基于T淋巴细胞分化的miR-451转录调控研究凉血解毒方治疗银屑病血热证的作用机理

Psoriasis is a T cell mediated chronic inflammatory skin disease. Removing pathogenic heat and toxin from the blood is the principle for treating blood-heat type psoriasis in TCM, which works effectively in clinical. However, its molecular mechanism is still need to be demonstrated in further. Recently, an expanding microRNA world has been associated with psoriasis. They may serve as the biomarker for the disease or therapy responses. miR-451, as a regulating microRNA, plays crucial roles in inhibiting both T cell activation and inflammatory response of cells. Our preliminary data showed that its expression in plasma was lowered with a negative correlation trend to psoriasis area and severity index (PASI). LiangXueJieDu decoction (LXJD), a basic TCM formula for blood-heat type psoriasis with roles to remove pathogenic heat and toxin from the blood, could improve skin lesions, lower Th1 and Th17 subsets, and increase miR-451 level, accompanied with changes in PBMC expression of transcription factors (TFs). The results suggested that LXJD therapy psoriasis in partially through miR-451. In the study, following the rule of miR-451 transcriptional regulation, we will observe the roles of LXJD in regulating TFs enrichment and epigenetic modification on the promoters and enhancers of miR-451 during Th1 and Th17 cell differentiation; and we will study their roles in cell phenotypes and functions. Meanwhile, we will study the changes in the genes associated with miR-451 regulation of blood-heat type psoriasis during LXJD treatment so as to discover its functional mechanisms. As a new method to study the mechanism of TCM medicines, the study will provide experimental evidence for optimizing psoriasis treatment and will shed light on the TCM theory of "to therapy psoriasis from the view of blood".

银屑病为T淋巴细胞参与的慢性炎症性皮肤疾病。凉血解毒为银屑病血热证的基本治法，疗效明确，其作用的分子机制有待深入。 miR-451作为调控的枢纽分子，具有抑制T淋巴细胞活化、抑制细胞炎性反应的作用，其在银屑病血浆表达下降，且与皮损严重程度相关。凉血解毒方在改善血热证患者皮损、降低Th1、Th17淋巴细胞水平的同时，升高miR-451，伴随转录因子表达改变，提示凉血解毒方可以通过影响miR-451的表达来影响疾病进程。本研究拟以miR-451的转录调控为线索，通过观察Th1、Th17淋巴细胞分化过程中，凉血解毒方对miR-451调控元件上的转录因子结合、表观遗传修饰改变，及对细胞表型和功能的干预作用，以及观察临床银屑病血热证患者miR-451调控链上相关基因在治疗前后的表达差异，来揭示该方的作用机制。本研究从新的视角和研究策略揭示中医药“从血论治”的分子机理，为中医治疗银屑病提供科学依据。

银屑病为T淋巴细胞参与的慢性炎症性皮肤疾病。凉血解毒为银屑病血热证的基本治法，疗效明确。microRNA作为基因表达调控的枢纽分子，影响细胞的表型和功能。为研究miR-451参与银屑病的病理环节、揭示银屑病血热证在“血”的分子特征以及凉血解毒治疗作用机理，本研究通过比较血热证患者在凉血解毒治疗过程中，伴随皮损发生、缓解到消退过程的症状体征、皮损严重程度(PASI)、T淋巴细胞亚群的动态变化，以及治疗前后PBMC基因表达和表观遗传修饰的变化，进行逐层深入研究。1）建立与临床疗效相结合的网络药理学研究模式，对银屑病血热证及其在凉血解毒治疗过程中伴随皮损改善的基因表达变化予以系统生物学角度的阐释，发现不仅存在与树突、T淋巴细胞活化相关的炎性基因高表达，同时存在与NK、B淋巴细胞防御功能相关的基因低表达，抑制T淋巴细胞活性是凉血解毒的作用环节之一；在转录组水平揭示了血热证在“血”的分子特征、以及对凉血解毒中药复方治疗的应答。2）同时，对凉血解毒治疗过程中的T淋巴细胞亚群变化进行动态观察, 发现在不同患者群，凉血解毒可降低过高的Th1-Th17、升高过低的Th1-Th17水平，其通过直接或间接机制对T辅助细胞发挥双向调节作用。3）进一步，结合PASI、Th1、Th17水平，分析银屑病患者血浆、CD3+T淋巴细胞的miR-451表达及DNA甲基化水平，采用体外诱导分化、细胞转染等实验，观察T淋巴细胞分化与miR-451的关系，发现凉血解毒治疗可影响microRNA及Th1转录因子的表观遗传修饰，其中血浆miR-451水平与PASI评分负相关；在miR-451基因和miR-155基因分别鉴定了与T淋巴细胞分化以及银屑病相关的CpG甲基化位点。本研究不仅为揭示中医证型分子基础、中药复方作用机理提供研究策略，为临床潜方用药提供实验依据，也为银屑病等自身免疫疾病的治疗提供药物作用的潜在靶点。