SIRT3在硫化氢抗血管内皮氧化应激损伤中的作用机制研究

Vascular endothelial dyfunctions initialize various cardiovascular diseases, with oxidative stress reported as one of its main mechanisms. Hydrogen sulfide (H2S) exerts significant anti-oxidative characteristic with different regulative mechenisms in different tissues and cells. SIRT3(sirtuin3) is a NAD-dependent deacetylase, which regulates deacetylation of related proteins to enhance anti-oxidative capacity. Our prior experiments indicated that H2S could significantly increase SIRT3 promoter activity and SIRT3 expression in vascular endothelial cells suffering oxidative stress induced by H2O2, which is possibly related to AP-1 binding sites in the promoter of SIRT3. It suggested that SIRT3, serving as an important molecular target, may play a critical role in protection against oxidative damage in vascular endothelium. Based on oxidative stress models in vascular endothelial cells and animals, hypothesis has been raised as follows: H2S directly impacts on SIRT3 through NAD, or regulates SIRT3 expression at transcriptional level, so as to further adjust relative downstream anti-oxidative proteins to exert anti-oxidation effect. The result of the project will provide evidence that H2S protects against oxidative stress in impaired vascular endothelial cells through SIRT3, and new ideas for prevention and treatment of diseases associated with endothelial dysfunction.

血管内皮功能障碍是多种心血管疾病发生发展的始动环节，氧化应激是重要机制之一。硫化氢（H2S）对多种组织细胞具有抗氧化应激作用，但调节机制不同。SIRT3（sirtuin3） 是一种NAD依赖的去乙酰化酶，可通过去乙酰化相关蛋白质提高机体抗氧化能力。我们的预实验提示：H2S可以明显逆转H2O2诱导血管内皮细胞氧化应激过程中SIRT3表达的下降，提高SIRT3启动子活性（可能与该启动子区域上AP-1结合位点有关），提示SIRT3可能作为一个重要的分子靶点，在H2S抗血管内皮氧化应激损伤中发挥关键作用。因此，本项目拟利用整体动物和血管内皮细胞氧化应激损伤模型，验证科学假说：H2S可通过NAD直接影响SIRT3，或在转录水平调控SIRT3的表达，进而调节SIRT3下游抗氧化靶蛋白，揭示H2S通过SIRT3保护血管内皮氧化应激损伤的新机制，为血管内皮功能障碍相关疾病防治提供新思路。

血管内皮功能障碍是多种心血管疾病发生发展的始动环节，氧化应激是重要机制之一。硫化氢（H2S）对多种组织细胞具有抗氧化应激作用，但调节机制不同。本研究发现：硫化氢供体GYY4137增加H2O2诱导下EA.hy926内皮细胞存活率，降低细胞培养液中的乳酸脱氢酶水平，抑制内皮细胞凋亡和氧化应激，改善线粒体呼吸功能障碍，增加线粒体膜电位，提高超氧化物歧化酶2（SOD2）和异柠檬酸脱氢酶2（IDH2）表达，抑制促分裂原活化蛋白激酶（MAPK）磷酸化，表明GYY4137对内皮细胞氧化应激损伤具有保护作用。进一步研究发现H2S明显逆转H2O2诱导下内皮细胞去乙酰化酶3（SIRT3）表达的下降；RNA干扰下调SIRT3表达后，GYY4137不能抑制H2O2诱导下内皮细胞细胞凋亡、氧化应激和MAPK磷酸化，不能增加SOD2和IDH2蛋白表达。荧光素酶报告基因检测发现GYY4137增强H2O2诱导下内皮细胞SIRT3启动子活性；SIRT3启动子截短至-491 bp和-242bp时GYY4137均能（但截短至-161bp后不能）增强 H2O2诱导下内皮细胞SIRT3启动子活性，提示-242bp 到-161bp 区域存在的活化蛋白-1（AP-1）结合位点在影响 GYY4137上调SIRT3 转录中发挥重要作用。染色质免疫共沉淀证实GYY4137增强H2O2诱导下AP-1与SIRT3启动子的亲和力；加入AP-1抑制剂后，GYY4137不能增强H2O2诱导下内皮细胞SIRT3启动子活性。整体研究发现：GYY4137明显改善野生型小鼠百草枯诱导后胸主动脉和肠系膜动脉内皮依赖性舒张功能，抑制胸主动脉氧化应激；但GYY4137对SIRT3基因敲除小鼠中上述指标无明显调节作用。研究证实H2S依赖SIRT3抗血管内皮氧化损伤，揭示了H2S抗血管内皮氧化损伤的新机制，为血管内皮功能障碍相关疾病防治提供新思路。