治疗白癜风新靶点TSPO的研究

Vitiligo is a common skin disease for which the pathology is not yet fully evaluated. The present targets which are used to treat vitiligo are key enzymes and immune system. The efficacy of present therapeutic methods which have two principal treatments including regulating agents of key enzymes in the downstream pigmentation system and immune modulators for about two decades, are very less. These decades old therapeutic methods were still in use due to the lack of identification of new clinically successful targets during the past two decades. Therefore, development of a novel treatment method is necessary to treat vitiligo to improve the quality of life of the vitiligo patients. By screening the herbs which are used to treat vitiligo, Butin was identified as small, pigment regulating molecule. Multiple preliminary experiments demonstrated that the pigment regulatory effect of Butin is due to its binding to TSPO (Translocator protein 18 kDa) which can be considered as new potential target to treat vitiligo. The aim of this study is to systematically verify the efficiency of targeting TSPO to treat vitiligo on skin samples from vitiligo patients, Tspo knockout and over expression cell lines, tspo knockout zebrafish and mice models. The active functional domain of TSPO in regulating pigment metabolism will be elucidated by various techniques and methods, such as MST and co-crystallization method and will be compared with different ligands that are related to melanin synthesis. Finally the active functional domain will be reverse verified by using site-directed mutation technique which leads to the elucidation of active functional domain of TSPO to regulate pigment metabolism. The final objective is to lay down the foundation for designing and developing new drug for the treatment of vitiligo.

白癜风是临床常见的皮肤疑难症，由于目前病理机制仍有不明，治疗药物主要集中在针对下游色素合成系统关键酶的调节剂和免疫调节剂两大类，临床疗效不确切。因此急需打破白癜风治疗靶点20多年未有突破的瓶颈，促进新药开发。课题组前期通过对临床有效植物药研究，发现具有色素调节活性且不同于现有药物作用机理的小分子化合物紫铆素。通过多角度实验筛选分析，发现了调控色素合成的潜在新靶标TSPO，并完成了初步确认。本项目拟在前期基础上，通过白癜风病人皮损区皮肤样本、Tspo基因敲除及过表达细胞系模型、tspo基因敲除小鼠及斑马鱼动物模型进一步系统性地确证靶点。从已报道的TSPO配体中筛选与色素代谢相关且结构不同的配体，通过MST技术、共结晶技术，解析相关活性功能结构域。并通过位点突变的方法进行反向验证，阐明TSPO调节色素代谢的活性功能结构域。旨在能为白癜风治疗药物的设计和开发奠定基础。

白癜风是临床常见皮肤疑难症，安全有效的新靶点研究无突破不能给药物设计提供理论依据，也成为白癜风的药物研发的瓶颈。课题组前期研究发现紫铆素促进黑色素合成与线粒体膜蛋白TSPO相关，需明确TSPO是否可成为候选靶点。本研究以紫铆素及TSPO激动剂Ro54864和拮抗剂PK11195为工具分子，利用微量热泳动技术以及表面等离子共振技术确证紫铆素可直接作用于TSPO。Ro54864亦可显著促进黑色素的合成， PK11195可抑制黑色素合成。但在构建的tspo基因敲除B16F10细胞以及斑马鱼模型上紫铆素及Ro54864仍可显著促进黑色素合成，由此判断紫铆素是TSPO的配体，TSPO不能直接调控黑色素合成。通过利用蛋白质组学的方法比对分析TSPO敲除前后给与紫铆素等B16F10细胞中蛋白的表达差异，发现其与胆固醇代谢、细胞凋亡、细胞增殖、炎症调控以及氧化应激等白癜风致病因素密切相关，未影响色素调控通路。基于TSPO 与细胞内类固醇激素的合成密切相关，进一步探索发现胆固醇和胆固醇合成酶抑制剂辛伐他汀均能促进色素的合成，对色素调控有显著影响的糖皮质激素未见显著改变，提示TSPO不是通过类固醇通路影响色素合成。同时在tspo基因敲除斑马鱼模型上验证了TSPO参与调控氧化应激，并与抑郁相关，临床上的数据也提示白癜风患者的疾病发生和发展与精神应激相关。课题组从精神应激角度发现TSPO在神经递质5-HT促进黑色素合成中扮演重要角色，作用机制为5-HT首先通过5-HT2A/5A受体激活Gi/o和Gq，与Gβγ解离后激活下游PLCβ和Ca2+，激活下游PKCε促进TSPO蛋白表达，通过氧化应激参与调控黑色素的合成。综上所述TSPO参与色素代谢的调控与神经调控密切相关，紫铆素可能通过调控TSPO间接发挥促黑色素功效。TSPO在白癜风的治疗过程中不能作为药物设计的直接靶点。目前在白癜风的治疗中还未见通过神经调控进行干预的深入研究，需要进一步加强在精神应激下TSPO在白癜风疾病发生发展中的角色研究。本项目的研究结果为后续治疗白癜风药物的开发提供了很有意义的理论参考。