ERK1/ERK2磷酸化在西地那非抑制猪肺动脉平滑肌细胞增殖中的作用

Role of phosphorylation of ERK1/ERK2 in antiproliferative effect of slildenafil on porcine pulmonary artery smooth muscle cells

目的探讨丝裂原激活的蛋白激酶-44/蛋白激酶-42（ERK1/ERK2）磷酸化在西地那非抑制猪肺动脉平滑肌细胞增殖中的作用。方法体外原代培养猪肺动脉平滑肌细胞。细胞培养到3～5代后用于实验。随机分为4组：对照组（C组）、血小板衍生生长因子（PDGF）组（P组）、西地那非干预组（PS1组和PS2组）,P组细胞用20 ng/ml PDGF孵育,PS1组和PS2组在加入PDGF前20 min分别加入24、96μmol/L西地那非。于加入PDGF后1 h测定ERK1/ERK2磷酸化水平;于加入PDGF后24 h掺入5-溴脱氧尿嘧啶核苷（5-BrdU）,计算5-BrdU阳性细胞百分率,反映细胞DNA合成水平,并测定增殖细胞核抗原（PCNA）蛋白表达;于加入PDGF后3 d测定细胞增殖程度。结果与C组比较,P组、PS1组和PS2组ERK1/ERK2磷酸化水平、PCNA表达、5-BrdU阳性细胞百分率及细胞增殖率增加（P＜0．05或0．01）,PS1组和PS2组ERK1/ERK2磷酸化水平、PCNA表达、5-BrdU阳性细胞百分率及细胞增殖率低于P组（P＜0．05或0．01）,各组间总ERK1/ERK2水平差异无统计学意义（P＞0．05）。结论西地那非通过抑制ERK1/ERK2的磷酸化,下调PCNA的表达,抑制了DNA的合成,从而抑制了PDGF诱导猪肺动脉平滑肌细胞的增殖。

Objective To investigate the underlying mechanism of the antiproliferative effect of sildenafil on the pulmonary artery smooth muscle cells （PASMCs）. Methods Primary cultured porcine PASMCs after 3-5 passages were randomly divided into 4 groups-Ⅰ control group ; Ⅱ PDGF group in which PASMCs were incubated with platelet-derived growth factor （20 ng/ml） ;Ⅲ and Ⅳ sildenafil groups in which sildenafil 24 and 96 μmol/L were added to the cells respectively 20 min before the cells were incubated with PDGF. The phosphorylation of ERK1/ERK2 was measured by Western blot at 1 h after PDGF was added. DNA synthesis and expression of proliferative cell nuclear antigen （PCNA） protein were assessed by 5-BrdU incorporation assay and Western blot at 24 h after addition of PDGF. MTT assay was used to assess cell proliferation after the cells were incubated with PDGF for 3 days. Results The proliferation index, level of DNA synthesis, expression of PCNA protein and level of phosphorylation of ERK1/ERK2 were significantly increased in PDGF group and 2 sildenafil groups as compared with control group and meanwhile they were significantly lower in the 2 sildenafil groups than in PDGF group. The difference in expression of total ERK1/ERK2 was not significant among the 4 groups. Conclusion Sildenafil can inhibit proliferation of porcine PASMCs induced by PDGF by suppression of ERK1/ERK2 phosphorylation and down-regulation of PCNA expression.