一种新型的抗cGVHD天然提取物- - 芒果甙调控Tregs、Bregs、免疫炎症反应的研究

It is essential to explore a novel anit-chronic graft-versus-hose disease (cGVHD) strategy without impairing graft-versus-leukemia (GVL) effect. We have identified phytochemical mangiferin activates Nrf2 antioxidant pathway and reduces oxidative stress injury in hematopoietic cells. Recent studies revealed that Nrf2 could become a novel target against GVHD. Activating Nrf2 antioxidant pathway resulted in increase of regularoty T cells (Tregs) and mitigation of GVHD in murine models. Regularoty B cells (Bregs) participates in autoimmunity and cGVHD. Immunosuppressive activity of Bregs depends largely on production of interleukin-10. Nrf2 activation resulted in alleviation of autoimmune disease and initiation of interleukin-10 transcription in animal models. Therefore Nrf2 may enhance immunosuppressive activity of Bregs. We also found that Tregs, Bregs and Nrf2 levels significantly decreased in cGVHD patients after allo-HSCT. Therefore natural Nrf2 activator mangiferin could prevent and treat cGVHD through regulating Tregs, Bregs and immuno-inflammatory responses. We also proved that mangiferin not only has anti-leukemia activity, but also does not reduce chemosensitivity of leukemia cells. Thus mangiferin may not impair GVL. The project is proposed to study effects of natural Nrf2 activator mangiferin on cGVHD and GVL, and explore regulative mechanisms of mangiferin on Tregs, Bregs and immuno-inflammatory responses in vitro and in vovo. The project will lay the foundation to clarify new pathogenesis of cGVHD and seek novel anti-cGVHD strategy without imparing GVL. It will contribute to improving long-tern survival and life quality of patients post allo-HSCT.

探索防治cGVHD而不损伤GVL的新策略是亟待解决的重要难题。我们发现天然提取物芒果甙可在造血细胞中激活Nrf2抗氧化通路、减轻氧化应激损伤。Nrf2是新发现的GVHD防治靶点，激活Nrf2可提高小鼠Tregs活性、减轻GVHD。Bregs参与自身免疫及cGVHD发病，其活性与分泌IL-10密切相关。激活Nrf2可抑制自身免疫、启动IL-10表达，激活Nrf2可能提高Bregs活性。预实验显示cGVHD患者Tregs、Bregs、Nrf2降低。因此芒果甙很可能通过激活Nrf2调控Tregs、Bregs、免疫炎症反应，防治cGVHD。我们也证实了芒果甙有抗白血病活性、不影响白血病细胞化疗敏感性，提示芒果甙可能不影响GVL。本项目拟在体内外模型中研究天然Nrf2激活剂芒果甙对cGVHD和GVL的影响、及其调控Tregs、Bregs、免疫炎症反应的新机制，探索不损伤GVL的cGVHD防治新策略。

Nrf2是新发现的GVHD 防治靶点，但其是否调控Bregs、减轻cGVHD尚不明确。芒果甙（mangiferin,MA）是天然Nrf2 激活剂，MA能否通过激活 Nrf2防治 cGVHD，以及其调控Bregs、免疫炎症反应的机制亦不明确。本项目在临床患者、体外实验、动物模型不同层次对Nrf2、Bregs参与cGVHD发病机制、芒果甙影响cGVHD及作用机制进行了研究。在allo-HSCT的患者中，我们发现cGVHD患者Nrf2抗氧化通路明显下调，Tregs、Bregs水平均明显降低，外周血单个核细胞（PMSCs）中氧化应激水平明显增加。在动物体内实验研究中，我们成功建立了半相合移植Scl-cGVHD小鼠模型，发现与空白对照组（control）、同基因移植组（SynBMT）相比，异基因移植组（AlloBMT）小鼠Nrf2抗氧化通路明显下调，Bregs水平明显降低，IL-10水平明显下降。相比于AlloBMT组，MA给药组（即AlloBMT+MA）小鼠Nrf2抗氧化通路上调，Bregs水平升高，cGVHD严重程度及纤维化程度均减轻。在细胞体外实验中，我们发现MA可抑制小鼠脾脏单个核细胞（MNCs）凋亡，上调MNCs中Bregs水平,通过激活JAK2/STAT3、ERK、TLRs/MyD88信号通路，促进MNCs分泌IL-10,抑制促炎因子IL-2、INF-γ表达；不影响MNCs中Tregs水平及血浆中TGF-β的水平。因此，我们的结果提示cGVHD患者Bregs活性降低、Nrf2信号通路活性下调；在cGVHD小鼠体内，MA可激活Nrf2信号通路，提高Bregs水平及活性、并显著抑制靶器官免疫反应、减轻cGVHD；在体外研究中，MA能通过激活JAK2/STAT3、ERK、TLRs/MyD88信号通路，提高Bregs水平及活性。本研究所需的基因敲除小鼠Nrf2-/-C57BL/6、 Nrf2+/-Balb/c已完成制备，Nrf2-/-Balb/c将于近期完成制备，我们随后将采用Nrf2基因敲除小鼠确认Nrf2参与cGVHD发病机制、以及MA激活Nrf2通路、防治cGVHD的作用机制。我们的研究将有助于揭示芒果甙调控Bregs的新机制，探索有效、安全、新颖的cGVHD防治手段，为改善allo-HSCT受者长期疗效和生活质量提供新策略。