新型内皮素受体拮抗剂ETP-508对低氧培养大鼠肺动脉平滑肌细胞增殖的影响

Effects of ETP-508,a novel endothelin receptor antagonist,on the proliferation of pulmonary arterial smooth muscle cells of rat cultured in hypoxia environment

目的探讨新型内皮素受体拮抗剂ETP-508对低氧培养大鼠肺动脉平滑肌细胞(PASMCs)增殖的影响。方法贴壁原代培养的PASMCs分为4组:常氧组(氧浓度21%),低氧组(氧浓度2%),低氧+BQ-485组(BQ-485的终浓度分别为10-6、10-7、10-8、10-9mol/L),低氧+ETP-508组(ETP-508的终浓度分别为10-6、10-7、10-8、10-9mol/L)。4组细胞均分别培养24、48、72h后,采用MTT法(波长492nm)检测BQ-485和ETP-508对PASMCs增殖的影响。按上述分组培养细胞48h,采用流式细胞仪测定细胞周期;放免法测定培养上清液中内皮素-1(ET-1)的含量。结果24h时各实验组A值差异不明显;48h时低氧组A值明显增加(P<0.01),低氧+BQ-485组、低氧+ETP-508组在10-8、10-7、10-6mol/L时A值下降,与低氧组比较有统计学意义(P<0.01);72h时低氧组A值仍高于常氧组,但较48h时略下降(P<0.05)。48h时低氧组G2、S期细胞比率、DNA合成增加,与常氧组比较有统计学意义(P<0.01,P<0.05);与低氧组比较,低氧+BQ-485组、低氧+ETP-508组G2、S期细胞比率下降(P<0.05),G1期细胞增多(P<0.05),DNA合成减少。48h时低氧组ET-1水平增高(P<0.01),给予BQ-485、ETP-508后ET-1水平降低,10-7mol/L BQ-485、10-9mol/L ETP-508可明显降低ET-1水平(P<0.01)。结论ETP-508作为一种新型内皮素受体拮抗剂能抑制低氧培养PASMCs的增殖,减少ET-1的生成,且其作用较BQ-485强。

Objective To study the effects of ETP-508, a novel endothelin receptor antagonist, on the proliferation of pulmonary arterial smooth muscle cells （PASMCs） of rat cultured in hypoxia environment. Methods Primary culture of rat PASMCs was prepared by the method of tissue block anchorage, and they were assigned into four groups： normoxia group （21% O2 ）, hypoxia group （2% O2 ）, hypoxia＋BQ-485 group （10^-6, 10^-7, 10^-8, 10^-9mol/L） and hypoxia ＋ ETP-508 group （10^-6, 10^-7, 10^-8, 10^-9mol/L）. MTT （492nm） assay was used to detect the A value of the four groups after cells were cultured for 24, 48 and 72h. Flow cytometry and mdioimmunoassay were respectively used to detect the cell cycle and ET-1 content at 48h time point. Results MTT assay demonstrated that A value of each group did not significantly differ at 24h time point. At 28h time point, A value of hypoxia group was markedly increased （ P〈0. 01）. Meanwhile, A values of both hypoxia ＋ BQ-485 group and hypoxia ＋ ETP-508 group were statistically different compared with hypoxia group （P〈20. 01）, and it declined when the concentration of BQ-485 or ETP-508 was 10^-8, 10^-7 and 10^-6mol/L. A value of hypoxia group was still higher than that of the normoxia group at 72h time point, but lower than that at 28h time point （P〈20. 05）. Flow eytometry demonstrated that at 48h time point, G2, S cell rates and DNA synthesis of hypoxia group increased compared to normoxia group （ P〈2 0. 01, P〈20. 05）. Compared with hypoxia group, Gz, S cell rates （ P〈0. 05） and DNA synthesis decreased, and Cn cells rates increased （ P〈20. 05） in hypoxia ＋ BQ-485 group and hypoxia ＋ ETP-508 group. ET-1 content of hypoxia group elevated at 48h time point, and significantly decreased after being treated with BQ-485 （10^-7mol/L） and ETP-508 （10^-9mol/L, P〈0. 01）. Conclusion ETP-508, as a novel endothelin receptor antagonist, can inhibit the proliferation of PASMCs, reduce ET-1 generation, and has a stronger effect than the classical endothelin receptor antagonist, BQ-485.