脂多糖调控PI3K/Akt通路增强中性粒细胞吞噬功能

Lipopolysaccharides enhances neutrophil phagocytosis of E. coli via PI3K/Akt pathway

目的:探讨脂多糖对中性粒细胞细菌吞噬功能的影响及可能机制。方法:取健康人静脉血中性粒细胞,重悬分为对照组、脂多糖10 min、20 min、30 min组,按1∶50加入大肠埃希菌GFP(25922GFP),孵育30 min,荧光显微镜和流式细胞术检测中性粒细胞的吞噬功能;另取中性粒细胞重悬分为对照组、脂多糖组、LY294002(PI3K抑制剂)组、LY294002+脂多糖组,一部分细胞行丝状肌动蛋白(filamentous actin,F-actin)荧光染料染色,流式细胞术观察与分析细胞中F-actin,荧光显微镜观察中性粒细形态;另一部分细胞按1∶50加入25922GFP,孵育30 min,荧光显微镜和流式细胞术检测中性粒细胞的吞噬功能。免疫印迹法检测各组细胞蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、磷脂酰肌醇激酶(PI3K)和磷酸化磷脂酰肌醇激酶(p-PI3K)的表达。结果:与对照组比较,脂多糖10 min、20 min、30 min组中性粒细胞25922GFP荧光表达逐渐增强,p-PI3K和p-Akt表达逐渐增加;脂多糖组荧光强度和p-PI3K和Akt表达明显高于对照组(P均<0.05)。与对照组比较,脂多糖组F-actin表达增加,细胞形态改变,LY294002组和LY294002+脂多糖组细胞形态无明显改变,LY294002+脂多糖组F-actin表达明显低于脂多糖组(P<0.05);对照组、LY294002组和LY294002+脂多糖组p-Akt表达明显低于脂多糖组。结论:经脂多糖刺激后中性粒细胞吞噬功能增强,其机制可能与脂多糖促进PI3K/Akt通路磷酸化有关。

Objective：To investigate the effect of lipopolysaccharides（LPS）on neutrophil phagocytosis and its potential mechanism.Methods：（1 ）Neutrophils separated from the blood samples of healthy adult were divided into control group,LPS 1 0 min,20 min,30 min group;each group was treated with GFP-E. coli （25922GFP）in proportion as 1 ∶50 for 30 min.Neutrophil phagocytosis was detected by using flow cy-tometry and fluorescence microscope.（2 ） Neutrophils were randomly divided into control group,LPS group,LY294002（PI3K inhibitor）group and LPS＋LY294002 group;some of them were stained with fila-mentous actin（F-actin）fluorochrome and analyzed by flow cytometry and fluorescence microscope.In addi-tion,the neutrophil morphology and F-actin were also observed by fluorescence microscope.Each group was treated with GFP-E.coli （25922GFP）in proportion as 1 ∶50 for 30 min.Neutrophil phagocytosis was de-tected by flow cytometry and fluorescence microscope.Expression of Akt,PI3K,p-Akt and p-PI3K were determined by western blotting.Results：（1 ） Compared with control group,three LPS-treated groups showed higher expression levels of 25922GFP fluorescence and p-PI3K,p-Akt in neutrophils;the neutro-phil phagocytosis in three LPS-treated groups were higher than that in control group （both P〈0.05 ）;（2）Compared with control group,LPS group had higher levels of F-actin expression,changed cell mor-phology,while there were no special changes of cell morphology in LY294002 group and LPS＋LY294002＆nbsp;group;LPS＋LY294002 group showed lower levels of F-actin than that in LPS group （P〈0.05 ）.And there were no significant changes in expression of p-Akt among control goup,LY294002 group and LPS＋LY294002 group,while lower than LPS group.Conclusion：LPS could enhance the neutrophil phagocyto-sis,which was associated with the activated phosphorylation of PI3 K/Akt.