台灣紫芝多醣體免疫調節機轉與功能評估

Abstract

靈芝具有多種的藥理功能，在亞洲已有相當久遠的藥用歷史，台灣紫芝是台灣原生種靈芝，其藥用相關研究卻相當少，因此本研究以液態深層醱酵培養法生產台灣紫芝胞外多醣體，探討台灣紫芝多醣體之免疫調節功能、作用機制與功能性評估。 台灣紫芝醱酵液經酒精沉澱和膠體過濾層析純化可分離得PS-F1、PS-F2和PS-F3三個主要分劃，其各分劃佔比分別為16.24 ± 2.19 %、55.81 ± 2.97 %和27.95 ± 2.11 %。PS-F2多醣體分子量約14 KDa，主要單糖組成為mannose、galactose、glucose及fucose，分別佔44.91 %、38.64 %、8.26 %及8.02 %，而醣基鍵主要以6-Gal和t-Man鍵結為主。 PS-F2能夠刺激小鼠巨噬細胞RAW 264.7產生TNF-α和NO，並增強巨噬細胞的吞噬能力，並能刺激巨噬細胞增生。以PS-F2刺激小鼠腹腔能誘發急性發炎反應，吸引嗜中性球和單核球來到腹腔。另外PS-F2亦可刺激小鼠樹突細胞表面成熟標誌CD40、CD80、CD86及MHC II的表現增加，顯示PS-F2具有免疫調節之活性，能活化先天性免疫反應。 PS-F2作用的受體研究方面，PS-F2刺激巨噬細胞產生TNF-α受到anti-Dectin-1抗體、anti-CR3抗體和laminarin顯著性抑制，TLR4受體缺陷小鼠BMDM也降低了PS-F2刺激BMDM產生TNF-α的活性。以上結果顯示PS-F2能經由Dectin-1、CR3及TLR4受體刺激巨噬細胞產生TNF-α。 在PS-F2活化巨噬細胞作用機制部分， PS-F2刺激能使ERK、JNK和p38絲裂原活化蛋白激酶磷酸化、I-κB降解和NF-κB核移位反應。添加ERK、JNK、p38和NF-κB訊息傳遞途徑專一性抑制劑皆可顯著抑制PS-F2刺激RAW 264.7細胞產生TNF-α，確認PS-F2透過活化絲裂原活化蛋白激酶和NF-κB訊息傳遞途徑產生TNF-α。此外，以Syk抑制劑piceatannol處理後也會抑制PS-F2刺激RAW 264.7產生TNF-α、I-κB降解和ERK磷酸化，顯示PS-F2刺激RAW 264.7細胞會經由Dectin-1受體透過Syk活化下游NF-κB和ERK訊息傳遞途徑。 在功能性評估方面，C57BL/6小鼠以PS-F2每兩天管餵一次持續四週後，發現小鼠脾臟細胞之T淋巴球族群比例提高，血清中之IgM及IgG抗體濃度均有顯著增加，顯示PS-F2會活化C57BL/6小鼠體內非特異性免疫反應。 在抗腫瘤功能評估上，發現腹腔注射PS-F2可有效抑制Sarcoma 180、B16黑色素瘤細胞和C26腫瘤細胞之生長，抑制率分別達67.5 % 、23.7 %及43.3 %；以管餵方式給予小鼠PS-F2亦有抗腫瘤之效果。腹腔注射PS-F2使接種腫瘤小鼠脾臟細胞之T淋巴球族群比例顯著性提高，其中CD4與CD8之細胞比例均有增加，特別是CD4細胞；而表現CD49b的NK細胞比例及其胞殺活性有些微增加；而在血清中也發現I gM抗體濃度有顯著增加。在C26腫瘤細胞模式中，腹腔注射PS-F2小鼠CD4與CD8 T細胞活化標誌表現CD44high CD62Llow，顯示T細胞有顯著性活化；PS-F2在SCID小鼠模式不具有抗腫瘤活性，以及adoptive transfer試驗也都近一步確認CD4 T細胞和B細胞為主要影響抗腫瘤的免疫細胞。綜合以上結果顯示PS-F2能活化先天性免疫相關細胞及後天性免疫反應，透過活化細胞介導免疫反應和體液免疫反應使腫瘤生長受到抑制，證實PS-F2具有抗腫瘤活性。 本研究確認以液態深層醱酵培養台灣紫芝生產之胞外多醣體PS-F2具有免疫調節活性及抗腫瘤功能，確立台灣紫芝多醣體於機能性食品之用途與應用。

