雌激素促进血管内皮硫化氢释放的非基因效应机制及其功能研究

Estrogen has been proven to reverse vascular endothelial dysfunction and reduce the incidence of atherosclerosis in postmenopausal women, while the detailed mechanisms remain obscure. We have discovered a novel mechanism for estrogen's endothelial actions, that is, 17β-estradiol (E2) activates protein kinase G (PKG) and rapidly promotes the release of hydrogen sulfide (H2S) from vascular endothelial cells, resulting in the improvement of endothelial function. However, what is the exact nongenomic mechanism responsible for the above mentioned action? Does this nongenomic pathway provide the long-term anti-atherogenic effect? Further efforts are needed to answer these questions. The preliminary results of this project showed that E2 elevated cGMP level, which was blocked by Gi signaling inhibitor pertussis toxin. Based on these results, the present project aims to explore the following issues. Firstly, we plan to investigate the modulatory effect of membrane estrogen receptor α (ERα) on particulate guanylyl cyclase A activity and the subsequent activation of cGMP/PKG/H2S. Secondly, the critical role of Giα protein in this process will be confirmed. Finally, we will use animal model to observe the alteration of estrogen's anti-atherogenesis effect when this nongenomic action is activated or blocked. The present project aims to explore the nongenomic mechanism and function of E2-stimulated H2S release and it will shed light on the novel strategy of estrogen replacement therapy by the selective interference of nongenomic effects.

雌激素可逆转绝经后女性血管内皮功能失调，降低动脉粥样硬化（AS）发生率，但其机制尚未阐明。在国家自然青年项目资助下，我们首次报道：17β-雌二醇（E2）可激活蛋白激酶G（PKG），快速促进血管内皮细胞释放气体信号分子硫化氢（H2S），改善内皮功能。E2通过何种非基因机制激活PKG？此非基因效应能否产生长期的抗AS作用？上述问题均需进一步研究。预实验结果表明，E2可增加PKG上游cGMP的水平，该作用被Gi蛋白抑制剂所阻断。由此，本项目拟：1、探讨膜雌激素受体α（mERα）调控鸟苷酸环化酶A进而激活cGMP/PKG/H2S的效应。2、研究Giα蛋白在其中的关键作用。3、在动物模型中激活或阻断此非基因效应，明确其抗AS作用。本项目在原创性工作的基础之上，深入揭示雌激素调控H2S这一非基因效应的内皮机制，证实其长期功能，并将提示，对非基因效应进行选择性干预，可能成为激素替代疗法防治AS的新策略。

雌激素具有心血管保护效应，但其分子机制尚未阐明。我们的前期研究发现：17β-雌二醇（E2）可通过基因途径，上调血管内皮细胞胱硫醚γ裂解酶（CSE）蛋白表达，促进气体信号分子硫化氢（H2S），从而改善内皮功能。新近的研究表明，E2的非基因途径亦在其抗动脉粥样硬化作用中发挥重要影响。E2是否可通过非基因途径快速促进H2S释放？其具体分子机制如何？目前尚未有相关研究。本项目研究结果发现：在培养的脐静脉血管内皮细胞中，雌激素可时间依赖性及浓度依赖性快速促进H2S释放，此效应不被转录抑制剂（Act D）或蛋白合成抑制剂（CHX）所阻断，且不能通透细胞膜的大分子E2-BSA也能时间依赖性及浓度依赖性上调H2S水平。进一步结果表明，E2时间依赖性及浓度依赖性上调CSE蛋白丝氨酸/苏氨酸磷酸化的水平，这一作用被蛋白激酶G抑制剂所阻断。通过过表达和siRNA干扰技术，发现PKGⅠβ是参与E2促H2S快速释放的主要亚型。免疫共沉淀技术表明：E2可通过增加ERα与Gαi2/3及pGC A的交联，促进内皮细胞cGMP水平，从而激活PKG，导致CSE磷酸化增加。动物实验表明：CSE与pGC A均介导E2促血管舒张这一作用。本研究主要从非基因效应的分子机制出发，发现了E2调控H2S快速释放的分子机制，并为ERT合理用于延缓绝经后妇女心血管疾病提供新的理论基础。