降钙素基因相关肽抑制铜绿假单胞菌外膜囊泡致炎作用  

Calcitonin Gene-Related Peptide Inhibits Inflammatory Response Induced by Outer Membrane Vesicles of Pseudomonas Aeruginosa

作　　者：谢先强 朱宏亮 陈帆 易伟 杨志刚[1] 李冬盛[1] XIE Xian-qiang;ZHU Hong-liang;CHEN Fan;YI Wei;YANG Zhi-gang;LI Dong-sheng(Department of General Surgery,the 908th Hospital of Chinese PLA Joint Logistic Support Force,Nanchang 330002,China)

机构地区：[1]中国人民解放军联勤保障部队第九〇八医院普外科,南昌330002

出　　处：《南昌大学学报（医学版）》2020年第5期17-21,共5页Journal of Nanchang University：Medical Sciences

摘　　要：目的探讨降钙素基因相关肽(CGRP)对铜绿假单胞菌外膜囊泡(OMVs)诱导炎症反应的影响。方法培养铜绿假单胞菌并提取OMVs,取1、5、10μg·mL-1 OMVs刺激鼠巨噬细胞RAW264.7,采用ELISA法检测细胞中促炎性细胞因子[包括白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)]含量,采用CCK-8法及流式细胞术分别检测细胞增殖及凋亡;采用10μg·mL-1 OMVs与1、5、10 nmol·L-1 CGRP同时处理RAW264.7细胞,并检测细胞中促炎细胞因子水平;采用20μg·mL-1 OMVs与10 nmol·L-1 CGRP同时处理RAW264.7细胞,检测细胞增殖与凋亡;培养人中性粒细胞,采用脱颗粒实验检测CGRP对其分泌中性粒细胞明胶酶脂质运载蛋白(NGAL)及弹性蛋白酶活性的影响。结果不同浓度OMVs均能显著刺激RAW264.7细胞分泌TNF-α、IL-6和IL-1β(P<0.05),而CGRP则能够抑制RAW264.7细胞分泌促炎细胞因子,且呈剂量依赖性(P<0.05);20μg·mL-1 OMVs则能够显著降低细胞活性,并促进细胞凋亡(P<0.05),10 nmol·L-1 CGRP与OMVs共同处理能显著增加RAW264.7细胞活性,抑制凋亡发生(P<0.05);不同浓度OMVs均能提升NGAL表达水平以及弹性蛋白酶活性(P<0.05),而10 nmol·L-1 CGRP则能够显著抑制OMVs引起的脱颗粒效应(P<0.05)。结论CGRP可有效下调铜绿假单胞菌OMVs诱导的鼠巨噬细胞RAW264.7促炎因子分泌水平、减少凋亡率以及特异性中性粒细胞释放,进而抑制炎症反应。Objective To investigate the effect of calcitonin gene-related peptide(CGRP)on inflammatory response induced by outer membrane vesicles(OMVs)of Pseudomonas aeruginosa.Methods The OMVs were extracted from cultured Pseudomonas aeruginosa,and mouse macrophages RAW264.7 were stimulated with 1,5 and 10μg·mL-1 OMVs.The levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in RAW264.7 cells were measured by ELISA.Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry,respectively.The levels of proinflammatory cytokines were determined after RAW264.7 cells were co-treated with 10μg·mL-1 OMVs and 1,5 or 10 nmol·L-1 CGRP,and cell proliferation and apoptosis were measured after co-treatment with 20μg·mL-1 OMVs and 10 nmol·L-1 CGRP.In addition,the effects of CGRP on neutrophil gelatinase-associated lipocalin(NGAL)secretion and elastase activity were analyzed in cultured human neutrophils by degranulation test.Results All concentrations of OMVs stimulated the secretion of TNF-α,IL-6 and IL-1β,increased NGAL expression and enhanced elastase activity in RAW264.7 cells(P<0.05).However,CGRP inhibited the production of proinflammatory cytokines in RAW264.7 cells in a concentration-dependent manner(P<0.05).Furthermore,OMVs at 20μg·mL-1 reduced cell activity and promote cell apoptosis.Nevertheless,co-treatment with 10 nmol·L-1 CGRP increased cell activity,reduced cell apoptosis and inhibited OMVs-caused degranulation(P<0.05).Conclusion CGRP can effectively inhibit the secretion of proinflammatory cytokines,reduce apoptosis and release of neutrophil specific granules,and further suppress inflammatory response induced by OMVs of Pseudomonas aeruginosa in RAW264.7 cells.

关 键 词：铜绿假单胞菌 降钙素基因相关肽 外膜囊泡 炎症反应 

分 类 号：R633[医药卫生—外科学]