西地那非通过上调ERK1/ERK2磷酸化增强5-羟色胺的促肺动脉平滑肌细胞增殖作用

摘要

目的 探讨西地那非增强5-羟色胺引起的肺动脉平滑肌细胞增殖作用的可能机制.方法 体外原代培养猪肺动脉平滑肌细胞,取第3～5代细胞用于实验.细胞随机分为4组:对照组(CON)、5-羟色胺组(HT)、西地那非组(SIL)、西地那非和5-羟色胺联合干预组(S-HT).细胞传代培养的第3天,给予含0.2%胎牛血清的培养基饥饿72 h,CON组、HT组、SIL组分别给予PBS、10 μmol/L 5-HT、1 μmol/L西地那非干预;S-HT组提前20 min给予1 μmol/L西地那非干预,再给予10 μmol/L 5-羟色胺处理.细胞干预60 min后采用Western blot法测定各组细胞外信号调节激酶(ERK1/ERK2)的磷酸化水平;加入5-羟色胺24 h后,流式细胞法测定细胞周期并计算S期细胞占细胞总数的比例;加入5-羟色胺72 h后MTT法检测细胞增殖程度.结果 与CON组相比,HT组ERK1/ERK2的磷酸化水平、S期细胞比例及细胞增殖率明显增加(P<0.05),S-HT 组ERK1/ERK2的磷酸化水平、S期细胞比例及细胞增殖率较HT组进一步增加(P<0.05);各组间总ERK1/ERK2水平差异无统计学意义(P>0.05).结论 西地那非增强5-羟色胺引起的肺动脉平滑肌细胞DNA合成和增殖,可能与ERK1/ERK2的磷酸化水平上调有关.%Objective To explore the underlying mechanism of potential effect of sildenafil on porcine pulmonary artery smooth muscle cells (SMCs) proliferation induced by serotonin. Methods Porcine pulmonary artery SMCs at 3-5 passages were randomly divided into 4 groups: control group (CON), 5-HT group (HT), sildenafil group (SIL) and sildenfil-5HT combined group (S-HT). Pulmonary artery SMCs at exponential growth phase were serum starved with 0.2% FBS for 72 h, followed by sterile PBS, serotonin (10 μmol/L) and sildenafil (1 μmol/L) incubation in CON group, HT group and SIL group, respectively. In combined group (S-HT): pulmonary artery SMCs were serum starved and then exposed to sildenafil for 20 min, followed by serotonin incubation for indicated time. The phosphorylation of extracellular signal regulated kinase (ERK1/ERK2) was measured by Western blot at indicated time points. Flow active cell sorting (FACS) was used to evaluate the ratio of S phase cells in the cell cycle after 24 h of treatment. MTT color metric method was used to analyze SMCs proliferative index after 72 h of treatment. Results Compared with CON group, the phosphorylation of ERK1/ERK2, the percentage of cells in S phase, and the cell proliferation index increased remarkably after incubation with 5-HT (P<0.05). Preincubation with sildenafil followed by serotonin enhanced the phosphorylation of ERK1/ERK2 (p-ERK1/ERK2) and further elevated the percentage of cells in S phase and the cell proliferation index compared with that of HT group. While the total ERK1/ERK2 (t-ERK1/ERK2) was not statistically different among these groups. Conclusions Sildenafil potentiates the proliferative effect of serotonin on pulmonary arterial SMCs via upregulating phosphorylation of ERK1/ERK2.