牙龈卟啉单胞菌脂多糖对活性中性粒细胞胞外诱捕网形成的影响

Effects of Porphyromonas gingivalis lipopolysaccharide on the formation of vital neutrophil extracellular traps

目的:探讨牙龈卟啉单胞菌(P.gingivalis)脂多糖(LPS)对活性中性粒细胞胞外诱捕网(NETs)形成的影响及可能的调控机制。方法:采用25 ng/mL粒细胞-巨噬细胞集落刺激因子(GM-CSF)和0.3μg/mL P.gingivalis LPS刺激人中性粒细胞35 min,免疫荧光显微镜下观察活性NETs的形成,酶标法对胞外DNA定量,PCR法检测构成NETs的DNA来源。检测产生活性NETs的中性粒细胞感染P.gingivalis后胞内外的细菌数量,以明确NETs的杀菌效果;流式细胞术检测细胞产生的活性氧(ROS)水平;Western blot检测丝氨酸/苏氨酸蛋白激酶(Raf)、丝裂原活化的细胞外信号调节激酶(MEK)和细胞外信号调节蛋白激酶(ERK)的磷酸化水平。结果:GM-CSF+P.gingivalis LPS可促进线粒体DNA来源的活性NETs形成和胞外DNA数量的增加(P<0.05),增强中性粒细胞对P.gingivalis的杀菌活性(P<0.05)。与阴性对照组相比,GM-CSF+P.gingivalis LPS刺激的中性粒细胞ROS和Raf、MEK、ERK磷酸化水平均明显增高(P<0.05)。还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂DPI处理后,GM-CSF+P.gingivalis LPS组胞外DNA数量降低,ROS水平降低(P<0.01)。结论:GM-CSF+P.gingivalis LPS可能通过Raf、MEK、ERK磷酸化调控ROS生成,促进人中性粒细胞形成活性NETs,进而促进P.gingivalis的清除,调控牙周免疫炎症反应。

Objective:To observe the effects and mechanisms of Porphyromonas gingivalis(P.gingivalis)lipopolysaccharide(LPS)on the formation of vital neutrophil extracellular traps(NETs).Methods:Neutrophils were obtained from peripheral venous blood of healthy individuals by density gradient centrifugation and primed with 25 ng/mL GM-CSF and 0.3μg/mL P.gingivalis LPS for 35 min.Formation of vital NETs was observed by immunofluorescence staining and extracellular DNA was quantified by a microplate reader.DNA sources were explored by PCR.Quantities of extra-and intracellular P.gingivalis in neutrophils were determined to assay the bactericidal efficiency of vital NETs.Levels of reactive oxygen species(ROS)were detected by flow cytometry.Phosphorylation levels of protein kinase Raf,mitogen-activated extracellular signal-regulated kinase(MEK),and extracellular signal-regulated kinase(ERK)were explored by Western blot.Results:GM-CSF+P.gingivalis LPS stimulation led to the formation of vital NETs and the increased levels of extracellular DNA(P<0.05),which enhanced bactericidal activity of neutrophils(P<0.05).In this process,mitochondrial DNA was released instead of nuclear DNA.In addition,levels of ROS,p-Raf,p-MEK and p-ERK were significantly increased in the cells treated with GM-CSF+P.gingivalis LPS compared with negative group(P<0.05).In addition,DPI,an inhibitor of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase,pretreatment resulted in decreased levels of extracellular DNA and ROS(P<0.01).Conclusions:GM-CSF+P.gingivalis LPS might contribute to the formation of vital NETs depending on the production of ROS and the phosphorylations of Raf,MEK,ERK,which might contribute to the elimination of P.gingivalis and regulate periodontal inflammatory responses.