基于miR-30b-5p调控Notch信号通路探讨龙胆泻肝汤治疗葡萄膜炎大鼠的作用机制

Uveitis is an autoimmune eye disease that can seriously damage eyesight. The imbalanced Th17/Treg ratio is related to the pathogenesis of uveitis, and Notch signaling pathway can influence the differentiation of both Th17 and Treg cells, thereby regulating Th17/Treg ratio and influencing the occurrence and prognosis of uveitis. MicroRNAs (miRNAs) regulate the expressions of target genes via RNA interference, playing a pivotal role in the pathogenesis of uveitis. To date, the detailed mechanism that whether Notch signaling molecules influence the occurrence and prognosis of uveitis is still unknown. Our previous findings have shown that the miR-30b-5p levels were down-regulated in intraocular tissues, spleen and lymph node in experimental autoimmune uveitis (EAU) rats, whereas the levels of related genes involved in Notch-signaling-pathway molecules were up-regulated, and the Th17/Treg ratio was imbalanced in EAU rats, the administration of Longdan Xiegan decoction (LXD) was effective in treating uveitis. Based on these findings, we assume a hypothesis of double balance theory that LXD administration could modulate the ratio of Th17/Treg cells and restoring the balance status of both systemic immune system and local immune microenvironment of intraocular tissues via Notch signaling pathway via the regulation of miR-30b-5p. Using an EAU rat model, the present project aims to explore the effect of active Notch signaling pathway on the pathogenesis of uveitis, investigate the role of LXD administration in the differentiation and the ratio of Th17, Treg cells from EAU rats by gene knockout, RNA interference and lentivirus transfection, thereby elucidating the mechanisms of LXD administration in treating uveitis via regulating the balance of Th17/Treg ratio based on miR-30b-5p regulating Notch signaling pathway and illustrating the biological essence of LXD treatment restoring the balance of both systemic immune environment and intraocular local immune microenvironment based on holistic views and balance concept. Our investigations will present a theoretical base in treating uveitis with Traditional Chinese Medicine in clinical practice.

葡萄膜炎是一类严重危害视力的复发性自身免疫性眼病，Notch信号通路通过调控Th17、Treg细胞分化影响二者比例，对其发病与转归有重要影响，miRNAs以RNA干扰方式在葡萄膜炎发病中发挥作用。我们发现：葡萄膜炎大鼠眼、脾和淋巴结组织中miR-30b-5p表达降低，Notch通路相关基因表达升高，Th17/Treg比例失衡，龙胆泻肝汤治疗葡萄膜炎效果确切，提出“龙胆泻肝汤通过miR-30b-5p调控Notch通路均衡Th17/Treg比例，促进全身和眼内免疫环境双平衡治疗葡萄膜炎”的假说。本项目拟采用基因敲除、慢病毒转染等手段，研究Notch通路在葡萄膜炎发病中的作用，探讨龙胆泻肝汤经miR-30b-5p调控Notch通路促进Th17/Treg平衡、治疗葡萄膜炎的作用机制，从整体观和平衡观阐释龙胆泻肝汤促进葡萄膜炎大鼠全身和眼内免疫环境双平衡的生物学本质，为中药治疗葡萄膜炎提供理论依据。

葡萄膜炎是严重危害视力的自身免疫性眼病，常用激素和免疫抑制剂治疗，副作用大。本项目采用慢病毒转染、基因干扰等手段，探讨了Notch通路活化与葡萄膜炎之间的关系以及龙胆泻肝汤(LXD)通过提高miR-30b-5p水平治疗葡萄膜炎的作用机制。体内实验发现，实验性自身免疫性葡萄膜炎（EAU）大鼠miR-30b-5p低表达，Notch1和DLL4基因和蛋白高表达，Notch通路被活化，Th1和Th17水平升高，CD4+/CD8+和Th17/Treg比例失衡。LXD通过抑制EAU大鼠Notch通路活化从而有效降低Th1和Th17细胞分化，减少IL-17、IFN-γ等炎性细胞因子的表达，恢复CD4+/CD8+和Th17/Treg细胞平衡，调节全身免疫状态和眼内微环境恢复平衡，从而有效减轻眼部炎症，保护眼组织。此外，基于网络药理学和生物信息学分析平台筛选了LXD治疗葡萄膜炎的重要分子靶点，开展了实验验证，证实了EAU大鼠中miR-30b-5p水平与Notch通路活化以及Th细胞分化之间的关系。LXD含药血清可提高miR-30b-5p表达水平并阻断Notch1-DLL4轴的激活，降低Th1、Th17细胞的分化水平。同时，基因干扰实验发现，玻璃体腔注射Notch1-shRNA慢病毒可有效抑制Notch通路活化，降低Th1、Th17分化和IL-17等炎症因子水平以及Th1/Th2、Th17/Treg比例，恢复机体免疫稳态，进一步证实了LXD经miR-30b-5p抑制Notch通路活化治疗葡萄膜炎的作用机制。本项目通过研究Notch通路活化与葡萄膜炎发生之间的作用关系，从整体观和平衡观阐释了LXD经miR-30b-5p抑制Notch通路活化恢复Th17/Treg平衡、治疗葡萄膜炎的作用机制，建立了基于miRNA为作用靶点的有效新型治疗方法，为采用中医药治疗葡萄膜炎提供了重要的理论基础和研究思路。