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目的从细胞分子水平上研究15-酮基二十碳四烯酸(15-ketoeicosatetretraenoic acid,15-KETE)对大鼠肺动脉平滑肌细胞上ERK1/2的活性的影响。方法通过酶法分离、培养SD大鼠肺动脉平滑肌细胞(pulmonary arterial smooth cells,PASMCs),使用Western blot技术观察15-KETE对大鼠PASMCs上ERK1/2活性的影响。结果 1)10-6mol/L 15-KETE作用从0.5 h到24 h与对照组(未加15-KETE)比较,对ERK1/2总量没有明显的影响。2)与对照组比较,15-KETE处理0.5、1.0、2.0、4.0、8.0 h明显增强p-ERK1/2(P<0.05)。3)与对照组比较,10-8、10-7和10-6mol/L 15-KETE明显增强p-ERK1/2(P<0.05),随15-KETE浓度增大,p-ERK1/2增多。4)与15-KETE作用比较,ERK1/2抑制剂PD98059 20μmol/L、50μmol/L和U0126 0.1μmol/L、1μmol/L均能抑制15-KETE对ERK1/2的活化(P<0.05),并且随着抑制剂浓度增大,抑制作用加强。结论 15-KETE对大鼠肺动脉血管平滑肌细胞上的ERK1/2表达没有明显的影响,但能激活肺动脉血管平滑肌细胞ERK1/2,使其磷酸化,并呈时间-剂量依赖关系。ERK1/2通路的特异性抑制剂PD98059、U0126能抑制15-KETE对ERK1/2的磷酸化。
Objective To investigate the effect of 15-ketoeicosatetratraenoic acid (15-KETE) on the activity of ERK1 / 2 on rat pulmonary artery smooth muscle cells at the cellular and molecular level. Methods Pulmonary artery smooth muscle cells (PASMCs) of SD rats were isolated and cultured by enzymatic method. The effect of 15-KETE on the activity of ERK1 / 2 on PASMCs was observed by Western blot. Results 1) Compared with the control group (without 15-KETE), the effect of 10-6mol / L 15-KETE from 0.5 h to 24 h had no significant effect on the total amount of ERK1 / 2. 2) Compared with the control group, 15-KETE treatment significantly increased p-ERK1 / 2 at 0.5, 1.0, 2.0, 4.0 and 8.0 h (P <0.05). 3) Compared with the control group, p-ERK1 / 2 (P <0.05) was enhanced by 10-8, 10-7 and 10-6mol / L 15-KETE, . 4) ERK1 / 2 inhibitor PD98059 inhibited the activation of ERK1 / 2 (P <0.05) by 20μmol / L, 50μmol / L and U0126 0.1μmol / L and 1μmol / And as the inhibitor concentration increases, the inhibitory effect is strengthened. Conclusions 15-KETE has no obvious effect on the expression of ERK1 / 2 on pulmonary artery smooth muscle cells, but it can phosphorylate ERK1 / 2 in pulmonary artery smooth muscle cells in a time-and dose-dependent manner. The specific inhibitors of ERK1 / 2 pathway PD98059 and U0126 can inhibit the phosphorylation of ERK1 / 2 by 15-KETE.