鲍曼不动杆菌OmpA经ROS/NLRP3信号通路调控THP-1细胞自噬和凋亡

Autophagy and apoptosis induced by OmpA of Acinetobacter baumannii via ROS/NLRP3 signaling pathway in THP-1 cells

目的:探讨鲍曼不动杆菌外膜蛋白A(outer membrance protein A,OmpA)对人单核巨噬THP-1细胞自噬和凋亡的影响及其可能机制。方法:构建OmpA/pET-28a载体,转化大肠埃希菌BL21(DE3)表达纯化重组OmpA蛋白,并免疫家兔制备多克隆抗体;采用蛋白质印迹法检测呼吸机相关性肺炎患者发病前后血清中OmpA抗体水平变化。以不同浓度(1、10μg/mL)重组OmpA蛋白分别刺激THP-1细胞1、3、6 h,采用蛋白质印迹法检测自噬相关蛋白LC-3和Beclin-1表达,流式细胞术检测细胞凋亡率及活性氧产生,荧光实时定量PCR检测细胞含NLR家族Pyrin域NOD样受体家族3(NLRP3)、Caspase-1和IL-1β等mRNA表达。结果:自制多抗可以识别临床菌株OmpA蛋白。呼吸机相关性肺炎患者发病前后,血清中均可检出OmpA抗体(IgG型),且发病后OmpA抗体水平显著高于气管插管前(P<0.05)。重组OmpA蛋白刺激THP-1细胞后,细胞自噬相关蛋白LC3-Ⅱ和Beclin-1表达显著上升,细胞凋亡率显著升高,呈一定的时间和浓度依赖性(P<0.05)。与对照组比,在重组OmpA蛋白作用1 h时,细胞IL-1βmRNA表达和活性氧含量显著增加(P<0.05);3 h和6 h时,3种mRNA表达量和活性氧含量显著降低(P<0.05)。结论:OmpA通过激活THP-1细胞NLRP3炎症小体,释放活性氧和IL-1β等炎症介质,诱导THP-1细胞自噬和凋亡发生。

Objective:To investigate the effect of outer membrance protein A(OmpA)of Acinetobacter baumannii on autophagy and apoptosis of human monocyte macrophage THP-1 cells and its underlying mechanism.Methods:The OmpA/pET-28a vector was constructed and transformed into E.coli BL21(DE3)to express the recombinant OmpA(rOmpA),and the rabbits were immunized by rOmpA to prepare the polyclonal anti-OmpA antibody.The anti-OmpA antibody levels in serum of ventilator-associated pneumonia(VAP)patients were detected by Western blotting.THP-1 cells were treated with different concentrations(1μg/mL and 10μg/mL)of rOmpA for 1 h,3 h and 6 h;and the expression of autophagy-related proteins LC-3 and Beclin-1 were detected by Western blotting;the flow cytometry was used to detect the THP-1 cell apoptosis rate and ROS production;the mRNA expressions of NLRP 3,Caspase-1 and IL-1βin THP-1 cells were detected by qRT-PCR.Results:The OmpA expression in clinical A.baumannii strains could be detected by polyclonal anti-OmpA antibody,and the anti-OmpA antibody(IgG)in serum of VAP patients serum could be detected before and after the onset of VAP.The levels of anti-OmpA antibody in VAP patients′serum were significantly higher than that before using tracheal intubation(P<0.05).After treated with rOmpA,the expression of autophagy-related proteins LC3-Ⅱand Beclin-1 in THP-1 cells increased significantly,and the apoptosis rate increased vastly in a time-and concentration-dependent manner(P<0.05).Compared with the control group,when cells were treated by the rOmpA for 1 h,the IL-1βmRNA expression and reactive oxygen species were significantly up-regulated(P<0.05);when treated for 3 h and 6 h,the three kinds of mRNA expression had a significant decrease compared with the control group(P<0.05).Conclusion:OmpA induces THP-1 cells autophagy and apoptosis by activating NLRP3 inflammatory cytokines and releasing inflammatory factors such as ROS and IL-1β.