灰树花多糖D组分对PM_(2.5)引起肺泡巨噬细胞损伤的拮抗作用

Antagonistic effects of Grifolan D-fraction on damage of alveolar macrophages induced by fine particulate matter

目的探讨灰树花多糖D组分（D-fraction）对大气细颗粒物PM2.5染毒后的大鼠肺泡巨噬（NR8383）细胞损伤的拮抗作用。方法分别以不同浓度的SRM 2786悬液（31.25、62.5、125、250和500μg/ml）、不同浓度的D-fraction溶液（1.56、3.125、6.25、12.5、25、50、100、200及400μg/ml）及SRM 2786溶液（浓度为125μg/ml）与不同浓度的D-fraction溶液（3.125、6.25、12.5、25μg/ml）暴露NR8383细胞24 h,同时,设置空白对照（F-12K培养基）组,采用MTT法检测细胞存活率。将125μg/ml SRM 2786悬液分别与不同浓度（3.125、6.25、12.5、25μg/ml）的D-fraction溶液联合暴露NR8383细胞24 h后,采用酶联免疫法（Elisa）检测细胞上清液肿瘤坏死因子α（TNF-α）、白介素6（IL-6）及白介素1β（IL-1β）三种炎性因子的释放。结果与空白对照组相比,各浓度SRM 2786暴露组NR8383细胞的存活率均较低,差异有统计学意义（P〈0.01）;且随着SRM 2786暴露浓度的升高,NR8383细胞的存活率呈下降趋势。与空白对照组相比,1.56~25μg/ml D-fraction暴露组NR8383细胞的存活率均升高,差异有统计学意义（P〈0.05）;而50~400μg/ml D-fraction暴露组NR8383细胞的存活率均无明显改变。与125μg/ml SRM 2786暴露组相比,125μg/ml SRM 2786＋各浓度D-fraction暴露组NR8383细胞的存活率均明显增高;细胞上清液中的TNF-α、IL-6和IL-1β含量均较低,除125μg/ml SRM 2786＋3.125μg/ml D-fraction暴露组外,差异有统计学意义（P〈0.01）。且随着D-fraction暴露浓度的升高,125μg/ml SRM 2786暴露NR8383细胞的存活率及细胞上清液中的TNF-α和IL-1β含量均呈逐渐下降趋势,IL-6含量呈波浪性下降的趋势。结论 PM2.5可显著降低细胞存活率和促进炎性因子的释放,而D-fraction可改善由PM2.5引发的NR8383细胞损伤。

Objective To investigate the antagonistic effect of Grifolan D-fraction on damage of pulmonary macrophages（NR8383） induced by fine particulate matter（PM2.5）. Methods SRM 2786（31.25,62.5,125,250 and 500 μg/ml）, Grifolan Dfraction（1.56,3.125,6.25,12.5,25,50,100,200 and 400 μg/ml）,and SRM 2786（125 μg/ml） with the doses of Grifolan Dfraction（3.125,6.25,12.5,25 μg/ml） were selected to treat NR8383 for 24 h respectively, cell survival rates were measured by MTT method. Then NR8383 cells were exposed to both of 125 μg/ml SRM 2786 and Grifolan D-fraction（3.125,6.25,12.5,25μg/ml） for 24 h. ELISA kits were applied to examine cytokine release（TNF-α,IL-6,IL-1β） from the NR8383 cells supernatant. Results SRM 2786 significantly decreased the cell survival rate in contrast to the control group（P〈0.01）; The Grifolan D-fraction（1.56-25 μg/ml） significantly improved NR8383 cells survival rates in contrast to control group（P〈0.05）,yet, no obvious changes in cell survival rates were detected when the concentrations of Grifolan D-fraction were 50-400 μg/ml;Compared with SRM 2786-treated group（125 μg/ml）,the combination of SRM 2786（125 μg/ml） and Grifolan D-fraction（1.56,3.125,6.25,12.5,25,50,100,200 and 400 μg/ml） significantly increased NR8383 cells survival rates; The levels of inflammatory cytokines（TNF-α,IL-6,IL-1β） in supernatant were significantly decreased（P〈0.01） except the group that treated by 125 μg/ml SRM 2786 and 3.125 μg/ml D-fraction. In addition, with the increased concentrations of Grifolan D-fraction, the levels of TNF-α and IL-1β in supernatant declined gradually, while the level of IL-6 in supernatant decreased undulately. Conclusion PM2.5can obviously reduce NR8383 cells survival rate and promote the release of different inflammatory cytokines, yet D-fraction can effectively improve the damages of NR8383 cells induced by PM2.5.