机械牵张诱导大鼠PMVECs黏附能力增强时内皮素-1与p38MAPK和PI3K/Akt信号通路的关系

摘要

Objective To evaluate the relationship between endothelin-1 (ET-1) and p38 mitogenactivated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathways during mechanical stretch-induced enhancement of adhesion of rat pulmonary microvascular endothelial cells (PMVECs).Methods Rat PMVECs were seeded in the culture plate at a density of 0.5×105 cells/ml (2 ml/well) and divided into 5 groups (n=24 each) using a random number table:control group (group C),mechanical stretch group (group MS),mechanical stretch plus specific PI3K inhibitor LY294002 group (LY group),mechanical stretch plus specific p38 MAPK inhibitor SB203580 group (SB group),and mechanical stretch plus selective ETA receptor blocker BQ123 group (BQ group).Cells were exposed to 20％ cyclic stretch at 0.3 Hz for 4 h using a sine wave.In LY,SB and BQ groups,LY294002,SB203580 and BQ123 at the final concentration of 10 μmol/L were added,respectively,after mechanical stretch,cells were incubated for 10 min,and then extracted and purified rat polymorphonuclear neutrophil leukocytes (PMNs,5× 105 cells/well) were added and co-incubated with PMVECs for 30 min and then washed out.The concentrations of ET-1 and interleukin-6 (IL-6) in the culture medium were determined using enzyme-linked immunosorbent assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated Akt (p-Akt) was detected by Western blot.Adhesion of PMNs was measured by immuno-histochemistry,and the adhesion rate was calculated.The expression of P-selectin mRNA was detected using real-time polymerase chain reaction.Results Compared with group C,the concentrations of IL-6 and ET-1 in the culture medium were significantly increased,the expression of p-p38 MAPK,p-Akt and P-selectin mRNA was up-regulated,and the adhesion rate of PMNs was increased in the other four groups (P＜0.05).Compared with group MS,the concentration of IL-6 in the culture medium was significantly decreased,the expression of p-Akt and P-selectin mRNA was down-regulated,and the adhesion rate of PMNs was decreased in LY,SB and BQ groups,the concentration of ET-1 in the culture medium was significantly decreased in group BQ,and the expression of p-p38 MAPK was significantly down-regulated in SB and BQ groups (P＜0.05).Conclusion The signaling mechanism underlying ET-1-mediated enhancement of rat PMVEC adhesion may be related to activating p38 MAPK and PI3K/Akt signaling pathways.%目的 评价机械牵张诱导大鼠肺微血管内皮细胞(PMVECs)黏附能力增强时内皮素-1(ET-1)与p38丝裂原活化蛋白激酶(p38 MAPK)和磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路的关系.方法 将PMVECs以0.5×105个/ml的密度接种于培养板(2 ml/孔),采用随机数字表法分为5组(n=24):正常对照组(C组)、机械牵张组(MS组)、机械牵张+PI3K特异性抑制剂LY294002组(LY组)、机械牵张+p38 MAPK特异性抑制剂SB203580组(SB组)、机械牵张+ET受体A阻断剂BQ123组(BQ组).机械牵张:拉伸幅度20％,频率0.3 Hz,波形为正弦波,时间4h.LY组、SB组和BQ组在机械牵张后分别加入终浓度均为10 μmol/L的LY294002、SB203580和BQ123,孵育10 min;随后加入提取的PMNs(5×105/孔)作用30 min.采用ELISA法检测培养液ET-1和IL-6浓度.采用Western blot法测定磷酸化p38 MAPK(p-p38 MAPK)和磷酸化Akt(p-Akt)表达水平.采用免疫组化法确定PMNs黏附率.采用real-time PCR法测定P-选择素mRNA的表达水平.结果 与C组比较,其余4组培养液IL-6和ET-1浓度升高,p-p38 MAPK、p-Akt和P-选择素mRNA表达上调,PMNs黏附率升高(P＜0.05).与MS组比较,LY组、SB组和BQ组培养液IL-6浓度降低,p-Akt和P-选择素mRNA表达下调,PMNs黏附率降低,BQ组培养液ET-1浓度降低,SB组和BQ组p-p38 MAPK表达下调(P＜0.05).结论 ET-1介导大鼠PMVECs机械牵张后黏附能力增强的信号机制可能与激活p38 MAPK和PI3K/Akt信号通路有关.