CN113694061A - 一种采用色氨酸代谢物抑制银屑病病发的方法 - Google Patents
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Abstract
本发明公开了一种采用色氨酸代谢物抑制银屑病病发的方法，属于银屑病治疗技术领域，所述方法采用喹啉酸治疗银屑病，所述喹啉酸通过激活芳香烃受体AhR抑制NLRP3炎症小体抑制银屑病，具体的说，皮肤表面色氨酸代谢的代谢物喹啉酸通过激活细胞核内的AhR负调控NLRP3炎性体通路的激活，从而抑制炎症细胞因子的分泌参与银屑病的发病。
Description
技术领域
本发明涉及银屑病治疗技术领域，尤其涉及一种采用色氨酸代谢物抑制银屑病病发的方法。
背景技术
大量微生物群栖息在皮肤中，与宿主共享营养，并产生参与调节皮肤健康状态和免疫功能的代谢物。微生物群产生的色氨酸(Trp)代谢物因其在免疫调节，尤其是免疫抑制中的作用而受到广泛关注。多项研究表明，它们在免疫组织的成熟和炎症性疾病的微调中发挥作用，如炎症性肠病、类风湿性关节炎、代谢综合征、神经精神特征等。与肠道相比，皮肤处于干燥、营养贫乏和酸性的环境中，因此皮肤微生物群的代谢功能与肠道有很大不同。表皮的角质层富含来自死角化细胞和破损角蛋白的氨基酸，为皮肤微生物群的色氨酸代谢提供了丰富的底物。喹啉酸(QA)是色氨酸通过犬尿氨酸为代谢途径下游的重要代谢物，在多种精神疾病中发挥重要作用，例如抑郁症和压力相关疾病。在过去的几十年里，许多关于QA的研究都集中在它与神经系统疾病的联系上。在治疗银屑病领域中，是否能利用代谢物QA是值得关注的事情。
银屑病是一种多基因遗传背景下的免疫异常疾病，因其具有发病率较高和反复发作等特点而在皮肤科临床和科研领域倍受重视。NLRP3炎症小体是固有免疫系用识别受体家族组要成员，在银屑病皮损处高表达，除此之外，NLRP3通路相关基因(如NLRP3本身、caspase-1(含半胱氨酸的天冬氨酸水解酶)和IL-1β)可能携带导致组成性促炎模式的功能获得性突变。在银屑病治疗中多项研究表明，与健康受试者相比，银屑病患者皮肤微生物群的群落多样性发生了显着变化，例如链球菌和金黄色葡萄球菌。然而，这些研究大多集中在银屑病病变和健康皮肤之间细菌丰度的变化，皮肤微生物群的代谢仍然很大程度上未知，这阻碍了对皮肤微生物群与宿主相互作用的功能重要性的全面理解，尤其是在皮肤微生物群代谢与NLRP3炎症小体之间的调节关系，也阻碍了突破常规技术发展新型银屑病治疗方案的进程。
发明内容
因此，本发明的目的是提供一种采用色氨酸代谢物抑制银屑病病发的方法，通过研究皮肤微生物群产生的色氨酸代谢产物QA对银屑病的影响来抑制银屑病病发。QA在银屑病的发病中以AhR依赖性方式发挥调节功能抑制NLRP3炎症小体，能明显减轻银屑病炎症，抑制皮肤炎症中细胞因子的表达，进而抑制银屑病病发。
本发明通过以下技术手段解决上述技术问题：
一种采用色氨酸代谢物抑制银屑病病发的方法，采用喹啉酸治疗银屑病。
进一步，所述喹啉酸是皮肤菌群色氨酸的代谢产物。
进一步，所述喹啉酸通过激活芳香烃受体AhR抑制NLRP3炎症小体抑制银屑病，具体的说，皮肤表面色氨酸代谢的代谢物喹啉酸通过激活细胞核内的AhR负调控NLRP3炎性体通路的激活，从而抑制炎症细胞因子的分泌参与银屑病的发病。
进一步，所述喹啉酸通过抑制角质形成细胞中IL-1β蛋白和IL-18蛋白的分泌来抑制银屑病。
进一步，在抑制银屑病病发时，所述喹啉酸的用药浓度0.1μg/ml-1mg/ml。
进一步，所述喹啉酸与药学上可接受的辅料混合制成药剂用于抑制银屑病。如果人体用药，最佳的选择为参照制药基质做成乳膏使用，如凡士林、羊毛脂、醇类等常见乳剂/乳膏/擦剂型基质。
有益效果：
本发明的皮肤菌群色氨酸代谢产物喹啉酸QA可明显抑制银屑病表型，抗细胞增殖及炎症，抑制皮肤炎症中细胞因子的表达，进而抑制银屑病病发。同时使用QA治疗银屑病具有良好的药用前景，QA的化学性质较为稳定，可以制成乳膏、擦剂使用。
附图说明
图1：液相色谱法检测结果图；
