延髓头端腹外侧垂体中叶素在自发性高血压大鼠交感中枢调控中的作用与机制研究

Sympathetic activation plays a vital role in the pathogenesis and development of hypertension. Inhibiting the sympathetic activation has become an important strategy in preventing and treating hypertension. It has been reported that bilateral microinjection of intermedin (IMD) into the paraventricular nucleus decreases the renal sympathetic nerve activity and arterial blood pressure in hypertension rats. So far, the regulative mechanism of IMD in the rostral ventrolateral medulla (RVLM) has not been reported. Preliminary studies in our lab have shown that the IMD protein in RVLM was decreased in spontaneously hypertensive rats（SHR）compared with WKY rats. Microinjection of IMD produced decreases in renal sympathetic nerve activity, mean arterial pressure, and heart rate in SHR. No changes in behavior were observed in WKY rats. Moreover, nitric oxide (NO) level was increased in the RVLM. Based on our previous study, we would adopt SHR animal model of rats as research object, combining with in vivo and in vitro experiment, to investigate the molecular and neural regulative mechanism of IMD on the regulation of cardiovascular function in the RVLM. In order to explore the neural signaling network involved in the regulative effects of IMD on cardiovascular function in the RVLM, we consider NO as research access and adopt multiple electrophysiological, molecular experimental research methods such as gene overexpression, si-RNA, brain area microinjection, RT-PCR, western blot, extracellular nerve discharges recording, immunohistochemistry. We focused on the mechanism of IMD and its receptors in the RVLM in the sympathetic activation and the key targets for treating the hypertension in SHR, especially investigating the anti-hypertensive effects of the intervention of these targets. The significance of this project is to clarify the neural and molecular mechanisms of the IMD in SHR. It is hopeful to provide a novel approach and an experimental foundation for treating hypertension.

交感神经过度激活在原发性高血压发生发展中起重要作用，抑制交感神经过度激活是防治原发性高血压的重要策略。研究表明在高血压大鼠室旁核注射垂体中叶素(IMD)可以降低过度激活的交感神经和血压。但IMD在延髓头端腹外侧（RVLM）的调控机制未见报道。我们发现：自发性高血压大鼠（SHR）RVLM区IMD表达减少；RVLM区注射IMD降低了SHR过度激活的交感神经活动和血压，并且上调了一氧化氮水平。基于这一重要发现，本项目应用基因过表达、RNA干涉、在体记录交感神经放电、蛋白免疫印记等电生理及分子生物学手段，从不同角度探讨IMD系统在SHR交感神经激活中的机制，特别是探讨RVLM区IMD系统与一氧化氮/一氧化氮合酶系统之间的相互效应，找出调控交感神经活动的关键靶点，研究干预这些关键靶点对原发性高血压的治疗作用。该研究有望阐明IMD系统在原发性高血压发病程中的作用与机制，为防治原发性高血压提供新思路。

IMD属于降钙素/降钙素基因相关肽（CGRP）家族成员，与该家族成员共享由降钙素受体样受体（CRLR）和受体活性修饰蛋白（RAMPs）组成的受体系统。IMD在外周系统和中枢系统均有表达。外周系统，IMD具有扩张血管，改善心脏功能，降低血压的作用；IMD在中枢对心血管也有调控作用。在正常大鼠侧脑室和孤束核（NTS）显微注射IMD可引起交感兴奋和血压升高，在室旁核（PVN）注射IMD可降低两肾一夹大鼠及心衰大鼠过度激活的交感神经活性。在本课题中我们发现在RVLM区下调IMD的表达或使用IMD受体拮抗剂，可使高血压大鼠的血压下降和交感神经活动减弱。本项目为临床高血压的治疗提供了理论依据。具体研究内容有以下几个方面：1.SHR RVLM区的IMD基因及蛋白的表达特点。 观察SHR的RVLM区的IMD蛋白及其基因的表达是否发生了变化，从蛋白水平和基因水平证明IMD为参与SHR交感神经过度激活的分子机制的物质基础。2. 增加和抑制IMD功能对SHR过度激活的交感神经活动的影响 观察外源性的IMD及其受体拮抗剂分别对SHR过度激活的交感神经活动的影响。3.增加和抑制IMD表达对SHR过度的交感神经兴奋的影响。腺病毒作为载体，利用RNA干扰技术，将RVLM区的IMD表达下调，观察到SHR过度兴奋的交感神经活动减弱，证明了IMD在SHR中调节交感神经和血压中发挥作用。4.RVLM区IMD与NO/NOS信号通路在SHR交感神经激活中的相互效应。观察IMD是否对SHR RVLM区NO/NOS系统产生调节作用。RVLM区NO的生成量、NOS酶及其基因表达和GABA水平与IMD含量的改变相关联，从蛋白水平和基因水平证明NO/NOS系统参与了IMD对SHR过度兴奋的交感神经活性和血压调控的机制。