由金針菇（Flammulina velutipes）子實體經由陽離子交換樹脂CM-52純化後所得到的蛋白：FIP-fve（Fungal immunomodulatory protein），是具有免疫調節功能的蛋白質。由之前的實驗得知：FIP-fve可以使正常人類週邊血液的淋巴球（human peripheral blood mononuclear cells；HPBMCs）以及Balb/c老鼠的脾臟細胞誘發IFN-γ的產生，並且對細胞有proliferation的能力。而由FIP-fve所誘發的IFN-γ是屬於Th1 細胞所誘發的細胞激素，可以抑制Th2 細胞所產生的細胞激素，例如：IL-4、IL-5、IL-6、IL-10及IL-13，因此在免疫系統上具有降低由Th2 cytokine調控IgE、mast cell等所引起的過敏反應，包括慢性氣喘、呼吸道發炎、紅斑性狼瘡等等。由以往的文獻得知IFN-γ可以經由p38 mitogen activated protein kinase pathway所調控，並且會進一步誘發下游基因iNOS的生合成，因此本篇研究以Western blot方式證明：以FIP-fve（100μg/ml）刺激HPBMCs 在10分鐘時可以使p38及ERK（extracellular signal-regulated protein kinase）磷酸化最明顯，但不會活化JNK（the c-Jun NH2-terminal kinase），並且預先處理p38 MAP kinase的抑制劑：SB203580（10μM）可以抑制p38的磷酸化。同時用ELISA方式證明以FIP-fve（100μg/ml）處理HPBMCs 48小時會明顯產生IFN-γ，而且誘導也會受到SB203580、LY294002（PI3-kinase的抑制劑）以及U0126（MEK1/2的抑制劑）所抑制。另外以Griess reagent assay方式測NO的分泌量，發現FIP-fve並不會調控NO的產生。除了由金針菇子實體純化的FIP-fve蛋白有誘發IFN-γ的能力以外，重組的FIP-fve融合蛋白（recombinant FIP-fve）也有FIP-fve將近一半的活性。
本實驗說明了FIP-fve調控IFN-γ的機制是可以透過p38 MAP kinase pathway及ERK的活化途徑，而不是經由活化JNK途徑。雖然FIP-fve不會調控NO的產生，但所誘發高量的IFN-γ卻可以藉由抑制TH2細胞所產生的細胞激素的生成，並期望將來可以應用在基因免疫的預防治療上。
A fungal immunomodulatory protein（FIP-fve）has been isolated and purified from the edible golden needle mushroom（Flammulina velutipes）. It has recently been reported FIP-fve stimulates the proliferation of human peripheral blood lymphocytes and Balb/c mice spleen cells and induces the expression of IFN-γ. In contrast, IFN-γ, secreted by T cells of the Th1 lineage, represses the development of Th2 cells and inhibits the class-switching event mediated by IL-4, IL-5, IL-6, IL-10, and IL-13. IFN-γ has inhibitory action on IgE and mast cell production by IL-4 and enhancement of Th1 immune responses by appropriate vaccines may be therapeautically beneficial against asthma, SLE, and allergic diseases. Recent studies on molecular mechanisms regulating IFN-γexpression have shown the importance of p38 mitogen-activated protein kinase（MAPK）pathway and induces iNOS expression. In the present study we demonstrate that FIP-fve activates p38 MAPK and ERK（extracellular signal-regulated protein kinase）, but not JNK（the c-Jun NH2-terminal kinase）. Our results indicate that p38 MAPK and ERK were activated by FIP-fve（100μg/ml）stimulated peaked at 10min, and 10μM SB203580（p38 MAP kinase inhibitor） decrease FIP-fve-induced p38 MAPK activity by Western blot. Cell culture supernatants were tested for IFN-γproduction by ELISA were significantly inhibited by both SB203580, LY294002（PI3-kinase inhibitor）, and U0126（MEK1/2 inhibitor）for 48 hours. The supernatants were tested for low levels of NO production by Griess reagent assay. The recombinant FIP-fve has induced IFN-γactivity as FIP-fve. Moreover, FIP-fve activates p38 MAPK and ERK, but not JNK. FIP-fve dose not induce NO expression. The requirement for p38 MAPK and ERK in FIP-fve function suggests that this pathway may be an important in vivo target for the anti-inflammatory actions and gene therapy of IFN-γ.