图2：QA抑制小鼠银屑病病发效果图；
图3：蛋白质印迹法检测结果图；
图4：免疫荧光数据图；
图5：AhR特异性siRNA治疗效果图；
图6：小鼠耳朵组织切片染色图。
具体实施方式
以下将结合具体实施例对本发明进行详细说明：
实施例1：银屑病患者皮损处和非皮损处QA水平降低
为了验证QA参与银屑病的发病机制，对银屑病患者皮损与非皮损除处的皮肤进行QA水平分析。
1、使用凝胶贴片方法收集健康受试者(健康皮肤)和银屑病患者皮肤(皮损处和非皮损处)表面的代谢物，获得皮肤表面微生物群样本；
2、对采集的样本使用液相色谱-串联质谱分析法分析比较银屑病患者和健康受试者皮肤表面的Trp代谢物水平；
得到的结果如图1所示，结论如下：
在Trp水平代谢物中，QA在银屑病患者的皮肤病变(皮损)和非病变皮肤(非皮损)中的水平显着低于健康受试者。此外，QA水平与银屑病面积和患者严重程度指数(PASI)之间存在显着的负线性相关性，表明银屑病患者越严重，QA在皮肤表面的表达越低。以上这些数据表明，银屑病患者皮肤表面存在丰富的微生物Trp代谢物，并且银屑病患者皮肤表面QA水平下降，表明QA可能参与银屑病的发病机制。
实施例2：QA减弱了IMQ诱导的小鼠银屑病样皮炎和炎症细胞浸润
基于实施例1的实验，将QA用于抑制银屑病病发，减弱炎症细胞浸润。利用常规的IMQ诱导的银屑病样小鼠模型来进行实验。
实验组：在小鼠耳朵上涂抹使用IMQ，浓度为5％，25mg/只耳朵/天，涂抹IMQ 60分钟后，将浓度为10μg/ml(二甲基亚砜稀释)QA涂抹于IMQ涂抹处的小鼠皮肤上，连续使用7天，于第5天、第7天观察小鼠上药处皮肤；
对照组：在小鼠耳朵上单独使用IMQ处理，浓度为5％，25mg/只耳朵/天，连续使用7天，于第5天、第7天观察小鼠上药处皮肤。
得到的结果如图2所示，得到的结论如下：
(1)用IMQ和QA混合处理的小鼠的耳朵厚度显着低于单独用IMQ处理(对照组)的小鼠的耳朵厚度，并且用IMQ加QA处理的耳朵的可见鳞屑和发红程度明显减少。
(2)苏木精和伊红染色显示IMQ加QA组小鼠耳朵的表皮厚度减少和炎症细胞浸润减少。
基于上述实验结果，采用流式细胞检测方法进行分析，结果显示：IMQ+QA组小鼠皮肤CD4+T细胞或Ly6G+细胞明显少于IMQ组，CD8+T细胞数量无差异；
实时PCR定量以检测细胞因子转录物(IL-1β、IL-6、IL-17、IL-18、IL-23、TNF-α、S100A8、CXCL1和CCL20)的表达，结果显示：与IMQ组相比，IMQ加QA组的IL-1β、IL-6、IL-18、S100A8和CXCL1显着降低。
综上，上述这些数据表明，局部应用QA减轻了炎症细胞的浸润并抑制了IMQ诱导的皮肤炎症中细胞因子的表达，对抑制银屑病病发有明显作用。
基于上述实验证明了QA可抑制银屑病发病。同时对上述实验组的小鼠样品进行蛋白质印迹法分析，得到的结果如图3所示，结果显示：在用混合细胞因子(20ng/mlIL-1α、20ng/ml IL-17,20ng/ml IL-22,50ng/ml TNF-α，20ng/ml Oncostatin M)加QA刺激后，角质形成细胞中NLRP3的表达显着下调；然而，NLRC4和AIM2的表达没有改变。此外，通过蛋白质印迹确定了QA对角质形成细胞中NLRP3炎性体的影响，发现在用混合细胞因子加QA刺激后，角质形成细胞中ASC、caspase-1、成熟型IL-1β和IL-18的蛋白水平均降低，这与免疫荧光数据一致。因此，我们的结果再次表明QA抑制了NLRP3炎症小体的激活以及角质形成细胞中IL-1β和IL-18的分泌，使用QA抑制银屑病病发是有效果的。
实施例3：激活芳香烃受体AhR抑制NLRP3炎症小体抑制银屑病
基于上述实施例，验证了采用QA抑制银屑病病发是有效果的，对于QA是如何抑制NLRP3炎症小体仍然需要进一步探究。
根据现有的技术培养HaCaT细胞，使用浓度为100μg/mLQA处理角质形成细胞后，结果如图4所示：结果表明，QA处理的角质形成细胞表现出AhR和CYP1A1(AhR激活的主要下游分子)的mRNA和蛋白质水平显着增加，表明AhR信号在角质形成细胞中被QA激活，免疫荧光数据进一步证实了这一结果。
