铜绿假单胞菌外膜囊泡经TLR4/NF-κB通路诱导巨噬细胞炎症反应

Pseudomonas aeruginosa outer membrane vesicles induces the inflammatory response in macrophages via the TLR4/NF-κB pathways

目的:初步探讨铜绿假单胞菌外膜囊泡(outer membrane vesicles,OMVs)对巨噬细胞炎症反应的调节作用及其分子机制。方法:体外培养铜绿假单胞菌(P.aeruginosa)提取OMVs,不同浓度OMVs刺激RAW264.7细胞后,酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)、实时荧光定量PCR(quantitative real time PCR,qRT-PCR)分别检测细胞上清中白细胞介素(interleukin,IL)-8及其mRNA的表达水平,并通过Western blot法检测细胞内核因子-κB(nuclear factor-κB,NF-κB)抑制蛋白(inhibitor of NF-κB,IκB)磷酸化水平;siRNA沉默Toll样受体4(Toll-like receptor 4,TLR4)后,检测细胞中IL-8的分泌水平;最后通过髓样分化因子(myeloid differentiation factor88,MyD88)抑制剂以及NF-κB抑制剂BAY11-7082预处理后OMVs刺激细胞,并检测细胞中IL-8的变化水平。结果:不同浓度的OMVs(1、5、10μg/mL)刺激处理RAW264.7细胞后,细胞内IL-8及其mRNA表达水平较未刺激组(0μg/mL OMVs)均明显增高[IL-8(1、5、10μg/mL OMVs):(31.54±4.25)pg/mL、(82.04±10.20)pg/mL、(136.30±16.03)pg/mL比(16.42±6.14)pg/mL,t值分别为7.23,8.92和10.76,P值均<0.05;mRNA(1、5、10μg/mL OMVs):(1.25±0.09),(2.34±0.12)、(4.25±0.43)比(1.00±0.07),t值分别为5.24,7.98和9.63,P值均<0.05],且呈一定的剂量依赖性。siRNA沉默TLR4后细胞中IL-8分泌水平较对照组显著降低[(46.07±18.23)pg/mL比(115.50±22.02)pg/mL,t=9.25,P<0.05]。而通过MyD88抑制剂以及NF-κB抑制剂预处理后,RAW264.7细胞内IL-8的分泌水平较处理前也明显减少[MyD88抑制剂预处理后:(54.04±12.02)pg/mL比(134.02±18.01)pg/mL,t=9.72,P<0.05;NF-κB抑制剂预处理后:(51.03±17.01)pg/mL比(142.01±22.02)pg/mL,t=7.21,P<0.05]。结论:铜绿假单胞菌OMVs能够通过TLR4以及NF-κB途径诱导巨噬细胞RAW264.7分泌IL-8。

Objective To investigate the effects of Pseudomonas aeruginosa outer membrane vesicle(OMVs)on the regulation of inflammatory response and its molecular mechanism in macrophages.Methods OMVs was extracted from the Pseudomonas aeruginosa after cultivating in vitro.After stimulation with different concentrations of OMVs on RAW264.7 cell,the expression levels of IL-8 in the supernatant and its mRNA were detected by enzyme linked immunosorbent assay(ELISA)and quantitative real time PCR(qRT-PCR)respectively.The phosphorylation level of inhibitor of NF-κB(IκB)was detected by Western blot.The secretion level of IL-8 in cells was detected after silencing Toll-like receptor 4(TLR4)by siRNA.Finally,after pretreatment with myeloid differentiation factor88(MyD88)inhibitor and nuclear factor-κB(NF-κB)inhibitor BAY11-7082,the levels of IL-8 was detected in the cells after stimulation with OMVs.Results After the treatment with OMVs of 1,5 and 10μg/mL in RAW264.7 cells,the levels of IL-8 and mRNA in the cells were significantly increased compared with the unstimulated group(0μg/mL OMVs)[IL-8(OMVs with 1,5,10μg/mL,respectively):(31.54±4.25)pg/mL,(82.04±10.20)pg/mL,(136.30±16.03)pg/mL vs(16.42±6.14)pg/mL,t values were 7.23,8.92 and 10.76 respectively,all P values<0.05;mRNA(OMVs with 1,5,10μg/mL,respectively):(1.25±0.09),(2.34±0.12),(4.25±0.43)vs(1.00±0.07),t values were 5.24,7.98 and 9.63 respectively,all P values<0.05],which showed a certain dose dependence.After silencing TLR4 with siRNA,the secretion level of IL-8 in the cells was significantly reduced compared with that in the control group[(46.07±18.23)pg/mL vs(115.50±22.02)pg/mL,t=9.25,P<0.05].At the same time,after pretreatment with MyD88 inhibitor and NF-κB inhibitor,the secretion level of IL-8 in RAW264.7 cells were also significantly reduced compared with unstimulated group of[pretreatment with MyD88 inhibitor:(54.04±12.02)pg/mL vs(134.02±18.01)pg/mL,t=9.72,P<0.05;pretreatment with NF-κB inhibitor:(51.03±17.01)pg/mL vs(142.01±22.02)pg/mL,t=7.21,P<0.05].Conclusion Pseudomonas aeruginosa OMVs could induce the secretion of IL-8 via the TLR4 and NF-κB pathway in macrophage RAW264.7.