COX2抑制剂对大鼠肺泡巨噬细胞生长及炎症反应的影响及机制研究

Effect of COX2 inhibitor on growth and inflammatory response of rat alveolar macrophages and its mechanism

目的探讨环氧合酶2(COX2)抑制剂塞来昔布对肺泡巨噬细胞凋亡、炎症反应及吞噬能力的影响。方法四甲基偶氮唑盐(MTT)比色法检测0.1μmol/L、1μmol/L、5μmol/L、10μmol/L、20μmol/L塞来昔布对大鼠肺泡巨噬细胞株NR8383的细胞毒性,选择合适的塞来昔布浓度。将NR8383细胞随机分为3组,即对照组、LPS组和LPS+塞来昔布组。对照组:细胞培养板加等体积磷酸盐缓冲液(PBS);LPS处理组:细胞用终浓度为1μg/ml的脂多糖(LPS)处理;LPS+塞来昔布组:10μmol/L的塞来昔布处理细胞1 h后,加1μg/ml的LPS处理细胞24 h。流式细胞术检测细胞凋亡率;Western blotting检测NF-κBp65、IκBα、Bax和Bcl-2蛋白表达;qRT-PCR检测炎症因子TNF-αmRNA表达;激光共聚焦技术检测巨噬细胞的吞噬能力。结果0.1~10μmol/L塞来昔布处理对NR8383细胞无细胞毒性,20μmol/L塞来昔布处理可抑制NR8383细胞活力。LPS组与对照组比较,细胞凋亡率升高,NF-κBp65和Bax蛋白表达升高,IκBα和Bcl-2蛋白表达降低,TNF-αmRNA表达升高,细胞吞噬能力降低(P<0.05),LPS+塞来昔布组与LPS组比较,细胞凋亡率降低,NF-κBp65和Bax蛋白表达降低,IκBα和Bcl-2蛋白表达升高,TNF-αmRNA表达降低,细胞吞噬能力升高(P<0.05)。结论塞来昔布可通过下调NF-κB信号抑制LPS诱导的NR8383细胞凋亡及炎症反应,并维持吞噬功能,从而对肺损伤起保护作用。

Objective To investigate the effects of COX2 Inhibitor Celecoxib on apoptosis,inflammatory response and phagocytosis of alveolar macrophages.Methods Cytotoxicity of 0.1μmol/L,1μmol/L,5μmol/L,10μmol/L,20μmol/L celecoxib on rat alveolar macrophage cell line NR8383 was detected by MTT colorimetric assay.The proper concentration of celecoxib was selected.NR8383 cells were randomly divided into 3 groups,namely,control group,LPS group and LPS+celecoxib group.Control group:cell culture plate plus equal volume of phosphate buffered saline(PBS);LPS treatment group:cells treated with lipopolysaccharide(LPS)at a final concentration of 1μg/ml;LPS+celecoxib group:10μmol/L After celecoxib treatment of cells for 1 h,cells were treated with 1μg/ml of LPS for 24 h.Flow cytometry was used to detect the apoptosis rate of cells.Western blotting was used to detect the expression of NF-кBp65,IкBα,Bax and Bcl-2 protein,and qRT-PCR was used to detect the expression of inflammatory factors TNF-αmRNA,and the phagocytosis of macrophages was detected by laser confocal technique.Results 0.1~10μmol/L celecoxib treatment had no cytotoxicity on NR8383 cells,and 20μmol/L celecoxib treatment could inhibit NR8383 cell viability.Compared with the LPS group and the control group,the apoptosis rate increased,the expression of NF-кBp65 and Bax protein increased,the expression of IкBαand Bcl-2 protein decreased,the mRNA expression of TNF-αincreased,and the phagocytosis of the cells decreased(P<0.05).Compared with LPS+celecoxib group and LPS group,the apoptosis rate,the expression of NF-кBp65 and Bax protein decreased,the expression of IкBαand Bcl-2 protein increased,the expression of TNF-αmRNA decreased,and the phagocytosis of cells increased(P<0.05).Conclusion Celecoxib can inhibit the apoptosis and inflammatory response of NR8383 cells induced by LPS by down regulation of NF-кB signal,and maintain the phagocytic function,thus protecting the lung injury.