EPO通过JAK2/STAT5信号通路减轻大鼠血管平滑肌细胞钙化

Erythropoietin inhibits rat vascular smooth muscle cell calcification via JAK2/STAT5 signaling pathyway

目的研究促红细胞生成素(EPO)减轻血管钙化的信号通路。方法为了分别探索JAK2/STAT5、PI3K/Akt和ERK三条信号通路在EPO减轻血管钙化中的作用,将大鼠血管平滑肌细胞(VSMCs)各分为4组:对照组、钙化组、钙化+EPO组和钙化+EPO+各信号通路阻断剂组(AG490或LY294002或PD98059)。采用高磷诱导细胞钙化。Western blot法检测VSMCs收缩表型标志分子平滑肌蛋白(Smoothelin)和钙结合蛋白(Calponin)以及成骨表型标志分子骨桥蛋白(OPN)的蛋白表达;碱性磷酸酶(ALP)活性、钙含量检测和茜素红染色检测钙化水平。结果与对照组相比,钙化组Smoothelin和Calponin蛋白表达水平降低(P<0.01),而OPN蛋白表达水平上升(P<0.05)。与钙化组相比,钙化+EPO组Smoothelin和Calponin蛋白水平显著升高(均P<0.01),OPN蛋白表达降低(P<0.05);ALP活性、钙含量和钙盐沉积均减少(均P<0.01)。与钙化+EPO组相比,钙化+EPO+AG490组Smoothelin和Calponin蛋白水平降低(P<0.05),而OPN蛋白水平显著上升(P<0.01);ALP活性、钙含量和钙盐沉积均增加(P<0.01或0.05)。与钙化+EPO组相比,钙化+EPO+LY294002组和钙化+EPO+PD98059组的钙含量和ALP活性均无明显变化。结论JAK2/STAT5通路可能介导了EPO减轻VSMCs钙化的作用。

Objective To study the mechanism of erythropoietin(EPO)inhibiting the calcification of rat vascular smooth muscle cells(VSMCs).Methods In order to explore the role of JAK2/STAT5,PI3K/Akt or ERK signaling pathway in EPO reducing vascular calcification,rat vascular smooth muscle cells were divided into four groups:control group,calcification group,calcification+EPO group,and calcification+EPO+signaling pathway inhibitor(AG490 or LY294002 or PD98059)group.High phosphorus was used to induce the calcification of VSMCs.The protein levels of Smoothelin,Calponin and osteopontin(OPN)were detected by Western blot.Alkaline phosphatase(ALP)activity,calcium content and alizarin red S staining were used to detect the calcification.Results Compared with control group,the protein levels of Smoothelin and Calponin were decreased in calcification group(all P<0.01),while the protein level of OPN was increased(P<0.05).Compared with calcification group,the protein levels of Smoothelin and Calponin in calcification+EPO group were significantly increased(all P<0.01),and OPN protein level was decreased(P<0.05);ALP activity,calcium content and calcium salt deposition were also decreased(all P<0.01).Compared with calcification+EPO group,the protein levels of Smoothelin and Calponin in calcification+EPO+AG490 group were decreased(all P<0.05),while the protein level of OPN was increased significantly(P<0.01);ALP activity,calcium content and calcium salt deposition were increased(P<0.01 or 0.05).Compared with calcification+EPO group,there were no significant changes in calcium content and ALP activity in calcification+EPO+LY294002 group or calcification+EPO+PD98059 group.Conclusion JAK2/STAT5 pathway might mediate the effect of EPO reducing VSMCs calcification.