同时使用小干扰RNA来干涉角质形成细胞中的AhR表达，然后添加混合炎性细胞因子来模拟体外银屑病模型。QA的加入显着抑制了NLRP3炎性体及其相关分子的表达。尽管如此，在干扰AhR后，QA对NLRP3炎性体以及IL1-β、IL-18和caspase-1分泌的抑制作用均不同程度恢复。以上这些结果表明，AhR是QA在银屑病发病机制中对NLRP3的抑制作用所必需的，采用小鼠模型进一步炎症。
将2.5nmol的AhR-siRNA与5mg乳膏基质混合得到AhR特异性siRNA(以上混合物为1只耳朵的用量)局部应用于IMQ治疗小鼠的耳部皮肤，使用时隔天涂抹于小鼠耳部，共涂抹4次。
得到的结果如图5所示，结果表明：使用上述药物后，有效地加剧了银屑病样病变，显示耳部厚度增加和红斑加重。用AhR特异性siRNA局部处理后小鼠耳朵组织切片的苏木精和伊红染色显示出明显的表皮变化，厚度和增生增加。说明AhR表达干涉后加剧了IMQ诱导的银屑病表型。
当敲低AhR后重复上述实验。通过蛋白质印迹法和RT-PCR分析显示，如图5所示：AhR的敲低导致了NLRP3、ASC、成熟caspase-1和IL-1β在mRNA和蛋白质水平上的表达回复，说明敲低AhR会影响NLRP3炎症小体和相关分子的表达水平。
综上，这些结果表明，皮肤表面色氨酸代谢的代谢物QA通过激活细胞核内的AhR负调控NLRP3炎性体通路的激活，从而抑制炎症细胞因子的分泌参与银屑病的发病。
根据实施例1-实施例3的实验结果可知，QA通过激活芳香烃受体AhR抑制NLRP3炎症小体抑制银屑病病发，即色氨酸代谢物QA能明显减轻银屑病炎症，抑制皮肤炎症中细胞因子的表达，进而抑制银屑病病发。
实施例4：QA用药浓度
QA用DMSO(二甲基亚砜)稀释后可直接使用，当然得，QA也可以参照制药基质做成乳膏使用，如凡士林、羊毛脂、醇类等常见乳剂/乳膏型基质。
利用常规的IMQ诱导的银屑病样小鼠模型对QA用药浓度进行实验，按照实施例2的实验组进行实验，QA浓度分别为0.01ug/ml、0.1ug/ml、1ug/ml、10ug/ml，得到的结果如图6所示，根据结果显示，当0.1μg/ml≤有效浓度≤10ug/ml时，苏木精和伊红染色显示表皮厚度减少和炎症细胞浸润减少，同时小鼠耳朵的可见鳞屑和发红程度明显减少，有效浓度在0.1-10μg/ml的范围时能抑制银屑病病发，减缓银屑病病情。
同时对细胞给药浓度进行实验，最佳的细胞用药浓度为0.1mg/ml-1mg/ml。
以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。本发明未详细描述的技术、形状、构造部分均为公知技术。
Claims (7)
1.一种采用色氨酸代谢物抑制银屑病病发的方法，其特征在于，采用喹啉酸治疗银屑病。
2.根据权利要求1所述的一种采用色氨酸代谢物抑制银屑病病发的方法，其特征在于，所述喹啉酸是皮肤菌群色氨酸的代谢产物。
3.根据权利要求1所述的一种采用色氨酸代谢物抑制银屑病病发的方法，其特征在于，所述喹啉酸通过激活芳香烃受体AhR抑制NLRP3炎症小体抑制银屑病。
4.根据权利要求1所述的一种采用色氨酸代谢物抑制银屑病病发的方法，其特征在于，所述喹啉酸通过抑制角质形成细胞中IL-1β蛋白和IL-18蛋白的分泌来抑制银屑病。
5.根据权利要求1-4任一权利要求所述的一种采用色氨酸代谢物抑制银屑病病发的方法，其特征在于，在抑制银屑病病发时，所述喹啉酸的用药浓度为0.1μg/ml-1mg/ml。
6.根据权利要求5所述的一种采用色氨酸代谢物抑制银屑病病发的方法，其特征在于，所述喹啉酸与药学上可接受的辅料混合制成药剂用于抑制银屑病。
7.根据权利要求6所述的一种采用色氨酸代谢物抑制银屑病病发的方法，其特征在于，所述药剂为乳剂、擦剂。
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