氧化应激诱导内皮细胞衰老在肥胖增加急性肺损伤易感性中的作用以及运动干预的保护机制研究

Obesity is known to impair pulmonary vascular endothelial function and primes the lung for injury, thus being implicated in the pathogenesis and clinical outcomes of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In the preliminary study, we found that oxidative stress-induced senescence of pulmonary endothelial cells might contribute to the priming effect of obesity on acute lung injury. Treadmill exercise training for 6 weeks significantly decreased the elevated oxidative stress and endothelial cell senescence in lung tissues of ob/ob obese mice, thus attenuating the priming effect of obesity on acute lung injury. Moreover, the protective effects of treadmill exercise on obesity-associated lung disorders was, at least partly, due to increases in cystathionine beta synthase (CBS) expression and endogenous hydrogen sulfide (H2S) production in lung tissues of ob/ob obese mice stimulated by treadmill exercise. It has been reported that H2S can modulate protein functions by modifying protein cysteine residues through S-sulfhydration. Using maleimide labeling technique and mass spectrometry, we obtained a number of potential target proteins which might be basally sulfhydrated by H2S in endothelial cells. Among these potential target proteins, aldehyde dehydrogenase 2 (ALDH2), p47phox and murine double minute 2 (MDM2) have been known to play important roles in the development of oxidative stress and cellular senescence. On the basis of these results, the present proposal will first observe the effects of exercise training on obesity-induced pulmonary oxidative stress, endothelial cell senescence, as well as the increased sensitivity to ALI in ob/ob obese mice. Then we will investigate whether increased CBS expression and H2S production contribute to the pulmonary protective effect of exercise training. Next, the effect of H2S on S-sulfhydration of ALDH2, p47phox and MDM2 will be clarified. And then, the potential S-sulfhydration sites of these proteins will be detected by maleimide labeling technique and mass spectrometry. Finally, we will elucidate whether S-sulfhydration of the above-mentioned three target proteins are involved in the protective effects of exercise training against obesity-induced pulmonary oxidative stress, endothelial cell senescence, as well as the increased sensitivity to ALI. This study not only clarifies the mechanisms underlying how exercise training attenuates the priming effect of obesity on ALI, but also shares alternative non-drug interventions for prevention of acute lung injury in obese population.

肥胖可使机体对肺损伤的易感性增加，从而影响ALI/ARDS的病理发生和转归。我们的前期工作提示氧化应激诱导肺血管内皮细胞衰老可能是肥胖时急性肺损伤易感性增加的关键原因；运动锻炼则可能通过促进肺组织中内源性硫化氢（H2S）生成而发挥抗氧化应激和抗内皮细胞衰老的作用，从而降低肥胖机体对急性肺损伤的易感性。H2S可以通过S-硫巯基化修饰靶蛋白而调节其功能，我们采用质谱技术筛选了内皮细胞中可能被S-硫巯基化的靶蛋白，其中ALDH2、p47phox和MDM2已知与氧化应激和细胞衰老机制密切相关。在此基础上，本项目首先将明确H2S生成增多在运动干预抑制肥胖小鼠肺组织氧化应激和血管内皮细胞衰老、降低对急性肺损伤易感性中的关键作用。然后阐明H2S靶蛋白的巯基修饰是否参与了运动干预的肺保护效应。本项目旨在阐明运动干预减轻肥胖对急性肺损伤易感性的机制，由此为运动干预作为肥胖相关肺疾病的非药物防治手段提供依据。

肥胖可使机体对肺损伤的易感性增加，从而影响ALI/ARDS的病理发生和转归。我们的研究发现ob/ob肥胖小鼠肺组织氧化应激水平、炎症因子水平、巨噬细胞浸润以及肺血管内皮细胞衰老比例显著高于对照小鼠， 同时对LPS诱导的肺血管内皮功能障碍、急性肺损伤易感性增加。运动干预可以显著降低ob/ob肥胖小鼠肺组织氧化应激、炎症反应、巨噬细胞浸润以及肺血管内皮细胞衰老，同时减轻ob/ob小鼠的血管内皮功能障碍、降低对LPS诱导急性肺损伤的易感性。此外，运动干预还可减轻博来霉素诱导的肺上皮细胞衰老和上皮-间质转换（EMT），由此减轻肺纤维化。进一步研究表明，ob/ob肥胖小鼠和博来霉素诱导肺纤维化肺组织中H2S合成酶CBS表达和H2S生成较之对照小鼠明显减少，而运动锻炼则可促进肥胖小鼠和肺纤维化小鼠肺组织中CBS表达和H2S生成。在ob/ob肥胖小鼠模型中，H2S供体NaHS处理则可显著抑制小鼠肺组织炎症反应和肺内皮细胞衰老，同时减轻ob/ob小鼠的血管内皮功能障碍、降低对LPS诱导急性肺损伤的易感性。而在博来霉素诱导肺纤维化模型中，NaHS可以抑制肺上皮细胞衰老、抑制TGF/Smad和LRP-6/-catenin信号通路，从而减轻博来霉素诱导的肺纤维化。此外，我们的研究还发现运动锻炼显著增加ob/ob肥胖小鼠肺组织皮质酮含量。运动干预可以显著增加肥胖小鼠肺组织中糖皮质激素代谢酶11-HSD1表达，然而对11-HSD2表达没有显著影响。糖皮质激素受体阻断剂RU486可以阻断运动锻炼对肥胖小鼠肺组织的抗炎和减重效应。近年来研究表明H2S可通过修饰靶蛋白半胱氨酸上的巯基形成S-硫巯基化而调节其功能。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ALDH2、p47phox和MDM2这三种蛋白已知与氧化应激和细胞衰老机制密切相关。我们在肺血管内皮细胞和肺泡II型上皮细胞的研究结果均表明H2S处理确实可以使得MDM2蛋白发生S-硫巯基化，MDM2通过抑制p53/p21信号通路从而抑制肺血管内皮细胞和肺泡II型上皮细胞衰老，从而改善其功能。