赖型钩端螺旋体OmpA外膜蛋白Loa22通过Ca^(2+)信号通路介导A549细胞凋亡

The OmpA-like Protein Loa22 from Leptospira interrogans Serovar Lai Induces Apoptosis in A549 via Ca^(2+) Signal Pathway

目的研究赖型钩端螺旋体(简称钩体)外膜蛋白Loa22通过Ca2+信号通路对细胞凋亡的影响,探讨该基因在钩体黏附侵袭细胞并诱导其凋亡过程中可能的信号传导机制。方法以人肺腺上皮细胞(A549)为模型,用100μg/mL纯化Loa22成熟肽作用于细胞24 h,检测细胞凋亡及细胞内游离钙浓度([Ca2+]i),观察F-actin细胞骨架变化,检测钙调素(CaM)mRNA表达水平。同时设磷脂酶C(PLC)特异阻断剂U73122预处理+Loa22成熟肽组,观察上述指标变化。结果 Loa22成熟肽作用A549后,细胞凋亡率升高,并诱导[Ca2+]i增加,CaMmRNA表达水平及乳酸脱氢酶(LDH)活性上升,F-actin细胞骨架重排。而预先用U73122预处理后,细胞凋亡率、[Ca2+]i、CaMmRNA和LDH活性均降低(P<0.01),F-actin细胞骨架重排程度减轻。结论 Loa22成熟肽通过PLC信号通路触发细胞[Ca2+]i升高,导致F-actin细胞骨架重排,从而引发细胞凋亡,提示该基因参与钩体侵袭细胞引发细胞凋亡病理过程,可能是致病性钩体的一个重要毒力相关基因。

Objective To study the effect of Loa22 mature peptide on the apoptosis of A549 to explore the mechanism of pulmonary impairment in severe forms of leptospirosis.Methods Loa22 mature peptide（100 μg/mL） was administered to culture with human lung adenocarcinoma cell line（A549）.After 24 hours,the apoptosis,the concentration of calcium of the cells were evaluated.The F-actin cytoskeleton structure was observed and calmdulin（CaM） mRNA expression was also detected.At the same time,after the pretreatment of A549 with PLC specific inhibitor U73122,adding an appropriate amount of mature peptide of Loa22 to act on the cells for a period of time,then detection same index.Results Loa22 mature peptide could induce the increase of intracellular Ca^2＋ concentration（i）,CaM expression in mRNA level,the activity of LDH,and cytoskeleton rearrangement of F-actin.But after blocking the signal pathway of PLC,Loa22 mature peptide reduced the increase degree of i,apoptosis rate,the expression of CaM mRNA,the activity of LDH compared with the unblocked group.Conclusion These data suggest that Loa22 mature peptide involves in the pathological processes of L.interrogans invasion and increase apoptosis in A549 by increase of i through signaling pathway of PLC.