ST2负向调控铜绿假单胞菌外膜囊泡诱导肺上皮细胞分泌细胞因子

ST2 negatively regulates the cytokines secretion of pulmonary epithelial cells induced by outer membrane vesicles of Pseudomonas aeruginosa

目的研究受体分子ST2在铜绿假单胞菌外膜囊泡(OMVs)诱导肺上皮细胞分泌促炎细胞因子中的负向调控作用。方法将气道上皮细胞BEAS-2B分为对照组和低、中、高浓度实验组。对照组给予等体积磷酸盐缓冲液刺激;低、中、高实验组分别用1,10和30μg·mL^(-1)的OMVs刺激。用实时荧光定量聚合酶链反应和Western blot法分别检测ST2 mRNA和蛋白的表达水平,用Western blot法检测细胞核内p65、H3和H4乙酰化,用酶联免疫吸附实验法检测肿瘤坏死因子-α(TNF-α)和白细胞介素-8(IL-8)的分泌,用染色质共沉淀检测组蛋白和ST2启动子的结合,用荧光素报告基因测定核转录因子κB(NF-κB)的酶活性。结果BEAS-2B细胞经OMVs处理后,低、中、高浓度实验组均可导致ST2转录水平和翻译水平明显增高,并能上调细胞核内p65的表达水平以及细胞核内H3和H4乙酰化;同时,低、中、高浓度实验组的TNF-α和IL-8分泌水平也显著增高。染色质共沉淀结果显示,H3和H4组蛋白能和ST2启动子区域结合。报告基因结果也显示,ST2能和NF-κB启动子结合,并抑制NF-κB的活性。结论铜绿假单胞菌OMVs经NF-κB诱导ST2表达,后者可抑制NF-κB的活性以及TNF-α和IL-8分泌。

Objective To investigate the negative regulatory role of the receptor molecule ST2 in outer membrane vesicles(OMVs)of Pseudomonas aeruginosa-induced pro-inflammatory cytokines secretion in pulmonary epithelial cells and to study its negative regulatory mechanism on the inflammatory response induced by OMVs.Methods Airway epithelial cells BEAS-2 B were divided into control group and experimental-L,-M,-H groups.Cells in the control group were treated by equal volume of phosphate buffered saline,and the experimental-L,-M,-H groups were stimulated with 1,10 and 30μg·mL^(-1) of OMVs,respectively.The expression of ST2 mRNA and protein was detected by real-time fluorescent quantitative polymerase chain reaction and Western blot.The expression of p65 protein in nucleus and the acetylation of H3 and H4 were detected by Western blot.Enzyme linked immunosorbent assay was used to detect the secretion of tumor necrosis factorα(TNF-α)and interleukin-8(IL-8).The binding of histone to ST2 promoter was detected by chromatin coprecipitation.The activity of nuclear factor kappa-B(NF-κB)was measured by luciferase reporter gene assay.Results After treatment with OMVs,the transcription and translation levels of ST2 were significantly increased in the experimental-L,-M and-H groups,and the expression levels of p65,H3 and H4 histone in nucleus were significantly increased.In addition,the secretion levels of TNF-αand IL-8 were also significantly increased in the experimental-L,-M and-H groups.Chromatin coprecipitation showed that the H3 and H4 histone could bind to ST2 promoter region.Reporter gene assay also showed that ST2 could bind to the promoter of NF-κB and thus inhibited its activity.Conclusion The OMVs of Pseudomonas aeruginosa can induce ST2 expression,which further inhibit the activity of NF-κB and the secretion of TNF-αand IL-8.