基于OX40/OX40L介导T细胞-肥大细胞相互作用，研究黄连解毒汤治疗“湿热蕴肤”型特应性皮炎分子机制

Atopic dermatitis (AD) is a complicated, relapsing chronic allergic inflammatory skin disease. Since the pattern of dampness-heat encumbrance of skin usually occurs in children and teenagers, clearing heat and removing toxicity is regarded as one of major therapeutic strategies. A series of reported researches on the underlying mechanisms of its therapeutic effect were focused on T cell or mast cell regulation by these therapeutic strategies in AD, however, ignoring the interaction/balance between T cell and mast cell. It has been shown that OX40/40L is a costimulatory molecule mediating the interaction of T cell and mast cell, and its overexpression highly correlates with severity of lesional skin in children patients with acute AD. Our previous studies found that Huang-Lian-Jie-Du-Extract could regulate balance of Th1/Th2 in AD mice, and inhibit activation of mast cell. Therefore, we hypothesize that Huang-Lian-Jie-Du-Extract could regulate interaction/balance of T cell and mast cell by inhibiting OX40/OX40L expression, further to improve the microenvironment of skin for the treatment of AD. In this study, we would focus on interaction/balance of T cell and mast cell mediated by OX40/OX40L signalling, systematically investigate proliferation and differentiation of T cell and activation of mast cell in AD. In addition, Huang-Lian-Jie-Du-Extract would be selected as the representative to investigate the therapeutic effect of clearing heat and removing toxicity strategy on dampness-heat encumbrance of skin-type AD, further to elucidate the underlying mechanisms of interaction/balance of T cell and mast cell, T cell immune response, mast cell-mediated allergic response.

特应性皮炎(AD)是一种易复发、慢性炎症性疾病。湿热蕴肤型AD多见于儿童及青少年，清热解毒是主要治法。目前其机制研究限于单独对T细胞或者肥大细胞的调节，忽视了两者之间的相互作用/平衡关系。OX40/OX40L是介导T细胞与肥大细胞相互作用的共刺激分子；在儿童及青少年患者中，OX40/OX40L高表达与急性期AD严重程度高度相关。我们前期研究发现，黄连解毒汤能调节AD小鼠体内Th1/Th2平衡，抑制肥大细胞激活。基于此，我们推测黄连解毒汤可通过抑制OX40/OX40L，平衡T细胞与肥大细胞相互作用，改善皮肤微环境发挥治疗AD作用。本研究拟基于OX40/OX40L信号，以T细胞与肥大细胞相互作用为切入点，系统考察AD中T细胞增殖、分化及肥大细胞活化等变化。选择黄连解毒汤为代表药，考察清热解毒对湿热蕴肤AD的治疗作用，阐释清热解毒法干预T细胞免疫应答、肥大细胞介导的过敏反应及两者平衡的作用机制。

特应性皮炎(atopic dermatitis，AD)是一种具有遗传倾向的过敏性皮肤病，在婴幼儿中发病率较高。AD是以Th2、Th17亢进的皮肤炎症。Th2、Th17型细胞因子分泌增多可下调表皮屏障蛋白，如兜甲蛋白（Loricrin, LOR）的表达，并且可促进瘙痒相关因子TSLP、IL-13、IL-31分泌。目前，APC-OX40L-OX40-CD4+T细胞轴是目前治疗AD的新靶点，其中，OX40主要在T细胞上表达，其配体OX40L可在肥大细胞上表达。OX40-OX40L的连接可促进T细胞分化为Th1、Th2、Th17，抑制Treg的分化，促进T细胞增殖，抑制肥大细胞脱颗粒。黄连解毒汤广泛应用于各类皮肤病的治疗，尤其是AD及湿疹，且具有影响肥大细胞活化及T细胞分化的作用。因此，我们提出假说认为OX40-OX40L连接所介导的T细胞-肥大细胞相互作用是黄连解毒汤治疗AD的关键途径。本课题研究内容包括以下五个部分：1、黄连解毒汤改善AD小鼠红斑、结痂、渗出、干燥、苔藓化等皮损症状，降低临床评分，改善AD小鼠皮损部位炎性浸润，减小棘层厚度，上调表皮屏障蛋白LOR和AQP3表达；2、黄连解毒汤减少AD小鼠搔抓次数，缓解瘙痒症状，减轻肥大细胞浸润情况，下调瘙痒相关因子TSLP、IL-13、IL-31、H4HR mRNA表达情况；3、黄连解毒汤降低AD小鼠脾脏指数，减少AD小鼠脾脏T细胞向Th2、Th17分化的情况，上调Th1/Th2比例，下调Th17/Treg比例分化；4、黄连解毒汤上调AD小鼠OX40、OX40L表达水平；5、黄连解毒汤促进肥大细胞-CD4+T细胞共培养体系中CD4+T细胞凋亡，促进Treg分化，下调Th17/Treg比例。