| Background and Objectives:The view of atherosclerosis is a chronic inflammatory disease has been widely recognized, while the view of vascular adventitial inflammation has played a certain role in each process of the pathogenesis and development in atherosclerosis also attracts more and more attention. The proliferation and migration of vascular smooth muscle cells (VSMCs), a major cellular component of the arterial wall, have been considered to be a important reasons of the artery stenosis and restenosis after injury. After being stimulated, extracellular signal is transferred into the nuclear through the signaling system, and induces a series of gene expression related with proliferation and migration of VSMCs. The existing researches suggest that the Janus Kinase2-Signal Transducer and Activator of Transcription3(JAK2-STAT3) signaling pathway is the one of the pathways from the membrane to the nucleus in the process of the proliferation of VSMCs.In this study, cytokines interleukin-1β (IL-1β) was medicated through the arterial adventitia to partial simulate the process of adventitia inflammation to induce the proliferation and migration of VSMCs, the correlation between it and JAK2-STAT3signaling pathway was observed, and the change degree of proliferation and migration in VSMCs through inhibitting the function of JAK2was also observed in order to clarify the role of JAK2-STAT3signaling pathway in vascular proliferative diseases. Whether IL-6would participate in this process and the role of it was cleared as well.Methods:1. Left common carotid arteries of24SD male rats, aged6-8weeks, were separated. In experimental group (n=18), vascular adventitia was wrapped with sepharose suspension containing IL-1Beta (2.5μg), while in control group (n=6), vascular adventitia was wrapped with sepharose suspension without interleukin.2,8,24,48h and1week,2weeks after surgery,3rats in experimental group and1in control group were euthanatized at each time point respectively. HE staining of the treated specimen was performed for morphology, and immunohistochemical staining was to mark and identify the VSMCs;2. Total RNA and protein were extracted respectively from the carotid arteries specimens at each time point of the rat model. The mRNA expressions of JAK2, STAT3were detected by RT-PCR, and the protein expressions of JAK2, STAT3, p-JAK2and p-STAT3were detected by Western blot. Immunohistochemistry staining was used to locate p-JAK2and p-STAT3;3. In another9SD male rats, both sides of common carotid artery were separated. After a slow release gel of JAK2inhibitor AG490was dropped on the left side, and an equivalent blank gel on the right side, the same amount of IL-1β was wrapped on both sides.3rats on the time point8,48h and1week after surgery were killed. HE staining, RT-PCR and Western blot were performed as before;4. Realtime PCR was performed with the total RNA from the carotid arteries specimens that had been preserved in this research for detecting the mRNA expressions of IL-6.Results:1. The proliferation and migration to the direction of luminal of vascular medial cells can be observed at2h point after the arterial adventitia was wrapped by IL-1β, this performance is most evident in the postoperative8-48h, and the migration cells were moved back at2week point. These changed cells were suggested to be VSMCs for marking a a-actin positive change in immunohistochemical staining;2. After the adventitia was wrapped by IL-1β, the mRNA of JAK2and STAT3showed different expressions at different time point, RT-PCR and gray scale analasis showed that there was a significant difference among that (P<0.05); Western blot and gray scale analasis showed that there was no significant difference in the protein expressions of total JAK2and STAT3(P>0.05); And there was a significant difference of phosphorylation in JAK2and STAT3among different time points (P<0.05). The result of immunohistochemistry staining prompted that p-JAK2and p-STAT3expressed in VSMCs;3. After the additional application of inhibitor, there was no significant difference in the protein expressions of total JAK2and STAT3yet (P>0.05); But the mRNA expression and phosphorylation level of JAK2and STAT3were obviously inhibited (P<0.05), and the vascular smooth muscles cells changed less;4.After the adventitia was wrapped by IL-1β, the mRNA of IL-6showed a different expression among different time points (P<0.05), the mRNA expression of IL-6in the postoperative8h showed the highest peak; After the additional application of inhibitor, there was no significant difference in the mRNA expression of total IL-6between the treated side and control side in8h team (P>0.05).Conclusion:1. Medication of cytokines IL-1β through the arterial adventitia can induce vascular smooth muscle cell proliferation and migration;2. The change of the mRNA expression of JAK2and STAT3and the phosphorylation in the corresponding protein can be seen at the same time with the proliferation and migration of VSMCs, thus it can be concluded that there is a direct correlation between them;3. AG490, the inhibitor of JAK2, can inhibit the function of JAK2-STAT3signaling pathway, and affect the level of proliferation and migration of common carotid artery in rat induced by medication of IL-1β through the arterial adventitia, it further confirmed that medication of cytokines IL-1β through the arterial adventitia can induce vascular smooth muscle cell proliferation and migration, such a change has a direct correlation with JAK2-STAT3signaling pathway;4. Early in the process of vascular smooth muscle cell proliferation and migration induced by medication of IL-1β through the arterial adventitia, the transcriptional activity of IL-6can be activated, it has no direct correlations with the activity of JAK2-STAT3signaling pathway. |
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