同型半胱氨酸/胱硫醚beta合成酶/硫化氢代谢途径功能障碍在内毒素诱导肺纤维化过程中的作用及其机制研究

Cystathionine beta-synthase (CBS) is the ?rst, and rate-limiting enzyme in the transsulfuration metabolic pathway of homocysteine (Hcy), meanwhile it is one of the endogenous hydrogen sulfide (H2S)-generating enzymes. In our preliminary study, we found that CBS expression was significantly suppressed in lung tissues obtained from a mouse model of endotoxin-induced epithelial mesenchymal transition (EMT) and pulmonary fibrosis, accompanying by decreased endogenous H2S production and increased Hcy accumulation in lung tissues. In addition, the slow-releasing H2S donor, GYY4137, significantly attenuated endotoxin-induced EMT and pulmonary fibrosis. On the basis of these results, the present proposal will firstly verify that the local dysfunction of Hcy/CBS/H2S metabolic pathway does contribute to endotoxin-induced EMT and pulmonary fibrosis, while intervention therapies aim at restoring the metabolic homeostasis of Hcy/CBS/H2S pathway can attenuate endotoxin-induced EMT and pulmonary fibrosis. It has been found that oxidative stress and activation of TGFbeta signal transduction pathway play critical roles in the development of EMT and organ fibrosis. Thus, the present project will investigate whether endotoxin-induced EMT and pulmonary fibrosis is partly due to Hcy/H2S imbalance-induced oxidative stress and TGFbeta pathway activation. Finally, we will examine whether the endotoxin receptor TLR4 and its downstream signal molecules are involved in the endotoxin-induced down-regulation of CBS. The present study will provide reliable evidence to elucidate the impact of Hcy/H2S imbalance on the pathogenesis of endotoxin-induced pulmonary fibrosis. Restoring the metabolic homeostasis of Hcy/CBS/H2S pathway may become one of the future treatment options of endotoxin-induced pulmonary fibrosis.

CBS既是同型半胱氨酸（Hcy）的代谢酶，又是硫化氢（H2S）的合成酶。我们发现在内毒素诱导肺上皮间质转化（EMT）及间质纤维化过程中，肺组织CBS表达显著降低，由此导致内源性H2S减少和Hcy蓄积；而缓释型H2S供体则可抑制内毒素诱导的肺纤维化。在此基础上，本项目首先将证实肺组织Hcy/CBS/H2S代谢途径功能障碍是内毒素诱导肺纤维化的重要原因，而多种能够恢复这一代谢途径功能的干预措施则能够减轻肺纤维化症状。鉴于氧化应激和TGFbeta信号通路激活已被证实在EMT及器官纤维化发生过程中起重要作用，本项目将进一步明确Hcy/CBS/H2S代谢途径失调是否也可通过上述机制诱导肺纤维化。最后我们还将研究内毒素受体TLR4及其下游信号分子在内毒素抑制肺组织CBS表达中的作用。本项目研究将有助于阐明内毒素诱导肺纤维化的发生机制，并从改善Hcy/CBS/H2S代谢途径的角度为临床治疗提供新的思路。

CBS既是同型半胱氨酸（Hcy）的代谢酶，又是硫化氢（H2S）的合成酶。我们发现在内毒素诱导肺上皮间质转化（EMT）及间质纤维化过程中，肺组织CBS表达显著降低，由此导致内源性H2S减少和Hcy蓄积；而缓释型H2S供体则可抑制内毒素诱导的EMT和肺纤维化。上述结果表明Hcy/CBS/H2S代谢途径功能障碍在内毒素诱导 EMT和肺间质纤维化中起着关键作用。进一步研究表明，内毒素诱导急性肺损伤和肺纤维化过程中均存在肺组织氧化应激水平增加的表现，同时肺组织中TGFβ1表达显著增加、Smad通路激活。而给予H2S供体NaHS、GYY4137或抗氧化药物白藜芦醇和氢水在抑制内毒素（LPS）诱导的肺组织氧化应激同时，还可显著减轻内毒素诱导的急性肺损伤、EMT和肺间质纤维化。上述结果说明内源性H2S代谢途径功能障碍可以通过促进肺组织氧化应激从而激活TGFβ/Smad信号转导，最终诱导EMT和肺纤维化的病理发生。以往研究表明CBS启动子上的Sp1结合位点在CBS转录调节中起关键作用。我们发现突变小鼠CBS启动子Sp1位点可使CBS启动子荧光素酶报告基因转录活性显著降低；LPS可以显著抑制Sp1表达；LPS可以激活PKC和MAPK家族的ERK和p38通路，其中PKC阻断剂可以逆转LPS抑制Sp1和CBS表达的作用。上述结果提示LPS可以通过激活PKC通路而抑制Sp1表达，由此抑制CBS启动子转录活性，最终导致CBS表达下调。此外，近年来研究表明内皮损伤所导致的内皮间质转化（EndMT）在器官纤维化病理发生发展过程中同样发挥重要作用。因此课题组还与上海交通大学附属新华医院江来教授课题组合作研究了氧化应激-NLRP3炎性小体激活-EndMT通路在机械通气诱导肺纤维化过程中的作用及其分子机制。另一方面，鉴于H2S可以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。因此，我们进一步采用马来酰亚胺标签结合高通量蛋白质组技术筛选了H2S作用后，肺组织中可能发生巯基硫巯基化的靶蛋白。由此希望找到能被H2S硫巯基化修饰的EMT/EndMT关键分子进一步研究H2S抗肺纤维化的分子机制。

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