H2S/NO介导的线粒体靶蛋白S-硫巯基化/S-亚硝基化修饰对肾上腺皮质反应性的调节及其机制研究

Normal mitochondrial function was essential for maintaining glucocorticoid production in adrenal cortex. We found that both inhibition of endogenous H2S generation and NO donor treatment led to mitochondrial damage and adrenocortical insufficiency. These effects were significantly attenuated by the treatment of H2S donor. It has been reported that H2S and NO regulate protein function through covalently modifying protein cysteine residues through S-sulfhydration and S-nitrosylation, respectively. Using maleimide labeling technique and mass spectrometry, we obtained a number of potential target proteins which might be basally sulfhydrated by H2S in adrenal gland. Among these potential target proteins, F0F1-ATP synthase, VDAC1 and P450scc were well-known to play important roles in maintaining mitochondrial function and steroidogenesis. Meanwhile, NO was known to S-nitrosylate F0F1-ATP synthase and VDAC1 and inhibited their functions. On the basis of these results, the present project would firstly study the effects of H2S and NO on S-sulfhydration and S-nitrosylation of these three proteins, respectively. And then, the potential S-sulfhydration and S-nitrosylation sites of these proteins would be determined via maleimide labeling technique and mass spectrometry. By constructing mutation vector, whether these potential sites were modified by H2S and NO would be further confirmed. Finally, we would observe if S-sulfhydration and S-nitrosylation of these three proteins were connected to adrenal insufficicency during endotoxemia. In summary, this study would help clarify that S-sulfhydration and S-nitrosylation of mitochondrial protein paly critical roles in maintaining mitochondrial function and adrenocortical responsiveness in adrenal cortex. It could also provide new clinical therapeutic ideas for treatment of relative adrenal insufficiency.

肾上腺维持稳定的糖皮质激素分泌首先要保证线粒体功能正常。我们发现阻断内源性硫化氢（H2S）生成或给予一氧化氮（NO）均能抑制线粒体功能和肾上腺皮质反应性，而给予H2S则能逆转上述现象。H2S和NO都可修饰靶蛋白半胱氨酸上的巯基，分别形成S-硫巯基化和S-亚硝基化蛋白而调节其功能。我们采用质谱技术获得了可能被S-硫巯基化的靶蛋白信息，其中F0F1-ATP合酶、VDAC1和P450scc这三种蛋白已知在线粒体功能维持和激素合成中起关键作用。NO已被证实可通过S-亚硝基化修饰F0F1-ATP合酶和VDAC1、并抑制其功能。在此基础上，本项目首先将明确H2S和NO对三种蛋白半胱氨酸巯基的修饰作用，并阐明其作用位点。最后我们将观察三种蛋白的巯基修饰水平变化是否参与内毒素诱导的肾上腺损伤。本研究旨在阐明H2S和NO通过线粒体蛋白巯基修饰参与肾上腺皮质功能调节的机制，并为肾上腺功能不全的治疗提供新思路。

脓毒症会导致全身多个器官功能受损，而肾上腺功能不全是影响其预后的关键因素之一，然而其具体发生机制尚未阐明。本课题以我们已建立的内毒素血症小鼠为模型，首先采用外源性 ACTH刺激实验证实该模型确实存在肾上腺反应性低下、发生了肾上腺相对功能不全，同时硫化氢（H2S）缓释供体GYY4137能够显著改善上述现象。GYY4137同样能够逆转H2S合成酶CBS敲除小鼠肾上腺反应性低下的现象。进一步研究表明，内毒素血症小鼠和CBS敲除小鼠肾上腺中线粒体凋亡的水平显著升高，且Bax抑制肽和GYY4137均能够降低上述模型小鼠肾上腺中线粒体凋亡的水平。此外，近年来研究表明H2S可修饰靶蛋白半胱氨酸上的巯基，形成S-硫巯基化蛋白而调节其功能。我们进一步采用质谱技术获得了可能被S-硫巯基化的靶蛋白信息，其中ATP5A1、VDAC1和P450scc这三种蛋白已知在线粒体功能维持和糖皮质激素合成中起关键作用。因此我们进一步确认H2S是否是通过硫巯基化上述三种蛋白进而影响肾上腺中线粒体功能及肾上腺皮质合成糖皮质激素的功能。结果表明，在肾上腺皮质细胞中，ATP5A1确实发生硫巯基化修饰，而VDAC1和P450scc并未检测到硫巯基化修饰。随后研究发现内毒素血症小鼠肾上腺皮质细胞中ATP5A1的硫巯基化显著降低，外源性硫化氢显著改善此现象。ATP5A1硫巯基化位点分析结果显示，ATP5A1第244位半胱氨酸位点突变后，ATP5A1的硫巯基化修饰消失，且ATP5A1-C244突变体能够消除H2S对内毒素血症小鼠肾上腺皮质细胞中线粒体凋亡的抑制作用，同时也减弱H2S对内毒素血症小鼠肾上腺反应性低下的改善作用。上述结果提示内毒素降低ATP5A1的硫巯基化水平是内毒素血症小鼠肾上腺皮质细胞中线粒体凋亡增加、进而发生反应性低下的重要原因之一。肺功能损伤同样是影响脓毒症预后的关键因素，我们结果也显示H2S同样可以通过抑制氧化应激和硝化应激的水平改善内毒素血症小鼠肺的功能，此结果也为硫化氢治疗脓毒症提供了理论依据。