The fungus of Ganoderma, called ‘Reishi’, has been used as traditional medicine in Asia and has been shown to exhibit various pharmacological functions. In this study, we investigated the immunomodulating activities and antitumor functions of polysaccharides produced from the submerged culture of Ganoderma formosanum, a native species isolated in Taiwan. The crude polysaccharides could be separated into three main fractions according to their sizes after alcohol precipitation and gel filtration. These three fractions were designated as PS-F1, PS-F2 and PS-F3, and the yields were 21.9% ± 2.19 %, 55.81 ± 2.97 %, and 27.95 ± 2.11 %, respectively. The molecular weight of PS-F2 was about 14 KDa. The monosaccharide composition analyse by HPLC-PDA method demonstrated that PS-F2 was composed of mannose, galactose, glucose and fucose with the mole percentage of 44.91:38.64:8.26:8.02. The mainly glycosyl linkages were 6-Gal and t-Man. We found that PS-F2 could stimulate RAW 264.7 murine macrophage cells to produce TNF-α and nitric oxide, and to enhance the phagocytic activity and proliferation of macrophages. In vivo, PS-F2 challenge in mice triggered an inflammatory response and led to the recruitment of neutrophils and monocytes. In addition, treatment of bone marrow-derived dendritic cells with PS-F2 resulted in the enhanced cell-surface expression of CD40, CD80, CD86 and MHC II, suggesting that PS-F2 was able to induce the maturation of immature dendritic cells and activation of innate immune response. Studies on the immune receptors for PS-F2 stimulated, PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of anti-Dectin-1 Ab and anti-CR3 Ab blocking antibody or laminarin. Moreover, PS-F2 induced TNF-α production by BMDM from C3H/HeN mice, but not C3H/HeJ mice that have a mutated TLR4 molecule, suggesting that TLR4 was also involved in PS-F2-mediated macrophage activation. In conclusion, we demonstrated that PS-F2 induced TNF-α production through the Dectin-1, CR3, and TLR4 membrane receptors in murine macrophages. About signaling mechanisms of cytokine production upon PS-F2 stimulation, we found that PS-F2 stimulated the phosphorylation of MAP kinases (ERK, JNK and p38), degradation of I-κB and the nuclear translocation of NF-κB. TNF-α production was decreased in the presence of specific inhibitors of MAP kinases and NF-κB in RAW 264.7 cells, suggesting that MAP kinases and NF-κB pathways play a crucial role in activating TNF-α expression upon PS-F2 stimulation. In addition, treatment of macrophages with the piceatannol also inhibited TNF-α production, I-κB degradation and ERK phosphorylation, indicating that Syk functioned upstream of other signaling events. These results may have important implications for our understanding on the molecular mechanisms in immunomodulating activities of PS-F2. We further investigated the immunomodulating functions and antitumor activities of PS-F2 in vivo. We examined the effect of oral administration on the immune fnctions in C57BL/6 mice with PS-F2, 50 mg/kg PS-F2 once every two days for 4 weeks. PS-F2 administration resulted in increased in the splenic CD3 T lymphocyte populations and the IgM and IgG levels in serum. It demonstrated that oral administration of PS-F2 activated non-specific immune response in C57BL/6 mice. Furthermore, we examined the effect of PS-F2 administration on tumor growth in an allogeneic and two syngeneic tumor models. Intraperitoneal administration of PS-F2 markedly inhibited the growth of sarcoma 180, C26, and B16 melanoma in mice and the inhibition rates were 67.5 %, 23.7 %, and 43.3 %, respectively. PS-F2 administration resulted in increases in the splenic CD3, CD4, and CD8 T lymphocyte populations, as well as the serum IgM level. Although PS-F2 administration did not affect the frequency of NK population in the spleen, we observed a slight increase in cytotoxic activity in PS-F2-treated mice. In C26-bearing mice, PS-F2 induced a CD44high CD62Llow phenotype on CD4 and CD8 T cells isolated from spleen of treated mice, suggesting the splenic T cells activated in PS-F2-trated mice. We further used B16 tumor xenografted SCID mice model and adpotive transfer to find out whether transfer of immune cell from PS-F2-treated mice might be sufficient to prevent tumor development. We found that adoptive fransfer CD4 T cells and serum antibodies into B16 tumor bearing mice significantly inhibited B16 progress in recipient mice. Overall, our results using different tumor cell lines and different mouse strains indicated that PS-F2 administration could protect mice from tumor challenge. PS-F2 administration may exert the anti-tumor effect via modulating the functions of both T and B cells in mice and enhancing cell-mediated immunity and humoral inmunity. The studies demonstrated that PS-F2 exhibited immunomodulating activities and antitumor response form the submerged culture of G. formosanum. Therefore, the polysaccharides PS-F2 from G. formosanum have the potential to be used as an immunomodulating agent in functional food for tumor therapy.