重组嗜肺军团菌鞭毛蛋白A(rflaA)体外诱导小鼠RAW264.7巨噬细胞分泌IL-6和IL-1β

Recombinant Legionella pneumophila flagella protein A(rflaA) induces the secretion of IL-6 and IL-1β in RAW264.7 cells in vitro

目的探讨重组嗜肺军团菌鞭毛蛋白A(rflaA)对RAW264.7巨噬细胞分泌白细胞介素6(IL-6)、IL-1β的影响及其机制。方法采用(0.000、0.125、0.250、0.500、1.000、2.000、4.000、8.000)μg/mL rflaA处理RAW264.7细胞,并设细胞对照组,用CCK-8法确定rflaA的半数效应剂量(EC_(50))。采用(0.04、0.08、0.16)μg/mL rflaA处理RAW264.7细胞24、36、48 h,采用ELISA检测IL-6、IL-1β分泌水平;(0.04、0.08、0.16)μg/mL rflaA处理RAW264.7细胞6、12、24、36、48 h,收集细胞,实时定量PCR检测IL-6、IL-1β、NOD样受体蛋白3(NLRP3)、胱天蛋白酶1(caspase-1)mRNA的含量;Western blot法检测NLRP3、caspase-1蛋白水平。结果 rflaA可促进RAW264.7细胞因子分泌,RAW264.7细胞被不同剂量的rflaA作用24、36、48 h,随rflaA剂量的递增,IL-6、IL-1β水平增加,以0.16μg/mL剂量时作用最显著,36 h达高峰;rflaA可增加RAW264.7细胞的IL-6、IL-1β、NLRP3、caspase-1 RNA水平,0.16μg/mL rflaA作用最显著,12 h达高峰;RAW264.7细胞被不同剂量的rflaA作用6、12、24、36 h,随rflaA剂量增加NLRP3、caspase-1表达水平增加,以0.16μg/mL剂量时作用最显著,24 h达高峰。结论 rflaA通过刺激NLRP3、caspase-1而增加RAW264.7细胞IL-6、IL-1β的分泌。

Objective To investigate the effect of recombinant Legionella pneumophila flagella protein A（rflaA） on the secretion of interleukin-6（IL-6） and interleukin-1β（IL-1β） by RAW264.7 macrophage and the possible mechanism.Methods RAW264.7 cells were treated with 0.000,0.125,0.250,0.500,1.000,2.000,4.000 and 8.000 μg/m L rflaA to determine the EC50 of rflaA using CCK-8 assay.Secretion of IL-6 and IL-1β were measured by ELISA at 24,36 and 48 hours after treatment of the cells with 0.04,0.08 and 0.16 μg/m L rflaA.At 6,12,24,36 and 48 hours after treatment of the cells with 0.04,0.08 and 0.16 μg/m L rflaA,the expressions of IL-6,IL-1β,NOD-like receptor protein 3（NLRP3） and caspase-1 mRNAs were detected by quantitative real-time PCR,and the expressions of NLRP3 and caspase-1 proteins were tested by Western blotting.Results Rfla A enhanced the expressions of IL-6 and IL-1β,and the higher concentration of rflaA was more potential.The expressions of IL-6 and IL-1β reached peak when the cells were treated with 0.16 μg/m L rflaA for 36 hours.Treatment of RAW264.7 cells with rflaA promoted the expressions of IL-6 and IL-1β,NLRP3 and caspase-1 mRNA,and 0.16 μg/m L rflaA was the most potential at 12 hours after treatment.Expressions of NLRP3 and caspase-1 protein increased after treatment with rflaA,and 0.16 μg/m L rflaA induced the highest expression of both proteins at 24 hours after treatment.Conclusion Rfla A could enhance the secretion of IL-6 and IL-1β by promoting the expressions of NLRP3 and caspase-1 in RAW264.7 cells.