低氧激活ERK／JNK通路促进Smad2／3蛋白磷酸化诱导小鼠心脏成纤维细胞表型转换的实验研究

ERK/JNK pathway promotes Smad2/3 protein phosphorylation induced phenotype conversion of cardiac fibroblasts during hypoxia in mice

目的研究体外低氧诱导小鼠心脏成纤维细胞（cardiac fibroblasts，CFs）向心脏肌成纤维细胞（cardiac myofibroblasts，CMFs）表型转换过程中，促分裂素原活化蛋白激酶（mitogen-activated protein kinases，MAPK）信号通路与Smad2／3蛋白磷酸化的关系。方法新生小鼠第1代CFs低氧（37℃、3％O2）无血清培养48h，免疫荧光检测α-SMA蛋白；新生小鼠CFs给予3种MAPK（ERK、JNK和p38）特异性抑制剂常氧培养1h，进行低氧培养30min，免疫印迹检测ERK、JNK、p38和Smad2／3蛋白磷酸化水平。结果①低氧显著上调新生小鼠CFs中α-SMA蛋白的表达；②低氧能显著地促进ERK、JNK和P38蛋白磷酸化，PD98059（ERK特异性抑制剂）、SP600125（JNK特异性抑制剂）和SB203580（P38特异性抑制剂）能抑制低氧诱导的ERK、JNK和P38蛋白的磷酸化；③PD98059（ERK特异性抑制剂）和SP600125（JNK特异性抑制剂）能抑制低氧诱导的Smad2／3蛋白的磷酸化，SB203580（P38特异性抑制剂）无此作用。结论①低氧可诱导小鼠CFs细胞发生表型转化为CMFs；②ERK和JNK信号通路能调控Smad2／3蛋白的磷酸化表达；③MAPK和Smad信号通路可能参与低氧诱导的小鼠CFs细胞的表型转化。

Objective To investigate whether hypoxia promotes differentiation of cardiac fibroblasts （CFs） to myofibroblasts in vitro and the relationship between MAPK signaling pathway proteins and the phosphorylation of Smad2/3. Methods CFs isolated from neonatal mice were in vitro cultured under normoxic and serum - free conditions. The expression of α - SMA protein was detected by immunofuorescent staining. In the absence or presence of three MAPK （ ERK, JNK and p38） - specific inhibitors, CFs were cultured under normoxic, then extracellular signal - regulated kinase （ERK） , c -Jun NH2 -terminal kinase （JNK） , p38 MAP kinase and Smad2/3 protein expression was measured by immunofuorescent staining. Results （1） Hypoxia markedly upregulated α -SMA protein expression in neonatal mice CFs. （2） Hypoxia increased the phosphorylation levels of ERK, JNK, p38 MAP kinases. The inhibitors PD98059, SP600125 and SB203580 attenuated hypoxiainduced phosphorylation levels of ERK, JNK and p38 MAP kinases, respectively. （3） PD98059 and SP600125 inhibited the up - regulation of the phosphorylation of Smad2/3 protein level in response to hypoxia rather than SB203580. Conclusion （1） Hypoxia itself is able to induce differentiation of neonatal mice CFs into myofibroblasts. （2） The ERK and JNK pathways mediate phosphorylation of Smad2/3 protein. （3） MAPK and Smad signaling pathway may be involved in the phenotypic transformation of hypoxia induced CFs.