鲍曼不动杆菌通过活化血小板激活因子受体引起感染人支气管上皮细胞的氧化应激和细胞凋亡

Activation of platelet activating factor receptor by Acinetobacter baumannii results in oxidative stress and apoptosis in human bronchial epithelial cells

目的探讨血小板激活因子受体(PAFR)在鲍曼不动杆菌(A.baumannii)感染人支气管上皮(HBE)细胞过程中的作用。方法 HBE细胞分为对照组、银杏内酯B(GB)处理组、A.baumannii感染组、A.baumannii感染联合GB处理组。使用临床分离的A.baumannii感染HBE细胞,GB处理组分别用1×10~3CFU/m L、1×10~5CFU/m L和1×10~7CFU/m L的A.baumannii感染HBE细胞,A.baumannii感染联合GB处理组则先用10μmol/L GB阻断PAFR活性而后进行1×10~3CFU/m L A.baumannii感染。采用Western blot法检测HBE细胞PAFR、磷酸化的Janus激酶1(p-JAK1)、磷酸化的信号转导子与转录激活子1(p-STAT1)的蛋白水平,使用CCK-8法检测细胞增殖能力,使用超氧化物歧化酶(SOD)和丙二醛(MDA)试剂盒检测各组细胞氧化应激水平;异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)染色结合流式细胞术检测细胞凋亡。结果与对照组相比,A.baumannii感染的HBE细胞中PAFR蛋白水平显著升高,细胞活力显著下降,细胞内MDA水平和细胞凋亡明显增加,p-JAK1和p-STAT1蛋白水平增加。结论 A.baumannii感染的HBE细胞PAFR活化,激活JAK1/STAT1信号通路促进HBE细胞的氧化应激和细胞凋亡。

Objective To investigate the effect of platelet activating factor receptor（ PAFR） on human bronchial epithelial（ HBE） cells infected by Acinetobacter baumannii（ A. baumannii）. Methods HBE cells were divided into control group,ginkgolide B（ GB） group,A. baumannii infected group,A. baumannii infection and inhibitor group. HBE cells were infected with low dose（ 1 × 10~3 CFU/m L）,medium dose（ 1 × 10~5 CFU/m L） and high dose（ 1 × 10~7 CFU/m L） A. baumanni separated from clinical samples. The PAFR activity was blocked by the 10 μmol/L GB. The expression of PAFR was detected using Western blotting in HBE cel s. The proliferation ability of HBE cel s was detected using CCK-8 assay. The oxidative stress level was evaluated by superoxide dismutase（ SOD） and malondialdehyde（ MDA） kits. Apoptosis of HBE cel s was observed by annexin V-FITC-Ⅴ/PI staining. The phosphorylation level of PAFR and its downstream molecule JAK1/STAT1 in HBE cel s were examined by Western blot analysis. Results Compared with the control group,the expression of PAFR increased significantly in A. baumannii infected group. A. baumannii infection could decrease cell vitality,but increase intracellular oxidative stress,apoptosis,and JAK1/STAT1 phosphorylation. Conclusion PAFR is an important mediator molecule for A. baumanni infection in HBE cel s,and PAFR/JAK1/STAT1 signaling pathway plays an important role in the pulmonary infection of A. baumanni.