H2S介导Sp1硫巯基化修饰在急性肺损伤EPCs动员及再内皮化中的作用及机制研究

Acute lung injury (ALI) is important contributors to morbidity and mortality in the ICU, the capillary endothelial cells are notably damaged in ALI, leading to increased vascular permeability, pulmonary edema, hypoxia, and increased inflammation. In recent years, the focus of investigation has turned toward understanding the endogenous processes that promote resolution of lung inflammation and initiate repair. Impaired mobilization of endothelial progenitor cells (EPCs) was reported to be a main contributor to high mortality for ALI patients. Transplantation of autologous EPCs could improve ALI. Increased numbers of EPCs in ALI mice treated with NaHS (a hydrogen sulfide, H2S donor), however decreased numbers of EPCs in ALI mice treated with PAG (an inhibitor of endogenous H2S production) was found in our preliminary experiment. Moreover, NaHS treatment alleviated lung injury following severe limb trauma. and NaHS was also found to enhance vascular endothelial growth factor A(VEGF-A) expression which play a central role in mobilization and re-endothelialization of EPCs. Moreover specificity protein 1(Sp1) is vital to regulation of VEGF-A expression. H2S plays multiply biological roles via S-sulfhydration of signal molecules. Therefore we conclude that: H2S enhances VEGF-A expression via S-sulfhydration of Sp1, which plays important roles in mobilization and re-endothelialization of EPCs in ALI. Mass spectrometry, flow cytometry, electrophoretic mobility shift assay, S-sulfhydration assay, oxidative cysteine assay, point mutation and cell transfection, western blotting will be employed in this study. The aim of this study is to clarify the roles of S-sulfhydration of Sp1 in regulation of VEGF-A expression by H2S, which play a key role in enhancing mobilization of EPCs and that reduced endogenous H2S production is the main cause of impaired mobilization and re-endothelialization of EPCs in ALI. Maintaining H2S balance may be a new strategy for ALI treatment in clinic.

内皮祖细胞(EPCs)动员不足是ALI预后不良的重要原因，增加内源性EPCs动员是肺损伤内皮细胞靶向治疗的新策略。新近报道及前期研究发现H2S能增加内源性EPCs动员，其机制与增加VEGF-A表达有关。目前已明确H2S能引起包括Sp1在内的众多信号分子发生硫巯基化修饰，而Sp1是调控VEGF-A表达的重要转录因子。由此我们提出："H2S通过硫巯基化修饰Sp1,增强Sp1转录活性及VEGF-A表达"是ALI时H2S增加EPCs动员及再内皮化的关键环节；本课题拟采用定点突变、RNAi、质谱分析、硫巯基化蛋白检测、氧化蛋白检测、western blotting、EMSA、流式细胞等技术，深入研究Sp1硫巯基化修饰在H2S调控VEGF-A表达及其在ALI时EPCs动员及再内皮化中的作用，阐明内源性H2S产生减少引起Sp1氧化修饰增加是ALI时EPCs动员不足的重要原因。为ALI治疗提供新靶点。

急性肺损伤(acute lung injury, ALI)是失控性炎症反应在肺部的表现，虽然随着广谱抗生素的应用和机械通气模式的改进，ALI治愈率有所改善，但死亡率仍高达40%。因此寻找有效的ALI治疗措施是目前危重病医学亟待解决的问题。肺微血管内皮细胞(pulmonary micro-vascular endothelial cells, PMVECs)弥漫性损伤是ALI的病理基础，针对PMVECs靶向治疗是改善ALI预后的关键治疗措施。内皮祖细胞(endothelial progenitor cells，EPCs)是修复损伤血管内皮最主要的干细胞，在ALI发生、发展中具有重要作用；然而ALI时存在EPCs动员及再内皮化不足的现象。增加内源性 EPCs 动员是肺损伤内皮细胞靶向治疗的新策略。因此本课题采用定点突变、RNAi、质谱分析、western blotting、EMSA、流式细胞等技术，深入研究ALI时EPCs动员不足的原因及机制。本项目研究发现：① 内源性硫化氢代谢改变在脓毒症肺损伤发生发展中具有重要的调节作用，给予外源性H2S可通过：增加VEGF的表达、增加EPCs动员及在内皮化、促进损伤肺血管的修复；抑制iNOS表达，促进巨噬细胞MΦ向M2型转换，促进损伤肺组织修复、减轻脓毒症肺损伤。给予外源性硫化氢产生抑制剂，可显著恶化LPS诱导的肺炎症细胞浸润、抑制肺微血管增生修复。②肺微血管细胞和上皮细胞骨架重排在肺血气屏障通透性增高中的作用及调控机制：在给予LPS后，肺微血管内皮和上皮细胞E-cadherin、VE-cadherin和occludin表达降低，细胞骨架改变与炎症反应相互促进，加重微血管内皮细胞通透性增加，通过调控Actomyosin可影响LPS肺损伤的病程。③Nrf2出核转运增加导致内源性抗损伤机制受抑是LPS肺损伤发生发展的重要因素。在不同水平阻断Nrf2出核转运可显著减轻LPS肺损伤。本项目阐明内源性H2S产生减少引起VEGF-A表达显著降低，进而引起EPCs动员-再内皮化抑制、肺微血管内皮细胞和上皮细胞骨架改变；Nrf2出核增加导致内源性抗损伤能力不足是ALI/ARDS发生发展的重要原因，因此从增强内源性抗损伤能力及增强EPCs动员及再内皮化可能是未来ALI/ARDS治疗的新策略。

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