PAD在六价铬诱导人表皮细胞ROS产生中的应答机制与功能

The exposure to hexavalent chromium (Cr (Ⅵ)) leads to dermatological health damage by inducing ROS (reactive oxygen species) formation. Peptidylarginine deiminases (PADs) have been found as a family of post-translational modification enzymes that catalyze protein citrullination, which plays a crucial role in homeostasis and anti-oxidative stress damage in human epidermal keratinocytes, but the underlying functional mechanism in exposure to Cr (Ⅵ) is not clear yet. Hence, in the present study, this proposal intends to study epidermal PADs gene expression, their enzyme activities and citrullination of targeting proteins, which response to Cr (Ⅵ)-induced ROS formation in human epidermal keratinocytes. Based on the pervious results, we aim to clarify that regulatory mechanism of the release of calcium ion and activation of NF-κB induced by ROS formation, which result in abnormal PADs gene expression and enzyme activity, respectively. Then it intends to detect the functional role of PADs during toxicity effect in epidermal keratinocytes exposed to Cr (Ⅵ). The proposal is expected to clarify the mechanism of the PADs to response ROS formation in human epidermal keratinocytes, which may contribute to a better understanding of toxicity mechanism of Cr (Ⅵ)–induced oxidative stress, and pave a new way to prevention and therapy of its dermatological hazards for occupational population.

六价铬接触诱导ROS(活性氧)产生可对人体皮肤组织造成健康损害。PADs (Peptidylarginine deiminases)是一类蛋白质翻译后瓜氨酸化修饰酶，对皮肤表皮细胞的内稳态维持和抗氧化应激损伤等起重要作用，但在六价铬暴露下表皮PADs的详实功能尚未阐明。本项目拟研究六价铬诱导ROS的产生对人表皮角质形成细胞中PADs基因表达、酶活性和靶向蛋白的瓜氨酸化的应激影响；结合前期研究结果，探明ROS产生所引起的胞内钙离子释放和NF-κB激活分别对PADs基因表达和PAD酶活性的调控作用；并揭示PADs在六价铬暴露致表皮细胞毒性效应中的功能性角色。本项目预期阐明PADs在六价铬诱导人表皮细胞ROS产生中的应答机制及其功能。这不仅有助于深入了解六价铬的氧化应激毒性机制，对六价铬职业人群的皮肤学危害防治也提供了新的思路。

六价铬接触对人体皮肤、角膜等上皮细胞参与构成的组织或器官可造成健康损害。PADs是一类蛋白质翻译后瓜氨酸化修饰酶，对细胞稳态维持和抗氧化应激损伤等起重要作用，但参与应答六价铬健康损害机制尚未阐明。本研究发现，在二维细胞模型中六价铬离子可诱导体外人正常皮肤表皮和角膜上皮细胞的细胞毒性与氧化应激反应。同时，细胞内铬浓度变化和氧化应激指标ROS积累均与重铬酸钾暴露浓度存在关联性。体外细胞模型与体内研究都分别证实了六价铬暴露可造成不同程度的细胞DNA损伤，甚至可能阻滞细胞周期。另一方面，六价铬离子可以刺激NF-κB、HMGB1、白介素等细胞炎症相关因子表达或者分泌，并且促进PADI1-3基因的转录水平的提高，提示六价铬所致PAD应答可能与炎症相关信号通路的开启存在互作关系。进一步深入研究还揭示了PADs转录水平的提高可能与PAD基因簇成员的启动子活性上升以及细胞内钙稳态失调有密切关系。在三维表皮模型研究中，我们还观察发现六价铬可致皮肤替代模型在组织层次上发生结构紊乱，角质层的表皮细胞数量上也发生明显下降；同时六价铬还可诱导角质层的一部分表皮细胞的胞浆PAD2表达呈弱阳性，提示PAD可能参与应答六价铬所致的细胞毒性与氧化应激反应，但相关分子调控机制仍有待进一步阐明。基于上述结果，我们初步构建了适用于重金属毒性研究的表皮细胞模型，以期将来在职业卫生学基础研究和毒理学安全性评价上获得应用。综上所述，本研究不仅有助于深入了解六价铬的氧化应激毒性机制，而且为揭示表皮PADs在六价铬暴露致细胞毒性效应中的作用提供了科学数据和理论基础。