大鼠肺动脉平滑肌细胞Rho激酶对p-Smad1核迁移的作用及机制

Effects of Rho kinase on p-Smad1 nuclear translocation in rat pulmonary artery smooth muscle cells

目的:研究Rho激酶对p-Smad1核迁移的作用及信号转导途径,探讨它们在肺血管重构中的作用机制。方法:分离培养大鼠远端肺动脉平滑肌细胞,应用血小板源性生长因子BB(PDGF-BB)启动肺动脉平滑肌细胞Rho激酶信号通路,应用骨形态发生蛋白2(BMP-2)启动BMP信号通路,并用Rho激酶抑制剂Y-27632、MEK抑制剂U0126进行干预。培养细胞分成5组:(1)对照组;(2)BMP-2组;(3)BMP-2+PDGF-BB组;(4)BMP-2+PDGF-BB+Y-27632组;(5)BMP-2+PDGF-BB+U0126组。免疫荧光染色标记p-Smad1在细胞核内外的分布并计算p-Smad1核迁移阳性细胞百分数(即核迁移率)。分离平滑肌细胞核蛋白和胞浆蛋白,West-ern blotting分析p-Smad1在细胞内的总量和细胞核内外相对含量的变化。Kit-WST-8试剂盒检测细胞增殖。结果:BMP-2组p-Smad1在细胞内的总量、在细胞核中的相对含量和核迁移率明显高于对照组(P<0.05);BMP-2+PDGF-BB组p-Smad1的核迁移率和在细胞核中的相对含量明显低于BMP-2组(P<0.05),但是细胞内p-Smad1总量无明显改变(P>0.05);BMP-2+PDGF-BB+Y-27632组和BMP-2+PDGF-BB+U0126组细胞内p-Smad1的总量、在细胞核中的相对含量和核迁移率与BMP-2组基本相似(P>0.05)。BMP-2组的A值明显小于对照组(P<0.05);PDGF-BB+BMP-2组的A值明显大于BMP-2组(P<0.05)且大于对照组(P<0.05);BMP-2+PDGF-BB+Y-27632组和BMP-2+PDGF-BB+U0126组的A值均小于PDGF-BB+BMP-2组(P<0.05)。结论:在大鼠肺动脉平滑肌细胞,PDGF-BB激活的Rho激酶通过MEK/ERK1/2抑制BMP-2引起的p-Smad1核迁移,促进平滑肌细胞增殖。

AIM： To explore the mechanism of bone morphogenetic protein（BMP） and Rho kinase signal pathways on the proliferation of pulmonary artery smooth muscle cells.METHODS： Pulmonary smooth muscle cells were isolated from the rat distal pulmonary artery and cultured.BMP and Rho kinase pathways were activated by BMP-2 and platelet-derived growth factor BB（PDGF-BB）,respectively.Rho kinase inhibitor Y-27632 and MEK inhibitor U0126 were also used.Immunofluorescent staining was applied to observe p-Smad1 distribution across the nucleus,and the cells with positive p-Smad1 nuclear accumulation were counted and the nuclear translocation rate was calculated.The total p-Smad1 and its distribution across the nucleus were quantitatively determined by Western blotting.The cell proliferation was analyzed by CCK-8 assay.RESULTS： Exposure to BMP-2 significantly increased both the total amount of p-Smad1 and its nuclear accumulation in pulmonary smooth muscle cells.Pretreatment with PDGF-BB significantly decreased the nuclear accumulation of p-Smad1 induced by BMP-2 without decrease of total p-Smad1.However,pretreatment with Y-27632 or U0126 reversed the inhibitory effect of PDGF-BB on p-Smad1 nuclear accumulation.BMP-2 significantly inhibited the cell proliferation,but PDGF-BB blocked the effect of BMP-2 and significantly increased the cell proliferation.After pretreated with Y-27632 or U0126,the PDGF-BB-activated cell proliferation was suppressed.CONCLUSION： PDGF-BB-activated Rho kinase inhibits BMP-2-induced p-Smad1 nuclear translocation via MEK/ERK1/2,and increases pulmonary artery smooth muscle cell proliferation.