靈芝多醣 PSG 與靈芝蛋白 LZ8 活化小鼠腹腔巨噬細胞及促進第一型 T 輔助細胞免疫反應之功效

Effects of Polysaccharide and Immunomodulatory Protein, Ling Zhi-8 from Ganoderma lucidum on Activation of Mouse Peritoneal Macrophages and Promotion of Th1 Immune Response

陳肖芹(Hsiao-Chin Chen)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10228

摘要

靈芝 (Ganoderma lucidum (Fr.) Karst) 所含的蛋白 LZ8 和多醣
(polysaccharide, PSG)，皆為能夠調節人體免疫系統的活性成分，且兩者活化的免疫機制不同，但是對於兩者作用之差異、何者較能夠作為靈芝的活性指標，仍不是非常清楚。本研究分為體外和體內試驗兩部分，首先在體外試驗的部分，探討 LZ8 對小鼠腹腔巨噬細胞、脾臟細胞的結合是否與 TLR4 相關，再比較靈芝蛋白 LZ8、靈芝多醣 PSG及去蛋白靈芝多醣 dePSG 對於小鼠腹腔巨噬細胞及脾臟細胞的活化作用，檢視三種靈芝樣本活化免疫途徑之差異。體內試驗的部分，透過餵食 LZ8, PSG 及 dePSG 的方式，檢測三種靈芝樣本對 OVA 免疫或未免疫小鼠體內 Th1/Th2 趨化之影響。
體外試驗的結果發現，經 FITC 標定的 LZ8 仍可與 TLR4 缺失小鼠 (C57BL/10ScN) 之腹腔巨噬細胞的結合，LZ8 可活化 TLR4 缺失小鼠之腹腔巨噬細胞產生 IL-12，顯示兩者的結合與 TLR4 無關，亦不會透過 TLR4 來活化巨噬細胞；對於小鼠脾臟細胞的結合也具有相同表現。而 LZ8, PSG 及 dePSG 皆能夠促進巨噬細胞 CD86 及 MHC calss II 表現量增加，其中又以 LZ8 效果最顯著，PSG次之，dePSG 最低。另一方面，經過 LZ8 活化後的巨噬細胞能夠顯著增加正常小鼠或 TLR4 缺失小鼠之 CD4+ 及 CD8+ T 細胞 IFN-γ 和 IL-2 的產生；經過 PSG 活化後的巨噬細胞能夠些微刺激小鼠 CD4+ T 細胞產生 IFN-γ，而經過 dePSG 活化的巨噬細胞對小鼠 CD4+ 及 CD8+ T 細胞分泌 IFN-γ無顯著影響。此外，經過 PSG 活化的巨噬細胞亦能夠刺激正常小鼠或 TLR4 缺失小鼠 T 細胞產生 IL-2，但是經過 dePSG 活化的巨噬細胞對 TLR4 缺失小鼠 CD4+ 及 CD8+ T 細胞分泌 IL-2 則無顯著影響，推測此結果可能是由於 PSG 中含有 LZ8 的成分所致。進一步結果發現，LZ8 能夠直接刺激活化小鼠 CD4+ 及 CD8+ T 細胞，顯著提升其 IFN-γ 和 IL-2 的產生，同時也顯著提升了 T 細胞表面分子 CD25 和 CD44 的表現量，PSG 及 dePSG 皆無此效果，表示 LZ8 與 PSG, dePSG 活化免疫細胞作用不同，LZ8 對於 T 細胞活化較顯著。
體內試驗的結果發現，經過餵食 LZ8，能夠抑制小鼠血清中 IgG1 的含量與脾臟細胞 IL-5 的分泌、增加小鼠血清 IgG2a 的含量與脾臟細胞 IFN-γ 的分泌，然而餵食 dePSG 與 餵食 PBS 之小鼠無明顯差異，且餵食 LZ8 能夠提升 OVA 致敏小鼠體內 OVA 特異性 IgG2a含量，顯示 LZ8 能夠促使小鼠體內趨向第一型 T 輔助細胞免疫反應 (Th1 response)，而餵食 dePSG 則無法產生此效果，LZ8 的功效更勝於 dePSG。
綜合上述結果可知， LZ8 的免疫調節功效，尤其對於 T 淋巴細胞，更勝於 dePSG，同時 LZ8 是已知結構且較易定量之成份，因而較適合做為靈芝活性指標之來源。

