P38MAPK在低氧及炎症联合刺激诱导人肺微血管内皮细胞凋亡中的作用

Role of P38MAPK in human pulmonary microvascular endothelial cells apoptosis induced by hypoxia and inflammation combination

目的探讨P38MAPK在低氧及炎症联合刺激诱导人肺微血管内皮细胞凋亡中的作用。方法将人肺微血管内皮细胞株进行复苏及传代培养,分为空白对照组、低氧组(低氧6 h、12 h、24 h)、肿瘤坏死因子(TNF-α)刺激组(用10 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml的TNF-α进行刺激)、模型组(低氧24 h+TNF-α100 ng/ml联合刺激)及通路阻断剂组(加入通路阻断剂SB203580进行干预)。Western-blot法分析各组细胞中P38丝裂原活化蛋白激酶(P38MAPK)表达,流式细胞术分析各组细胞半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活力,流式细胞术和TUNEL法联合检测细胞凋亡。结果与空白对照组相比,模型组p-P38MAPK表达升高(P<0.05)。与低氧组(24 h)、TNF-α刺激组(100 ng/ml)相比,模型组(低氧24 h+TNF-α100ng/ml联合刺激)细胞活力均下降(P均<0.01);与模型组相比,通路阻断剂组细胞Caspase-3活力下降(P<0.01)。与模型组相比,通路阻断剂组细胞凋亡率及凋亡指数均下降(P<0.01)。结论 P38MAPK在低氧及炎症联合刺激诱导的人肺微血管内皮细胞中具有促凋亡作用,P38MAPK通路阻断剂对低氧及炎症联合刺激损伤的人肺微血管内皮细胞具有保护作用。

Objective To investigate the effect of p38mitogen-actvated protein kinase (P38MAPK) on theapoptosis of human pulmonary microvascular endothelial cells induced by hypoxia and inflammation. MethodsHuman pulmonary microvascular endothelial cell line was recovered, subcultured, divided into blank control group,hypoxia group (hypoxia 6 h, 12 h, 24 h) and TNF alpha stimulation group (10 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/mlTNF alpha), model group (hypoxia combined with 100 ng/ml TNF alpha ) and pathway blocker group (SB203580).P38MAPK expression in each group was detected by Western blotting. Caspase-3 activity in each group wasanalyzed by flow cytometry. Apoptosis were detected by combination of flow cytometry and TUNEL assay. ResultsCompared with the blank control group, p-P38MAPK expression of model group was increased (P<0.05). Caspase-3activity of model group (24 h hypoxia combined with 100ng/ml TNF alpha) was reduced compared with hypoxiagroup (24 h) and TNF alpha stimulation group (100 ng/ml) (P<0.01); Caspase-3 activity of pathway blocker groupwas reduced compared with model group (P<0.01). Apoptosis rate and apoptotic index were reduced in pathwayblocker group than model group (P<0.01). Conclusion P38MAPK plays a role in promoting in human pulmonarymicrovascular endothelial cells apoptosis induced by hypoxia and inflammation. P38MAPK pathway blocker(SB203580) has protective effect on hypoxia and inflammation in human pulmonary microvascular endothelial cells.