漆黄素对ACD小鼠Th17/Treg免疫平衡分析及调控机制研究

Th1/Th2 cell unbalance induced allergic contact dermatitis (Th1/Th2) is one of the key factors that cause pet eczema, and the discovery of Treg and Th17 cells has become an important complement to the unbalance theory. Immunosuppressive drugs are the first choice for the treatment of pet eczema in veterinary clinic, but the toxicity and recurrence are the major obstacle in the widely use of these drugs. In our present we found that fisetin had strong immunoregulatory activity on Th1/Th2 unbalance, and had the advantages of Chinese medicine. This project, which is based on the previous work, is to investigate the immunoregulatory effect of fisetin on Th17/Treg unbalance. The allergic contact dermatitis (ACD) model was established by DNFB, then hematoxylin-eosin staining was used to analysis the ear histopathology. Th17/Treg cell specific nuclear factors and cytokines were detected by Real-time RT PCR and ELISA method, the number of Th17/Treg cells was detected by FCM assay. In order to identify the target proteins in signal transduction pathway, we used high throughput proteomics to screen. These studies established the groundwork for further research the pathogenesis of Eczema and fisetin treatment of Eczema mechanism.

Th1/Th2细胞失衡诱发的变应性接触性皮炎是引起宠物湿疹的关键因素（之一），Treg和Th17细胞的发现成为Th1/Th2细胞失衡学说的重要补充。应用免疫抑制药物是兽医临床上治疗宠物湿疹的首选方案，但多数药物因副作用大或药效持续时间短等原因导致治疗效果不理想。课题组前期筛选发现漆黄素对Th1/Th2细胞失衡具有显著的调节作用，且具有中药安全无毒副作用的优势。本项目拟在前期基础上，以DNFB建立小鼠变应性接触性皮炎（ACD）模型，采用HE染色法进行耳廓病理组织检查，Real-time RT PCR和ELISA法检测Th17/Treg细胞特异性核因子及相关细胞因子的表达与含量，FCM法检测Th17/Treg细胞数量，研究漆黄素对模型小鼠Th17/Treg细胞失衡的影响。同时，采用高通量蛋白质组学筛选漆黄素调控信号通路的相关蛋白，确定靶点蛋白，为进一步研究漆黄素治疗湿疹的作用机制提供研究基础。

宠物湿疹是宠物皮肤病中的一大类型，常伴有剧烈瘙痒，严重时可导致皮肤大面积坏死和全层剥脱，影响宠物的健康。目前兽医临床上主要使用免疫抑制药物治疗宠物湿疹，但副作用大是使用这些药物广泛应用的主要障碍，因此，研究开发对宠物湿疹类皮肤病安全有效的药物意义重大。研究发现，Th17和Treg细胞之间平衡状态的打破是发病机制的关键环节，本研究通过DNFB建立小鼠变应性接触性皮炎（allergic contact dermatitis，ACD）模型，对漆黄素（Fisetin，FIS）治疗湿疹类疾病的作用进行了疗效评价和机理分析。结果显示：1）FIS对于ACD具有一定的治疗作用，降低了ACD小鼠脾脏和胸腺指数，缓解了耳廓水肿程度；2）FIS能够抑制Th17相关促炎因子IL-4、IL-6、IL-17、TNF-α和IFN-γ水平，上调Treg相关抑炎因子TGF-β和IL-10的表达；3）FIS能够显著抑制小鼠脾脏淋巴细胞中CD4+IL-17+Th17细胞的相对数量，相反上调CD4+CD25+Foxp3+Treg细胞的相对数量，有效恢复Th17/Treg细胞平衡；4）使用RT-RCR方法检测Th17/Treg细胞特异性转录因子Foxp3、ROR-γt的mRNA表达，也证明了FIS在湿疹发病中对Th17/Treg免疫失衡有调节作用；5）采用 western blot方法检测发现，FIS能够通过有效调节STAT通路，下调p-STAT3蛋白表达，上调p-STAT5蛋白表达，进而抑制p-STAT3/p-STAT5蛋白比值，从而恢复Th17/Treg细胞平衡。