關鍵字

靈芝蛋白 LZ8 ； 去蛋白靈芝多醣 dePSG ； 巨噬細胞活化 ； OVA 免疫 ； T 細胞分化

並列摘要

Two major bioactive ingredients of Ganoderma lucidium, a historic and well known medicinal fungus, were protein LZ8 and polysaccharide PSG. Both the LZ8 and PSG have been proven to be capable of modulating human immune responses through different molecular mechanisms. However, the difference between them and which one could be a better indicator of the activity of G. lucidum are still unclear. The aim of this study was to clarify the relevance between TLR4 and the binding of LZ8 with murine peritoneal macrophages or splenocytes in vitro, and to compare the difference in immunomodulatory activity among LZ8, PSG and deproteinized PSG (dePSG), and to determine a more appropriate candidate for bioactivity indicator of G. lucidium. The differences in the ability to activate macrophages and splenocytes among LZ8, PSG and dePSG were then determined in vitro. In the in vivo studies, BALB/c mice were orally administrated with LZ8 (1 mg/kG body weight), PSG (10 mg/kG body weight) and dePSG (10 mg/kG body weight) with or without OVA immunization for Th1/Th2 immune responses examination.
Based on the results of in vitro study, TLR4 was not required for the binding of LZ8 and macrophages or splenocytes, and LZ8 did not activate macrophages through TLR4. LZ8, PSG and dePSG all increased the expression of major histocompatibility complex (MHC) class II and CD86 molecules on peritoneal macrophages. Among them, LZ8 exhibited the strongest efficacy while dePSG was the least effective. LZ8-stimulated murine macrophages enhanced IFN-γ and IL-2 production by CD4+ and CD8+ T cells obtained from TLR4 deletion mice. However, PSG-stimulated murine macrophages could only slightly enhance IFN-gamma by CD4+ T cell but not IL-12 production, and dePSG-stimulated cells had no significant effect on cytokine production. The observation of which PSG (but not dePSG), activated macrophages could lead to an increase of IL-2 production by CD4+ and CD8+ T cells obtained from Balb/c or TLR4 deletion mice. If was suggested that the increased IL-2 production might be resulted from small amount of LZ8 in PSG. Furthermore, LZ8 could activate murine CD4+ and CD8+ T cells directly, significantly elevated the production of IFN-γ and IL-2 and enhanced the expression of surface markers CD25 and CD44, while PSG and dePSG had no effects. These data indicated that LZ8 showed more significant effects on the activation of T cell.
In the results of in vivo study, oral administration of LZ8 suppressed the level of serum IgG1 and T helper 2 (Th2)-associated cytokine secretion in BALB/c splenocytes, and enhanced serum IgG2a and Th1-associated cytokine secretion in BALB/c mouse spleen cell culture. However, none of these effects were found significantly in mice orally administrated with dePSG. Additionally, oral administration of LZ8 upon immunization mice with ovalbumin (OVA) also significantly increased OVA-specific IgG2a levels compared with those of PBS-treated mice, suggesting that LZ8 could suppress OVA-induced Th2 response to drive Th1 development. Oral administration of dePSG showed no effects as compared with those of PBS-treated mice, indicating LZ8 had more immune effects in vivo.
Taken together, these studies demonstrated that immunomodulatory effects of LZ8 were superior to dePSG, especially on T cells, making LZ8 a more preferable indicator for bioactivity of G. lucidium.

並列關鍵字

Ganoderma lucidum immunomodulatory protein LZ8 ； deproteinated polysaccharide of Ganoderma lucidum (dePSG) ； macrophage activation ； OVA immunization ； T cell development

參考文獻

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