高毒力肺炎克雷伯菌诱导人外周血中性粒细胞凋亡延迟及其影响核因子-κB的潜在机制

Delayed apoptosis of human peripheral blood neutrophils induced by hypervirulent clinical variant of Klebsiella pneumoniae and the potential mechanism in regulating the expression of nuclear factor-κB

目的了解高毒力肺炎克雷伯菌(hv KP)和普通肺炎克雷伯菌(non-hv KP)对人外周血中性粒细胞凋亡及呼吸爆发功能的影响与差异,探讨hv KP影响核因子-κB(NF-κB)诱导中性粒细胞凋亡的潜在作用机制。方法离体检测hv KP和non-hv KP感染患者外周血中性粒细胞的凋亡情况。取健康成人外周血分离中性粒细胞,设0.9%Na Cl溶液对照组、hv KP组和non-hv KP组,各组分别刺激中性粒细胞0、3、6、9 h后,采用FITC-Annexin V/PI荧光标记流式细胞术检测细胞凋亡情况,采用流式细胞术检测细胞的呼吸爆发功能,采用Western blot检测感染后中性粒细胞NF-κB的表达水平。结果 hv KP组各个时间点的中性粒细胞凋亡率显著低于non-hv KP组(P<0.01)。体外研究显示,non-hv KP组各个时间点的中性粒细胞凋亡率显著高于hv KP组和对照组(P<0.01);hv KP组中性粒细胞的呼吸爆发率显著低于non-hv KP组(P<0.01)。Western blot检测发现随着感染时间的增加,hv KP感染人中性粒细胞的p65蛋白表达量显著高于non-hv KP(P<0.01)。结论hv KP可通过上调p65蛋白表达从而抑制NF-κB信号途径,导致中性粒细胞凋亡延迟,但却可降低中性粒细胞的呼吸爆发能力,其或可在hv KP感染致病中发挥重要作用。

Objective To investigate the differential effect of hypervirulent variant of Klebsiella pneumoniae(hvKP)and nonhvKP on the apoptosis and respiratory burst of human peripheral blood neutrophils,and explore the role of nuclear factor-κB(NF-κB)in delayed apoptosis of neutrophils induced by the hvKP strain.Methods The apoptosis of neutrophils in peripheral blood was measured in vitro and compared between hvKP group and non-hvKP group.The neutrophils freshly separated from the blood samples of healthy adults were cultured with 0.9%NaCl solution(control),hvKP,and non-hvKP strains.After the neutrophils were stimulated for 0,3,6,and 9 hours,annexin V staining and flow cytometry were used to measure cell apoptosis and respiratory burst.The expression of NF-κB p65 in neutrophils was measured by Western blot.Results The apoptosis rate of neutrophils after hvKP stimulation was significantly lower than after non-hvKP stimulation at each time point(P<0.01).Compared with control group and hvKP group,apoptosis of neutrophils was significantly higher in non-hvKP group at each time point(P<0.01).The hvKPstimulated human neutrophils showed significantly lower respiratory burst in vitro than non-hvKP-stimulated neutrophils(P<0.01).The expression of NF-κB p65 in neutrophils was significantly higher in hvKP group than in non-hvKP group at each time point(P<0.01).Conclusions hvKP may delay apoptosis of neutrophils and reduce respiratory burst by upregulating the expression of NF-κB p65 protein,and so inhibiting NF-κB signaling pathway.Delayed apoptosis of neutrophils may play an important role in the pathogenesis of hvKP infection.