烟曲霉对人支气管上皮细胞DNA损伤和IL-33表达的影响及其机制

Effect of Aspergillus fumigatus on DNA damage and IL-33 expression in human bronchial epithelial cells and its mechanism

目的:探讨烟曲霉(Af)对人支气管上皮细胞DNA损伤和白细胞介素33(IL-33)表达的影响,阐明其相关作用机制。方法:采用不同浓度(1、5和10 mg·L^(-1))Af刺激支气管上皮BEAS-2B细胞,筛选合适的刺激浓度。采用N-乙酰半胱氨酸(NAC)和Af刺激BEAS-2B细胞时,将细胞分为对照组、Af组、NAC组和Af+NAC组。采用DNA双链断裂修复抑制剂NU7441和Af刺激BEAS-2B细胞时,将细胞分为对照组、Af组、NU7441组和Af+NU7441组。采用彗星实验检测各组细胞彗星尾部DNA百分率,采用免疫荧光法检测各组细胞中DNA损伤相关蛋白磷酸化组蛋白H2AX(γH2AX)表达水平,采用2,7-二氯荧光素二乙酸酯(DCFH-DA)荧光探针法检测各组细胞中活性氧(ROS)水平,采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中IL-33、胸腺基质淋巴细胞生成素(TSLP)和白细胞介素25(IL-25)mRNA表达水平,采用Western blotting法检测各组细胞中磷酸化核因子κB(p-NF-κB)、磷酸化共济失调毛细血管扩张突变(p-ATM)和γH2AX蛋白表达水平。结果:与对照组比较,1 mg·L^(-1) Af组细胞中彗星尾部DNA百分率和γH2AX表达水平差异无统计学意义(P>0.05),5 mg·L^(-1) Af组细胞中彗星尾部DNA百分率和γH2AX表达水平明显升高(P<0.01);与5 mg·L^(-1) Af组比较,10 mg·L^(-1) Af组细胞中彗星尾部DNA百分率和γH2AX表达水平均明显升高(P<0.01)。与对照组比较,1 mg·L^(-1) Af组支气管上皮细胞中ROS水平明显升高(P<0.05);与1 mg·L^(-1) Af组比较,5 mg·L^(-1) Af组细胞中ROS水平明显升高(P<0.01);与5 mg·L^(-1) Af组比较,10 mg·L^(-1) Af组细胞中ROS水平明显升高(P<0.05)。NAC处理后,与Af组比较,Af+NAC组细胞中彗星尾部DNA百分率(P<0.01)、γH2AX表达水平(P<0.05)和ROS水平(P<0.01)明显降低;NU7441处理后,与Af组比较,Af+NU7441组细胞中彗星尾部DNA百分率明显升高(P<0.01),γH2AX表达水平明显升高(P<0.01)。RT-qPCR检测,NAC处理后,与对照组比较,Af组细胞中IL-33 mRNA表达水平明显升高(P<0.05);与Af组比较,Af+NAC组细胞中IL-33 mRNA表达水平明显降低(P<0.05);NU7441处理后,与Af组比较,Af+NU7441组细胞中IL-33 mRNA表达水平明显升高(P<0.05)。Westernblotting法检测,NAC处理后,与对照组比较,Af组细胞中p-NF-κB、p-ATM和γH2AX蛋白表达水平明显升高(P<0.05);与Af组比较,Af+NAC组细胞中p-NF-κB、p-ATM和γH2AX蛋白表达水平明显降低(P<0.05)。NU7441处理后,与Af组比较,Af+NU7441组细胞中p-NF-κB、p-ATM和γH2AX蛋白表达水平明显升高(P<0.05)。结论:Af通过引起人支气管上皮细胞DNA损伤促进细胞中IL-33表达,其机制可能与激活ATM/NF-κB信号通路有关。

Objective:To discuss the effect of Aspergillus fumigatus(Af)on DNA damage and interleukin(IL)-33 expression in the human bronchial epithelial cells,and to clarify its related mechanism.Methods:Different concentrations(1,5,and 10 mg·L⁻¹)of Af were used to stimulate the bronchial epithelial BEAS-2B cells to select the appropriate stimulation concentration.When the BEAS-2B cells were treated with N-acetylcysteine(NAC)and Af,the cells were divided into control group,Af group,NAC group,and Af+NAC group.When the BEAS-2B cells were treated with DNA double-strand break repair inhibitor NU7441 and Af,the cells were divided into control group,Af group,NU7441 group,and Af+NU7441 group.The comet assay was used to detect the percentages of comet tail DNA of cells in various groups;immunofluorescence method was used to detect the expression levels of DNA damage-related protein phosphorylated H2AX(γH2AX)in the cells in various groups;2,7-dichlorofluorescein diacetate(DCFH-DA)fluorescence probe was used to detect the levels of reactive oxygen species(ROS)in the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of interleukih-33(IL-33),thymic stromal lymphopoietin(TSLP),and interleukih-25(IL-25)mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of phosphorylated nuclear factorκB(p-NF-κB),phosphorylated ataxia telangiectasia mutated(p-ATM),andγH2AX proteins in the cells in various groups.Results:Compared with control group,the percentage of comet tail DNA and the expression level ofγH2AX in the cells in 1 mg·L⁻¹Af group showed no significant difference(P>0.05),while the percentage of comet tail DNA and the expression level ofγH2AX in the cells in 5 mg·L⁻¹Af group were significantly increased(P<0.01);compared with 5 mg·L⁻¹Af group,the percentage of comet tail DNA and the expression level ofγH2AX in the cells in 10 mg·L⁻¹Af group were significantly increased(P<0.01).Compared with control group,the ROS levels in the bronchial epithelial cells in 1 mg·L⁻¹Af group was significantly increased(P<0.05);compared with 1 mg·L⁻¹Af group,the ROS level in the cells in 5 mg·L⁻¹Af group was significantly increased(P<0.01);compared with 5 mg·L⁻¹Af group,the ROS level in the cells in 10 mg·L⁻¹Af group was significantly increased(P<0.05).After treatment of NAC,compared with Af group,the percentage of comet tail DNA(P<0.01),the expression level ofγH2AX(P<0.05),and the ROS level(P<0.01)in the cells in Af+NAC group were significantly decreased;after treatment of NU7441,compared with Af group,the percentage of comet tail DNA and the expression level ofγH2AX in the cells in Af+NU7441 group were significantly increased(P<0.01).The RT-qPCR results showed that after treatment of NAC,compared with control group,the expression level of IL-33 mRNA in the cells in Af group was significantly increased(P<0.05);compared with Af group,the expression level of IL-33 mRNA in the cells in Af+NAC group was significantly decreased(P<0.05);after treatment of NU7441,compared with Af group,the expression level of IL-33 mRNA in the cells in Af+NU7441 group was significantly increased(P<0.05).The Western blotting results showed that after treatment of NAC,compared with control group,the expression levels of p-NF-κB,p-ATM,andγH2AX proteins in the cells in Af group were significantly increased(P<0.05);after treatment of NU7441,compared with Af group,the expression levels of p-NF-κB,p-ATM,andγH2AX proteins in the cells in Af+NAC group were significantly decreased(P<0.05);After treat ment of NU7441,compared with Af group,the expression levels of p-NF-κB,p-ATM,andγH2AX proteins in the cells in Af+NU7441 group were significantly increased(P<0.05).Conclusion:Af promotes the IL-33 expression in the human bronchial epithelial cells by causing DNA damage,and its mechanism may be related to the activation of ATM/NF-κB signaling pathway.