WP-17生物肽通过与TLR4相互作用抑制眼部炎症的作用机制

Uveitis is a common intraocular inflammatory disease, which is still lack of effective method in clinical treatment. Small peptides, owing to their advantages such as smaller molecule, less toxicity over proteins, are becoming promising agents in developing new therapeutics for ocular application. By means of bioinformatics methods and experimental models in vivo and in vitro, our study group has successfully screened out and testified novel peptides with potential anti-inflammatory effects, within which peptide WP-17 shows better anti-inflammatory activity. Furthermore, we found out that WP-17 could interact directly with TLR4, which is reported to play a critical role in ocular inflammation, by binding to the extracellular domain of TLR4. Moreover, WP-17 treatment could inhibit LPS-induced NF-κB phosphorylation and translocation in RAW264.7 cells. Based on these findings, we will firstly further investigate the mechanism under the interaction between WP-17 and TLR4, secondly explore the effect of WP-17 treatment on molecuar expressions related with TLR4 signaling pathway using both in vivo and in vitro assays, and finaly explore the effect of WP-17 treatment on activation of NF-κB and transcription of a variety of cellular genes downstream.The purpose of this study is to find out the potential target of WP-17 and its mechanism in inhibiting ocular inflammation so that we could find a potential alternative for the management of ocular inflammatory diseases in the future.

葡萄膜炎是一类临床常见、治疗棘手的眼部免疫原性疾病，小分子肽类药物凭借分子量小、毒性低等优势，成为治疗葡萄膜炎等新的潜在药物。本研究前期应用生物信息学技术和体内外实验模型筛选和论证，发现了具有抑制眼部炎症作用的新型生物肽，其中WP-17生物肽作用效果最佳。进一步研究发现WP-17能够结合可溶性TLR4胞外段蛋白，并抑制其下游信号通路NF-κB的激活，而TLR4在眼部炎症中发挥重要作用。本课题将以此为基础，深入探讨WP-17对TLR4的直接调节作用；通过体内外实验观察WP-17对TLR4相关分子表达的影响；利用双荧光素酶报告基因等技术，探讨WP-17对NF-κB活性和下游靶基因的影响，阐明其调控TLR4/MyD88/NF-κB信号通路的作用，进一步明确新型多肽WP-17抑制眼部炎症的潜在作用靶点和具体细胞分子机制，可望发现一种适于眼组织安全有效的新型炎症抑制剂，并为其向临床成果转化奠定基础。

本研究前期应用生物信息学技术和体内外实验模型筛选和论证，发现了具有抑制眼部炎症作用的新型活性肽，可明显减轻EIU大鼠眼部的炎症反应和眼球组织中炎性细胞的浸润和蛋白渗出，并可减少脂多糖（lipopolysaccharide, LPS）诱导的RAW264.7细胞中肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）和白介素-6（interleukin-6, IL-6）等促炎症细胞因子的表达。进一步研究发现其抑制眼部炎症的作用与TLR4密切相关。WP-17能够结合可溶性TLR4胞外段蛋白，并抑制TLR4下游信号通路NF-κB的激活和向细胞核内的转录，而TLR4在眼部炎症方面发挥重要的作用。因此，本课题以此为基础，首先采用流式细胞术（Flow cytometry, FCM）分析WP-17多肽与TLR4相互作用的具体机制：发现WP-17多肽能够与人单核细胞THP-1结合，且结合率呈浓度依赖性，LPS刺激能够增加WP-17多肽与THP-1细胞的结合，而这一结合是通过与THP-1细胞上表达的TLR4结合来实现的。其次研究WP-17多肽对TLR4信号通路相关分子表达的影响，发现WP-17对LPS诱导的THP-1细胞中MyD88、IRAK4和TRAF6的合成和释放有明显的抑制作用，且呈剂量依赖关系；最后，采用免疫荧光和Western Blot探讨WP-17多肽对NF-κB信号通路的影响，发现WP-17的干预不仅抑制了LPS诱导的NF-κB p65向RAW264.7细胞核内的转移，而且减少了细胞中磷酸化NF-κB p65的蛋白表达量、抑制了IκBα的降解，且作用呈剂量依赖性。从而进一步明确新型多肽WP-17抑制眼部炎症的潜在作用靶点和具体细胞分子机制：通过与LPS竞争性结合人单核细胞THP-1上表达的TLR4，从而阻断TLR4传递LPS信号通路，抑制TLR4信号通路主要相关分子MyD88/IRAK4/TRAF6的级联反应，进一步抑制NF-κB信号通路的活化，阻止多种细胞因子和炎性介质的过度释放，起到抑制眼部炎症反应的作用。从而期望发现一种适于眼组织的安全有效的新型炎症抑制剂，并为其向临床成果转化奠定基础。