SirT1介导硫化氢对自发性高血压大鼠血管平滑肌细胞增殖的抑制效应

It was reported that endogenous hydrogen sulfide played antihypertensive effect via inhibiting the abnormal proliferation of vascular smooth muscle cell (VSMC). However, the mechanism by which hydrogen sulfide inhibits VSMC proliferation has not been clear. In the present study, the applicant plans to explore the mechanisms by which hydrogen sulfide inhibits the VSMC proliferation, targeting on the SirT1/FOXO1 pathway. Firstly, based on the spontaneously hypertensive rats and ET-1-induced rat VSMCs, we are to treat the rats with hydrogen sulfide donor or overexpress cystathionine gamma lyase in VSMC to study the effect of hydrogen sulfide on the VSMC SirT1/FOXO1 pathway and proliferation during the development of hypertension. Secondly, we plan to explore the role of SirT1/FOXO1 pathway in inhibiting VSMC proliferation by hydrogen sulfide via repressing the SirT1 activity or downregulating the SirT1 expression. Finnally, we design to demonstrate the sulfhydration modification by hydrogen sulfide on the thiol group of cysteine in SirT1 with combination of thiol modified drugs, specific probe for sulfhydration modification and site-direct mutation on cysteine in SirT1,and then disclose the molecular mechanism by which hydrogen sulfide acts on the SirT1. The abovementioned studies might be a help to demonstrate the mechanisms by which hydrogen sulfide inhibits the abnormal proliferation of VSMC and deepen the pathogenesis of hypertension.

既往研究表明内源性硫化氢可通过抑制血管平滑肌细胞（VSMC）增殖发挥抗高血压作用，但是硫化氢抑制VSMC增殖的机制尚未完全阐明。本课题将从SirT1/FOXO1通路切入，探索硫化氢对VSMC增殖的抑制机制。首先在自发性高血压大鼠及内皮素-1诱导VSMC模型上，通过补充硫化氢供体及过表达胱硫醚r裂解酶上调内源性硫化氢水平，研究硫化氢对高血压发生中VSMC SirT1/FOXO1通路及VSMC增殖的影响；进一步抑制SirT1活性或表达，研究SirT1/FOXO1通路在硫化氢抑制VSMC增殖中的作用；结合巯基修饰工具药、巯基化修饰特异性探针及半胱氨酸定点突变，研究硫化氢对VSMC SirT1半胱氨酸巯基的巯基化修饰作用，揭示硫化氢调节SirT1的分子机制。通过上述结果，以期阐明硫化氢抑制血管平滑肌细胞异常增殖的分子机制，进一步深化高血压的发病机制。

既往研究表明内源性硫化氢（H2S）可通过抑制血管平滑肌细胞（VSMC）增殖发挥抗高血压作用，但是硫化氢抑制VSMC增殖的机制尚未完全阐明。本研究探讨内皮素-1（ET-1）对大鼠VSMC内源性气体信号分子H2S的影响及补充H2S后抑制细胞增殖的机制，以转录因子FOXO1为切入点，揭示了H2S通过对蛋白的巯基化修饰抑制细胞增殖效应。实验将VSMC细胞分为正常对照组和实验组，在实验组中分别加入ET-1，分别应用H2S探针原位检测VSMC中H2S的水平，采用敏感硫电极法检测细胞上清中H2S的水平。此外，外源性补充H2S，将VSMC细胞分为以下4组：对照组、ET-1组（10-7mol/L）、H2S（100µmol/L）组、ET-1+H2S组。转染H2S生成酶CSE过表达的慢病毒，使H2S内源性生成增加，将A7r5细胞分为以下4组：空载体（EV）组、EV+ET-1（10-7mol/L）组、CSE组、CSE+ET-1（10-7mol/L）组。研究发现ET-1下调VSMC内源性H2S体系。H2S抑制ET-1刺激下VSMC的增殖。给予H2S供体后，相较于对照组，ET-1组细胞PCNA和P-FOXO1蛋白表达明显增高，转录因子FOXO1向核外转移，FOXO1与PCNA启动子的结合明显降低。与ET-1组细胞相比，ET-1+H2S组细胞巯基化的FOXO1、PCNA和P-FOXO1蛋白表达明显增高，转录因子FOXO1向核外转移减少，FOXO1与PCNA启动子的结合明显增强。转染过表达CSE慢病毒后，与EV组细胞相比，EV+ET-1组细胞PCNA和P-FOXO1蛋白表达明显增高，转录因子FOXO1向核外转移，FOXO1与PCNA启动子的结合明显降低。与EV+ET-1组细胞相比，CSE+ET-1组细胞巯基化的FOXO1、PCNA和P-FOXO1蛋白表达明显增高，转录因子FOXO1留在核内，FOXO1与PCNA启动子的结合明显增强。结果表明ET-1刺激可以导致VSMC增殖并且使其内源性H2S体系下调。H2S通过转录因子FOXO1途径抑制细胞增殖，即通过对FOXO1的巯基化修饰，进而抑制FOXO1的磷酸化，抑制其向核外转移，同时增强其与PCNA启动子的结合，最终抑制VSMC增殖。通过上述结果，揭示了硫化氢抑制VSMC异常增殖的分子机制，进一步深化了高血压的发病机制。