Fas/FasL途径在PM2.5诱导肺泡上皮细胞凋亡中的作用及机制

Role of Fas/FasL pathway in PM2.5-induced apoptosis of alveolar epithelial cells and its mechanism

目的探讨Fas/FasL途径在PM2.5诱导肺泡上皮细胞凋亡中的作用机制。方法24只SD大鼠随机分为对照组、PM2.5低剂量组、中剂量组和高剂量组,每组6只,分别经气管内滴注0.5 ml PBS或者1,2,4 mg/ml的PM2.5混悬液;CCL149细胞分为对照组、PM2.5组、PM2.5+NOK-2(FasL中和性抗体)组和PM2.5+Z-IETD-FMK(Caspase-8特异性抑制剂)组,对照组1 ml培养基中加入1 ml PBS,其余各组1 ml培养基中加入1 ml 100μg/ml的PM2.5混悬液,PM2.5+NOK-2组和PM2.5+Z-IETD-FMK组在PM2.5暴露前1 h分别给予NOK-2、Z-IETD-FMK干预。HE染色观察大鼠肺组织病理学改变,TUNEL染色和流式细胞仪分别检测肺组织和CCL149细胞凋亡,Western blot检测肺组织和CCL149细胞中Fas、FasL、Caspase-8和Caspase-3蛋白的表达水平。结果与对照组比较,PM2.5中剂量组和高剂量组大鼠肺组织出现病理学改变和细胞凋亡增加(P<0.01),低剂量组无明显变化(P>0.05),中剂量组肺组织Fas和Caspase-3蛋白表达上调(P<0.01)。与对照组比较,PM2.5组CCL149细胞凋亡率增加,呈浓度和时间依赖性(P<0.05),Fas、FasL、Caspase-8和Caspase-3蛋白表达增加(P<0.01)。与PM2.5组比较,PM2.5+NOK-2组和PM2.5+Z-IETD-FMK组细胞凋亡率下降(P<0.01),Caspase-8和Caspase-3蛋白表达下调(P<0.01)。结论PM2.5暴露可以诱导大鼠肺组织损伤和肺泡上皮细胞凋亡,Fas/FasL途径介导了PM2.5诱导肺泡上皮细胞凋亡,Fas/FasL在PM2.5诱导肺损伤中具有重要作用。

Objective To explore the role and mechanism of Fas/FasL pathway in apoptosis of alveolar epithelial cells induced by PM2.5.Methods Twenty-four SD rats were randomly divided into control group,PM2.5 low dose group,middle dose group and high dose group(6 rats in each group).The rats in PM2.5 groups were intratracheally instilled with 0.5 ml suspension of 1,2,4 mg/ml PM2.5,respectively,and the rats in control group were treated with the same volume of PBS.CCL149 cells were divided into control group,PM2.5 group,PM2.5+NOK-2(FasL neutralizing antibody)group and PM2.5+Z-IETD-FMK(Caspase-8 specific inhibitor)group.PBS 1 ml was added to 1 ml medium in control group,and 1 ml suspension of 100μg/ml PM2.5 was added to 1 ml medium in the other groups.CCL149 cells in PM2.5+NOK-2 group and PM2.5+Z-IETD-FMK group were intervened with NOK-2 and Z-IETD-FMK one hour before PM2.5 exposure,respectively.The pathological changes of lung tissues were observed by HE staining.The cell apoptosis in rat lung tissues and CCL149 cells was measured by TUNEL staining and flow cytometry,respectively.The protein expression of Fas,FasL,Caspase-8 and Caspase-3 was analyzed by Western blot.Results Compared with control group,the pathological changes were observed,and the cell apoptosis in lung tissue of rats was increased significantly in middle and high dose groups of PM2.5(P<0.01),but there was no significant change in low dose group(P>0.05).Compared with control group,the expression of Fas and Caspase-3 proteins in rat lung tissue was up-regulated in middle dose group(P<0.01).Compared with control group,the apoptosis rate of CCL149 cells was increased in a concentration-and time-dependent manner in PM2.5 groups(P<0.05),and the expression of Fas,FasL,Caspase-8 and Caspase-3 proteins in CCL149 cells was increased in PM2.5 groups(P<0.01).Compared with PM2.5 group,the apoptosis rate and the expression of Caspase-8 and Caspase-3 proteins in CCL149 cells were decreased in PM2.5+NOK-2 group and PM2.5+Z-IETD-FMK group(P<0.01).Conclusion PM2.5 can induce the lung injury and the cell apoptosis.Fas/FasL pathway mediates PM2.5-induced apoptosis of alveolar epithelial cells,and plays an important role in PM2.5-induced lung injury.