脂多糖调控PI3K/Akt通路增强中性粒细胞吞噬功能

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。

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

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 10 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 cytometry 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 filamentous actin(F-actin) fluorochrome and analyzed by flow cytometry and fluorescence microscope. In addition, 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 detected 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 neutrophil 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 morphology, while there were no special changes of cell morphology in LY294002 group and LPS+LY294002 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. ConclusionLPS could enhance the neutrophil phagocytosis, which was associated with the activated phosphorylation of PI3K/Akt.