretreatment for 4 h),50μg/L TNF-αgroup,selenium pretreatment+50μg/L TNF-αgroup,100μg/L TNF-αgroup,and selenium pretreatment+100μg/L TNF-αgroup.After 24 h of culture,the cell culture medium or cells were collected for detection.Griess method was used to detect nitric oxide(NO)level in cell culture medium.Hoechst 33342 nuclear staining was detected by fluorescence inverted microscope and karyopyknosis ratio was analyzed.The mRNA expression levels of B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2 associated X protein(Bax)and inducible nitric oxide synthase(iNOS)were detected by real-time fluorescence quantitative PCR.The protein expression levels of nuclear factor-kappa B(NF-κB)p65 and inhibitoryκB-α(IκB-α)were detected by Western blotting.Results The levels of NO in the control group,selenium pretreatment group,50μg/L TNF-αgroup,selenium pretreatment+50μg/L TNF-αgroup,100μg/L TNF-αgroup and selenium pretreatment+100μg/L TNF-αgroup were(10.58±2.32),(9.07±0.73),(15.53±3.97),(12.05±1.11),(30.65±4.16)and(19.02±3.72)μmol/L,respectively.Selenium and TNF-αhad main effects on NO level(F=37.71,99.07,P<0.001)and interaction effect(F=11.80,P<0.001).Selenium and TNF-αhad main effects on karyopyknosis ratio and Bcl-2 and Bax mRNA expression levels(F=56.37,462.81,18.04,32.85,18.38,170.77,P<0.05).Selenium and TNF-αhad an interactive effect on both karyopyknosis ratio and Bax mRNA expression level(F=21.48,19.96,P<0.001).Selenium and TNF-αhad main effects on iNOS mRNA expression level(F=129.98,1051.76,P<0.001)and interaction effect(F=53.28,P<0.001).Selenium and TNF-αhad main effects on the protein expression levels of NF-κB p65 and IκB-α(F=73.65,145.49,710.20,105.66,P<0.001)and interaction effects(F=26.73,197.59,P<0.001).Conclusion Selenium may inhibits iNOS expression through NF-κB signal system and protects cardiomyocytes from TNF-αinduced inflammatory injury.