尼可地尔对肺动脉平滑肌细胞增殖、迁移能力和Hippo/YAP信号通路的影响

Effects of Nicorandil on the Proliferation,Migration Ability and Hippo/YAP Signaling Pathway of Pulmonary Artery Smooth Muscle Cells

目的:体外评价尼可地尔对肺动脉平滑肌细胞(PASMCs)增殖、迁移能力和Hippo/YAP信号通路的影响。方法:将人原代PASMCs分为正常对照组、模型组和尼可地尔低、中、高浓度组(50、100、200μmol/L),每组设3个复孔。除正常对照组外,其余各组细胞均接种在凝胶包被的培养板上,模拟肺动脉高压环境,以建立肺动脉硬化(AS)细胞模型;随后,各药物组加入相应药物,正常对照组和模型组加入同等体积生理盐水,培养48 h。采用CCK-8法检测各组细胞的增殖能力(以光密度计),Transwell方法检测细胞迁移能力,实时定量聚合酶链式反应技术检测细胞中YAP靶基因子[结缔组织生长因子(CTGF)、双向调节蛋白(AREG)]mRNA的表达水平,Western blotting法检测细胞中CTGF、AREG蛋白的表达水平。结果:与正常对照组比较,模型组细胞的光密度显著升高,每视野迁移细胞数显著增加,细胞中CTGF、AREG m RNA及蛋白的表达水平均显著增强(P<0.01)。与模型组比较,尼可地尔低、中、高浓度组细胞的光密度、每视野迁移细胞数和细胞中CTGF、AREG mRNA及蛋白的表达水平均显著降低,且成浓度依赖性(P<0.05或P<0.01)。结论:尼可地尔可抑制AS模型PASMCs的增殖和迁移,其作用机制可能与抑制Hippo/YAP信号通路有关。

OBJECTIVE:To evaluate the effects of nicorandil on the proliferation,migration ability and Hippo/YAP signaling pathway of pulmonary artery smooth muscle cells(PASMCs).METHODS:Human primary PASMCs were divided into normal control group,model group,nicorandil low,medium and high concentration groups(50,100,200μmol/L),with 3 holes in each group.In addition to the normal control group,the rest of the cells were inoculated on the gel coated medium to simulate the pulmonary hypertension environment,so as to establish AS cell model.Then,each drug group was added with corresponding drugs,and the normal control group and model group were added with the same volume of normal saline,and cultured for 48 h.CCK-8 assay and Transwell assay were used for the examination of cell proliferation(by light density)and migration ability,respectively.mRNA expression of YAP target factors(CTGF and AREG)were examined by qRT-PCR.Western blotting assay was used to detect the protein expression of CTGF and AREG.RESULTS:Compared with normal control group,light density of cells was increased significantly in model group;the number of migration cells per field of view increased significantly;mRNA and protein expression of CTGF and AREG were significantly increased(P<0.01).Compared with model group,light density,the number of migration cells per field of view,m RNA and protein expression of CTGF and AREG in nicorandil low,medium and high concentration groups were decreased significantly,in concentration-dependent manner(P<0.05 or P<0.01).CONCLUSIONS:Nicorandil can inhibit the proliferation and migration of PASMCs in AS model,the mechanism of which may be associated with the Hippo/YAP signaling pathway.