中性粒细胞颗粒蛋白对炎症巨噬细胞脂质运载蛋白2表达的影响  

Effects of neutrophilic granule protein on the expression of lipocalin 2 in inflammatory macrophages

作　　者：王静 程绩 鲍全伟 朱俊宇 梁华平 Wang Jing;Cheng Ji;Bao Quanwei;Zhu Junyu;Liang Huaping(Department of Emergency Medicine,Xinqiao Hospital,Army Medical University,Chongqing 400037,China;Department of Wound Infection and Drug,Daping Hospital,Army Medical University,State Key Laboratory of Trauma and Chemical Poisoning,Chongqing 400042,China)

机构地区：[1]陆军军医大学新桥医院急诊科,重庆400037 [2]陆军军医大学大坪医院战伤感染与特需药品研究室,创伤与化学中毒全国重点实验室,重庆400042

出　　处：《中华危重病急救医学》2024年第10期1033-1037,共5页Chinese Critical Care Medicine

基　　金：基础加强计划项目(2022-JCJQ-ZD-224-12,2021-JCJQ-JJ-1081);重庆市博士"直通车"科研项目(CSTB2022BSXM-JCX0009)。

摘　　要：目的探讨中性粒细胞颗粒蛋白(NGP)对炎症巨噬细胞脂质运载蛋白2(LCN2)表达的影响及其机制。方法体外培养NGP高表达巨噬细胞株RAW264.7细胞(NGP/RAW细胞)和阴性对照RAW264.7细胞(NC/RAW细胞);同时提取NGP高表达小鼠和野生型C57BL/6小鼠原代腹腔巨噬细胞,并体外培养。使用10 mg/L脂多糖(LPS)分别刺激上述细胞构建炎症模型(LPS组),并设磷酸盐缓冲液(PBS)对照组;采用酶联免疫吸附试验(ELISA)检测不同类型细胞中LCN2水平,采用蛋白质免疫印迹试验(Western blotting)检测细胞中磷酸化信号转导及转录激活因子1(p-STAT1)的蛋白表达。另取NGP/RAW细胞和NC/RAW细胞,分别给予10 mg/L的LPS、5 mg/L的STAT1通路抑制剂氟达拉滨+10 mg/L的LPS处理,并设PBS对照组;采用ELISA法检测细胞中LCN2水平。结果在不同类型细胞中,与PBS对照组相比,LPS组LCN2水平均显著升高,并在24 h达峰值(μmol/L:NC/RAW细胞为25.61±1.02比0.46±0.02,NGP/RAW细胞为74.51±2.14比0.25±0.04,野生型C57BL/6小鼠原代巨噬细胞为10.13±0.22比0.01±0.01,NGP高表达小鼠原代巨噬细胞为28.35±0.61比0.08±0.01,均P<0.05),说明炎症反应时巨噬细胞中LCN2水平发生变化。与NC/RAW细胞相比,给予LPS刺激后,NGP/RAW细胞中各时间点LCN2水平升高更为显著(μmol/L:6 h为8.32±0.22比3.12±0.11,12 h为23.12±0.86比8.12±0.32,24 h为74.51±2.14比25.61±1.02,均P<0.05),且p-STAT1蛋白表达亦明显上调;与野生型C57BL/6小鼠原代巨噬细胞相比,给予LPS刺激后24 h NGP高表达小鼠原代巨噬细胞中LCN2水平亦显著升高(μmol/L:28.35±0.61比10.13±0.22,P<0.05)。使用STAT1通路抑制剂预处理细胞后,NGP/RAW细胞中LCN2水平较LPS组明显下降(μmol/L:6.81±0.19比22.54±0.58,P<0.05),但抑制剂对NC/RAW细胞中LCN2的生成无明显影响,与LPS组比较差异无统计学意义(μmol/L:8.04±0.20比7.86±0.15,P>0.05),说明NGP可以通过促进STAT1活化,从而上调LPS刺激后巨噬细胞中LCN2的表达。�Objective To explore the effects of neutrophilic granule protein(NGP)on the expression of lipocalin 2(LCN2)in inflammatory macrophages and its mechanism.Methods NGP-high-expressed RAW264.7 cells(NGP/RAW cells)and negative control RAW264.7 cells(NC/RAW cells)were cultured in vitro.Primary peritoneal macrophages of NGP-high-expressed mice and wild-type C57BL/6 mice were extracted,then cultured in vitro.The cell inflammatory model was established by stimulating with 10 mg/L lipopolysaccharide(LPS,LPS group),and the phosphate buffer solution(PBS)control group was set up.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of LCN2 in different types of cells.The protein expression of phosphorylated signal transduction and activator of transcription 1(p-STAT1)was detected with Western blotting.Other NGP/RAW cells and NC/RAW cells were treated with 10 mg/L LPS,5 mg/L STAT1 pathway inhibitor(fludarabine)+10 mg/L LPS,respectively.The PBS control group was set up.ELISA was used to detect the level of LCN2.Results In different types of cells,the levels of LCN2 were increased significantly after LPS stimulation in the LPS group as compared with those in the PBS control group,and peaked at 24 hours(μmol/L:25.61±1.02 vs.0.46±0.02 in NC/RAW cells,74.51±2.14 vs.0.25±0.04 in NGP/RAW cells,10.13±0.22 vs.0.01±0.01 in primary macrophages of wild-type C57BL/6 mice,28.35±0.61 vs.0.08±0.01 in primary macrophages of NGP-high-expressed mice,all P<0.05),indicating that the expression of LCN2 in macrophages altered during inflammation reaction.The level of LCN2 in NGP/RAW cells was found significantly increased at different time points after LPS stimulation comparing with that in NC/RAW cells(μmol/L:8.32±0.22 vs.3.12±0.11 at 6 hours,23.12±0.86 vs.8.12±0.32 at 12 hours,74.51±2.14 vs.25.61±1.02 at 24 hours,all P<0.05),along with the expression of p-STAT1 was significantly up-regulated.The level of LCN2 in the primary macrophages of NGP-high-expressed mice was also significantly increased at 24 hours after LPS

关 键 词：中性粒细胞颗粒蛋白 巨噬细胞 脂多糖 脂质运载蛋白2 

分 类 号：R392[医药卫生—免疫学]