尼古丁诱导的腹主动脉瘤nAChRs-JNK-AP-1轴调控机制研究

In our recent study, it has been found that nicotine induced the formation of abdominal aortic aneurysms via the increased expression of matrix metalloproteinases and the upregulation of JNK phosphorylation in aortic tissue in the older C57BL/6J wild-type mice. Our another study showed that nAChRs might be the signal upstream of JNK, because of the participation of JNK in the VCAM-1 expression induced by nicotine through the activation of nAChRs in mouse macrophages. The above evidences show that the nAChRs-JNK signal axis may regulate the formation of abdominal aortic aneurysms. However, the signal transduction mechanism associated with the abdominal aortic aneurysms induced by nicotine remains to be investigated to define the signals upstream and downstream of JNK. As the specific receptor of nicotine, nAChRs was expressed on the resident cells in aortic tissue, such as endothelial cells, vascular smooth muscle cells and macrophages, although it have yet to be investigated whether the phosphorylating activation of JNK was regulated by nAChRs in other cells. Additionally, previous researches have been shown that AP-1, the classical signal downstream of JNK, has a increased expression in the tissues from the abdominal aortic aneurysms. Based on the above evidences, we suppose that the nAChRs-JNK-AP-1 signal axis regulates the formation of abdominal aortic aneurysms induced by nicotine. To verify the above hypothesis, our study will observe the effects of nAChRs and JNK on abdominal aortic aneurysms induced by nicotine via utilizing the gene knockout techniques in animal experiments, and observe the effects of nAChRs and JNK on the expression of matrix metalloproteinases, inflammatory and chemotactic factors induced by nicotine in vascular smooth muscle cells or macrophages, and observe the effect on the migration of macrophages to vascular smooth muscle cells. Finally, our study will clarify the molecular mechanism underlying abdominal aortic aneurysms induced by nicotine and help developing therapeutic drugs and orienting the gene targets.

课题组前期研究发现：尼古丁通过p-JNK上调诱导小鼠腹主动脉瘤形成；在巨噬细胞中，JNK参与了尼古丁激活nAChRs诱导的VCAM-1表达；提示nAChRs-JNK可能调控腹主动脉瘤形成。但尼古丁诱导腹主动脉瘤的信号转导机制仍有待深入探讨。作为尼古丁的特异性受体，nAChRs在血管细胞膜表达，但是否调控JNK磷酸化未见报道。AP-1是JNK的经典下游信号，研究显示其在腹主动脉瘤组织表达增加。因此假设：nAChRs-JNK-AP-1轴调控尼古丁诱导的腹主动脉瘤形成。为验证该假设，本项目通过基因敲除技术、RNAi等技术，体内和体外观察nAChRs-JNK-AP-1对尼古丁诱导的腹主动脉瘤影响和尼古丁诱导血管平滑肌细胞或巨噬细胞基质金属蛋白酶、炎症趋化因子等表达，以及平滑肌细胞对巨噬细胞趋化活性的影响。本项目将阐明尼古丁诱导AAA的分子机制，为临床药物和靶向治疗提供支持。

本研究按预设课题方案实施，验证科学假设得出结论：1.尼古丁能够通过nAChRs和JNK诱导腹主动脉瘤的形成。2. 尼古丁联合血管紧张素II可以通过调控基质金属蛋白酶表达和活性诱导腹主动脉瘤形成。3.尼古丁通过JNK通路诱导血管平滑肌细胞MCP-1和RANTES的表达，并可进一步调控巨噬细胞产生趋化活性，此过程可能在尼古丁诱导的主动脉瘤形成过程发挥关键作用。4.用α7-nAChR激动剂在巨噬细胞（RAW264.7细胞）中通过调节ERK1/2/AP-1信号传导来抑制尼古丁诱导的炎症因子和MMP上调，在平滑肌细胞（MOVAS细胞）中通过调节AP-1信号传导来抑制尼古丁诱导的炎症因子和MMP上调。因此证明AP-1确实在尼古丁诱发MMP上调，炎性因子趋化，腹主动脉瘤形成中起关键作用。前三结论为预期结果，而结论四为超出预期的发现。也为我们进一步研究尼古丁可能通过α1-nAChR起到增加炎性反应，进一步引起血管生物学改变提供假设基础。