雲芝蛋白 YZ-1 之純化與其促進調節性 B 細胞分化之研究 – 作用機制與抗發炎性腸病之應用

Abstract

雲芝 (Coriolus versicolor) 為本草綱目紀載之藥用真菌。雲芝萃取物具抗腫瘤與免疫調節功效，並於日本與中國大陸被核准為輔助抗癌藥物，雲芝萃取物中主要活性成分為雲芝醣肽 (polysaccharopeptide)，其本質上屬混合物，組成中 28±10% 為蛋白質。本研究期由雲芝乾燥子實體中純化具免疫調節活性之蛋白質，並探討此純化物之功效與作用機制。以離子交換管柱層析法由雲芝蛋白萃取液中分離出分子量約 14 kDa 之蛋白質 YZ-1，經 PAS 染色證實 YZ-1 為不含醣之純蛋白質。體外試驗證實 YZ-1 不需其他免疫細胞輔助即能直接刺激小鼠脾臟 B 細胞增生活化，並分化為 IgM 分泌型漿細胞 (plasma cell) 與 IL-10 分泌型調節性 B 細胞 (Breg)。以流式細胞儀分析IL-10 分泌型細胞之性狀，證實 YZ-1 具專一性地刺激 CD1d+CD23- marginal zone 與 CD1d+CD23+ transitional 2 marginal zone precursor B 細胞分化為 Breg 細胞。體外免疫抑制試驗證實受 YZ-1 活化之 B 細胞 (BYZ-1) 可抑制 LPS 誘發之巨噬細胞發炎反應。經動物試驗證實，將 BYZ-1 移植 (adoptive transfer) 入小鼠體內可顯著抑制葡聚糖硫酸鈉鹽引發之結腸炎，減緩小鼠體重下降、降低病情指數與結腸損害。以抗體抑制、基因缺陷小鼠與螢光標定試驗證實 YZ-1 透過 TLR4 及 TLR2 與 B 細胞作用，活化 TLR 下游信息路徑，啟動轉錄因子 NF-κB 與 B 細胞分化基因 Prdm1 表現，進而促使 B 細胞分化為漿細胞與 Breg 細胞。本研究以不含醣之雲芝純蛋白質 YZ-1 為標的，發現其具有作為細胞免疫療法 (cell-base immunotherapy) 之潛力，並闡明其可能作用機制為透過 TLR 信息路徑促進 B 細胞分化，此成果對雲芝活性成分與藥理作用等研究提供新的見解。

Coriolus versicolor is a medicinal fungus appreciated as a folk remedy worldwide. The polysaccharopeptide extracts of C. versicolor were considered the main anti-tumor and immune-potentiating agents; however, the polysaccharopeptides were mixtures of multiple constituents containing 28±10% proteins. The aim of this study was to isolate an immunomodulatory protein from C. versicolor and to assess its function. Through ion-exchange chromatography, a 14 kDa non-glycosylated protein was purified and named as YZ-1. The stimulating effect of YZ-1 on MACS-purified murine splenic CD19+ B cells was evaluated by flow cytometry, qPCR, and ELISA. YZ-1 activated B cell proliferation and differentiated B cells into IgM-secreting CD138+ plasma cells as well as IL-10-secreting CD1d+ regulatory B cells (Bregs). Furthermore, YZ-1-induced B cells (BYZ-1) inhibited TNF-α and IL-1β production by LPS-activated mice peritoneal macrophages in vitro. In a DSS-induced colitis murine model, adoptive transfer of BYZ-1 significantly reduced the disease activity, alleviated intestinal injuries, and suppressed colonic inflammatory cytokine expression. Blockage of toll like receptor (TLR) 4 and TLR2 by neutralizing antibodies limited YZ-1-induced B cell activation. In addition, the binding between YZ-1 and B cells was impaired in TLR4-/- and TLR2-/- mice. The expression of MYD88, TIRAP, TRAF6, NF-κB, and Prdm1 were induced in BYZ-1, suggesting that these cells were activated through a TLR-mediated signaling pathway. In conclusion, YZ-1 was a new protein purified from C. versicolor that could activate plasmacytic and Breg differentiation via a TLR4/TLR2-dependent mechanism. Moreover, BYZ-1 had immune-suppressive potential and showed efficacies in a cell-base immunotherapy against autoimmune disease. These findings provided insights to the study of C. versicolor regarding its active ingredient and immune-regulatory function.