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.

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 γ H2AX 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. [ABSTRACT FROM AUTHOR]