清热利湿饮通过调控miRNA抑制角质形成细胞JAK1-STAT3通路及其治疗银屑病的作用机制研究

Psoriasis is a lifelong, chronic, recurrent and inflammatory skin disease, which seriously undermines the life quality of patients and their families. Conventional therapies for psoriasis are ineffective, and are often accompanied by adverse side effects. Chinese medicine has been extensively used in treatment of psoriasis and produces promising clinical results, which shows long-time effect with less disease recurrence and less side effects. Based on the traditional Chinese medicine for psoriasis, we have developed Qingrelishi recipe, which was proved to be effective for treatment of psoriasis. However, the molecular mechanism of Qingrelishi recipe is poorly understood. Epidermal keratinocytes of psoriatic skin are characterized by over activated STAT3, and increased levels of cytokines and growth factors which induced STAT3 activation have been found within psoriatic lesions. Our latest experimental results showed that Qingrelishi recipe decreased the levels of JAK1 protein and phosphorylated STAT3 in keratinocytes, suggesting that JAK1-STAT3 passway might be one of the targets of Qingrelishi recipe. Moreover, our results also suggest that inhibition of JAK1 by Qingrelishi recipe is mediated by miRNA regulation. In this proposed study, we will characterize the role of Qingrelishi recipe in keratinocytes using cellular and mouse model in vitro and in vivo and further identify the role of JAK1-STAT3 by which Qingrelishi recipe regulates this process. In addition, we will also explore the mechanisms by which Qingrelishi recipe regulates JAK1-STAT3 passway. This study will advance our understanding of the molecular function and mechanisms and help to promote the modernization of Chinese medicine.

银屑病是一种常见的难治性的慢性、复发性、炎症性皮肤病。中医在银屑病治疗上具有改善病情、复发率低，副作用小，远期疗效好等独特的优势。我院开发出清热利湿饮这一有效中药组方，经过大量的临床观察疗效显著，但目前对其治疗银屑病的分子生物学基础尚不清楚。角质形成细胞内STAT3通路的过度活化是导致银屑病的原因之一。我们最新发现提示清热利湿饮可能通过调控miRNA抑制角质形成细胞内JAK1蛋白表达以及STAT3磷酸化水平，角质形成细胞JAK1-STAT3通路可能是该药方治疗银屑病的靶点之一。本研究拟从miRNA调控角度出发，利用表皮角质形成细胞，结合银屑病小鼠模型以及临床标本，系统地分析清热利湿饮对银屑病角质形成细胞的影响，明确JAK1-STAT3通路在其中的作用以及清热利湿饮抑制该通路的分子机制，为揭示中医药治疗银屑病的关键作用机制提供实验证据，为推动中医药现代化奠定理论基础。

中医在银屑病治疗上具有改善病情、复发率低，副作用小，远期疗效好等独特的优势，但目前对其治疗银屑病的分子生物学基础尚不清楚。在国家自然科学基金的资助下，我们系统地分析清热利湿饮对银屑病角质形成细胞的影响和分子调控机制。我们发现清热利湿饮可以显著降低HaCaT细胞内JAK1、JAK2及P-STAT3的表达，并且清热利湿饮可显著减轻咪喹莫特诱导的小鼠皮损PASI评分以及皮损角质形成细胞中Ki67、P-STAT3、JAK1、JAK2蛋白表达。进一部研究揭示清热利湿饮能够促进miR-17-5p的表达，进而抑制JAK1 3’UTR活性，抑制JAK1的翻译，降低JAK1的表达，进而抑制STAT3的磷酸化和其功能，从而发挥抑制角质形成细胞增殖和炎性因子分泌的功能。在本课题的资助下，我们还分析了清热利湿饮的基础方龙胆泻肝汤治疗银屑病的作用机制。我们发现龙胆泻肝汤处理导致人皮肤角质形成细胞细胞形态发生明显变化，凋亡比例明显高于未处理细胞组，细胞内Caspase-3和Bax 的表达明显上调，Bcl-2 蛋白表达下降，提示龙胆泻肝汤能明显诱导人角质形成细胞的凋亡，其机制可能与下调Bcl-2的表达，增高Bax和Caspase-3的表达有关。我们还发现龙胆泻肝汤还能够有效降低角质形成细胞内STAT3总蛋白以及磷酸化蛋白水平，抑制细胞内STAT3信号通路的活性，提示STAT3通路是龙胆泻肝汤治疗银屑病的重要靶点，龙胆泻肝汤可能通过抑制角质形成细胞内STAT3活性，抑制银屑病角质形成细胞增殖，诱导凋亡。另外，我们还揭示了传统中药黄连的活性成分小檗碱能够抑制角质形成细胞增殖、迁移以及免疫相关因子的分泌功能，并且可以有效缓解咪喹莫特诱导银屑病小鼠皮损的症状；分子机制分析显示小檗碱通过抑制STAT3磷酸化，进而抑制CDC6的表达，诱导角质形成细胞凋亡。这些结果为银屑病中医治疗提供了研究数据支撑，为推动中医药现代化奠定理论基础，为银屑病新药物开发提供了实验证据和理论依据。