H2S对缝隙连接的硫巯基化修饰在血压调节和血管舒缩中的作用机制研究

Protein S-sulfhydration by Hydrogen sulfide (H2S) and vasomotor regulation through gap junction play the pivotal role in the prevention and control of hypertension. Studies have confirmed protective effects of H2S on cardiovascular via changing the expression of connexins and gap junctional communication. However, whether H2S regulates blood pressure and vasomotor responses by physiologically modifies cysteines in the connexins by S-sulfhydration, which have been elusive. In the present project, the expression level of connexins and gap junction electrophysiological properties will be investigated in the thoracic aorta of the normal control group(Wistar-Kyoto rat ) and spontaneously hypertensive rat combined with treatment of NaHS and CSE inhibitor DL-propargylglycine (PAG) or the CBS inhibitor aminooxyacetic acid (AOA) using molecular and electrophysiologic techniques as well as biochemical method; The regulation of H2S treatment and inhibiton of its production on the expression and phosphorylation of connexins, gap junctional communication as well as vasomotion will be studied under normal and treated conditions by Real Time-PCR, immunoblotting, whole-cell patch clamp and pressure myograph; S-sulfhydration in the connexins between the groups above and the sulfhydrated positions of connexins will be studied here by biotin switch (S-sulfhydration) assay and tandem mass spectrometry (LC-MS/MS). The studies above will help us to dissect the molecular mechanism of crosstalk between the H2S and gap junction in blood pressure regulation to salt stress, which will provide important and valuable information for revealing the H2S how to resist against hypertension.

硫化氢（H2S）介导的蛋白硫巯基化修饰及缝隙连接调控的血管舒缩对高血压防治具有重要作用，研究已表明H2S可通过改变缝隙连接蛋白的表达及缝隙连接通讯对心脏起到保护作用，但H2S是否通过硫巯基化修饰调控缝隙连接通讯而调节血压和血管舒缩，其分子机理尚不清楚。本项目拟以Wistar-Kyoto大鼠和自发性高血压大鼠的胸主动脉为研究对象，结合硫氢化钠和硫化氢合成抑制剂处理，采用多种分子生物学、生化手段及电生理学技术，从不同水平研究H2S处理及其合成抑制对缝隙连接蛋白表达、磷酸化、缝隙连接电生理特性及血管舒缩活动的调控；解析H2S对不同连接蛋白的硫巯基化修饰及修饰位点，并通过体外实验揭示H2S对缝隙连接通讯的调控，最终阐明H2S和缝隙连接通讯在血压调节中的互作分子机制，为更深层次揭示H2S在高血压发生及防治中的作用提供重要而有意义的信息。

硫化氢（H2S）是一种具有心血管保护和血压调节作用的重要信号分子。然而，H2S对心血管系统保护调节的确切分子机制尚不完全清楚。因此，深入探究其心血管保护作用的分子机制至关重要。此外，心血管疾病与心脏、血管中缝隙连接蛋白（Connexins, Cxs）的表达和功能异常密切相关，但H2S是否可通过调节心血管系统中Cxs的表达及其功能而抑制心血管疾病的发生发展尚不清楚。本项目从心血管病变的独立危险因素-高血压入手，探究了H2S供体和缝隙连接阻断剂通过调节自发性高血压动物模型心肌、血管细胞及外周血T淋巴细胞上的Cxs表达或功能而发挥心血管保护和血压调节作用的分子机制。主要研究成果如下：① H2S供体硫氢化钠（NaHS）可通过增加自发性高血压大鼠（Spontaneously Hypertensive Rat, SHR）体内H2S含量来抑制自发性高血压引起的心室肥厚、心功能及血管舒缩功能异常，并降低其动脉血压。缝隙连接阻断剂18β-甘草次酸（18β-glycyrrhetinic acid, 18β-GA）亦能通过抑制SHR大鼠体内的缝隙连接而发挥心血管保护和降压作用；② NaHS和18β-GA可通过逆转SHR大鼠心脏或血管重构相关标志蛋白的异常表达来抑制自发性高血压诱导的心室和动脉血管重构；③ 首次全面系统地显示了高血压可导致SHR大鼠左心室、弹性动脉和阻力血管中不同种类Cxs的表达分布及Cx43磷酸化水平出现明显异常变化。而NaHS和18β-GA则可逆转SHR大鼠体内上述高血压受累靶器官中全部或部分Cxs的表达异常；④ 高血压介导的炎症应答可明显增强SHR大鼠外周血T淋巴细胞中Cxs的表达及其功能，而NaHS和缝隙连接阻断剂Gap27则可分别通过下调SHR大鼠外周T血淋巴细胞中Cxs的表达和功能而抑制高血压介导的炎症应答。. 综上所述，本研究成果表明H2S干预或抑制缝隙连接可通过调节高血压受累靶器官和外周血T淋巴细胞中Cxs表达或功能异常来抑制心室、血管重构及炎症应答，最终对高血压造成的心血管结构和功能损伤起到治疗作用。本研究具有重要的学术价值，并蕴藏着潜在的应用前景，既可为高血压病防治提供新的靶点，同时也为基于治疗心血管疾病的H2S供体和缝隙连接阻断药物的设计研发奠定了重要的理论基础。