Abstract:

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 factor κB （p-NF-κB）， phosphorylated ataxia telangiectasia mutated （p-ATM）， and γH2AX proteins in the cells in various groups. Results Compared with control group， the percentage of comet tail DNA and the expression level of γH2AX in the cells in 1 mg·L?1 Af group showed no significant difference （P>0.05）， while the percentage of comet tail DNA and the expression level of γH2AX in the cells in 5 mg·L?1 Af group were significantly increased （P<0.01）； compared with 5 mg·L?1 Af group， the percentage of comet tail DNA and the expression level of γH2AX in the cells in 10 mg·L?1 Af group were significantly increased （P<0.01）. Compared with control group， the ROS levels in the bronchial epithelial cells in 1 mg·L?1 Af group was significantly increased （P<0.05）； compared with 1 mg·L?1 Af group， the ROS level in the cells in 5 mg·L?1 Af group was significantly increased （P<0.01）； compared with 5 mg·L?1 Af group， the ROS level in the cells in 10 mg·L?1 Af 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 of γH2AX （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 of γH2AX 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， and γH2AX 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， and γH2AX 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， and γH2AX 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.

Key words: Aspergillus fumigatus, DNA damage, Interleukin-33, Bronchial epithelial cell, Reactive oxygen species