JAK_2-STAT_3信号通路在白介素-1β经动脉外膜给药致平滑肌细胞增殖迁移中的作用

Role of JAK_2 -STAT_3 signaling pathway in smooth muscle cell proliferation and migration induced by medication of IL-1β through arterial adventitia in mice

目的研究白细胞介素-1β经血管外膜给药导致动脉平滑肌细胞发生的改变及其与JAK2-STAT3信号通路的关系,明确JAK2-STAT3信号通路在血管增殖性病变中的作用。方法 6～8周龄SD雄性大鼠24只,分离左侧颈总动脉,实验组(n=18)血管外膜包裹含白介素-1β2.5μg的琼脂缓释悬液,对照组(n=6)包裹不含白介素的琼脂悬液,术后2、8、24、48 h,1、2周分别处死实验组大鼠3只,对照组1只,血管标本经切片HE染色观察形态,Western blot法检测JAK2、STAT3、磷酸化JAK2以及磷酸化STAT3的表达,免疫组化标记定位磷酸化JAK2及磷酸化STAT3;另取SD雄性大鼠9只,分离两侧颈总动脉,左侧滴加JAK2抑制剂AG490缓释凝胶,右侧滴加等量空白凝胶后两侧均包裹等量白介素-1β,术后8、48 h,1周处死动物分别行HE染色及Western blot检测。结果白介素-1β包裹血管外膜后出现血管平滑肌细胞的增殖、迁移,通道蛋白JAK2、STAT3在不同时间点出现不同程度的磷酸化,经Western blot检测并行灰度分析,p-JAK2相对灰度值对照组(0.337±0.216),8 h组(1.764±0.513),1周组(0.451±0.229);p-STAT3相对灰度值对照组(0.125±0.870),24 h组(1.909±0.309),2周组(0.448±0.516),各组间有明显差异(P<0.05)。加用抑制剂后,8 h组p-JAK2相对灰度值实验侧(0.085±0.031),对照侧(1.416±0.468),48 h组p-STAT3相对灰度值实验侧(0.460±0.065),对照侧(2.425±0.638),提示JAK2、STAT3的磷酸化水平被明显抑制(P<0.05),平滑肌细胞变化程度下降。结论炎性因子白细胞介素-1β经动脉外膜给药可致动脉平滑肌细胞增殖、迁移,这种改变与JAK2-STAT3信号通路有直接关系。

Objective To study the changes of arterial smooth muscle cells caused by medication of IL-1β through arterial adventitia and their correlation with JAK2-STAT3 signaling pathway,and to clarify the role of JAK2-STAT3 signaling pathway in vascular proliferative diseases.Methods Twenty-four SD male rats（6-8 weeks old） of which left common carotid arteries were separated were divided into an experimental group（n=18） and a control group（n=6）.In the experimental group,the rats＇ vascular adventitia was wrapped with Sepharose suspension containing interleukin-1β（IL-1β）（2.5 μg）,while in the control group（n=6）,the rat＇s vascular adventitia were wrapped with Sepharose suspension without IL.At each time point of 2,8,24,48 h,and 1,2 weeks after the operation,3 rats in the experimental group and 1 rat in the control group were sacrificed,respectively.In another 9 SD male rats,both sides of common carotid artery were separated.After a slow-release gel of JAK2 inhibitor AG490 was dropped on the left side of vascular adventitia and an equivalent blank gel on the right side of vascular adventitia,the same amount of IL-1β was wrapped on both sides.At each time point of 8 and 48 h and 1 week after operation,3 rats were sacrificed,respectively.HE staining was applied to observe the morphology of vascular specimens.The expressions of JAK2,STAT3,phospho-JAK2（p-JAK2） and phospho-STAT3（p-STAT3） were detected by Western blotting.Immunohistochemical staining was used to locate p-JAK2 and p-STAT3.Results The proliferation and migration of vascular smooth muscle cells were observed after the arterial adventitia was wrapped with IL-1β.Western blotting and gray scale analysis showed that relative gray scale values of p-JAK2 were 0.337±0.216 in the control group,1.764±0.513 in the 8 h group,and 0.451±0.229 in the 1 week group.The relative gray scale values of p-STAT3 were 0.125±0.870 in the control group,1.909±0.309 in the 24 h group,and 0.448±0.516 in the 2 weeks group.There were significant differences of JAK2 and STAT3 phosphorylation among different time points（P0.05）.After the inhibitor treatment,the relative gray scale values of p-JAK2 were 0.085±0.031 in the treated side,and 1.416±0.468 in the control side in the 8 h group.The relative gray scale values of p-STAT3 were 0.460±0.065 in the treated side,and 2.425±0.638 in the control side in the 48 h group.The phosphorylation of JAK2 and STAT3 were significantly inhibited（P0.05）,and the changes of smooth muscle cells decreased.Conclusion Medication of cytokine IL-1β through arterial adventitia can induce arterial smooth muscle cell proliferation and migration,which are directly associated with JAK2-STAT3 signaling pathway.