松杉靈芝對過敏免疫反應之調節研究

The Regulatory Effects of Ganoderma tsugae on Allergic Immune Responses

陳妙齡(Miaw-Ling Chen)

指導教授 ： 林璧鳳

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.01775

摘要

本研究首先為建立具有調節過敏免疫反應之食材的研究篩選模式，並評估其功效及可能的作用機轉。已知多數菇菌類食材對於免疫反應具有活化及調節之作用，故本實驗首先採用巴西洋菇、茯苓、樟芝與松杉靈芝等菇菌類為測試樣品，先以RBL-2H3肥大細胞株釋放組織胺能力、初代T細胞或TCR基因轉殖鼠分離出的T輔助細胞 (簡稱OVA特異性T細胞)分泌細胞激素能力，評估調節免疫功效。結果顯示，巴西洋菇、茯苓與松杉靈芝對組織胺的釋放無顯著影響，樟芝則促進組織胺釋放。此外，菇菌樣品均可促進T輔助細胞生成IL-2，且會抑制引發過敏疾病之Th2細胞激素的分泌。再以巴西洋菇、茯苓與松杉靈芝樣品餵食OVA致敏小鼠，結果顯示，三種菇菌樣品均可以降低呼吸道嗜伊紅白血球的聚集、IL-5的分泌與總蛋白質的濃度，對於脾臟細胞IL-4與IL-5的分泌，亦有抑制作用。因此，評估食材對過敏免疫反應之調節作用，以未分化的OVA特異性T輔助細胞，加入待測食材培養，分析分化過程中Th1與Th2細胞激素分泌量，為較準確且省時的篩選方式。
接著以細胞培養的方式初步探討松杉靈芝有效減緩過敏反應之成分為何。先以RAW264.7巨噬細胞分泌TNFα、RBL-2H3 肥大細胞釋放組織胺、EL4 小鼠T 細胞株或OVA 特異性T 細胞分泌細胞激素、及含IL-4 promoter 質體轉染之T 細胞報導基因表現等，評估松杉靈芝多醣類與三萜類區分物對過敏免疫反應之調節。結果顯示，松杉靈芝
多醣與三萜類可以顯著的活化RAW264.7 分泌TNFα。多醣類對於肥大細胞釋放組織胺
無顯著影響，但三萜類可以顯著抑制組織胺的釋放。松杉靈芝在T 細胞分化過程中，多
醣類對Th1 細胞的活化能力高於三萜類，三萜類對引發過敏疾病之Th2 免疫反應的抑制
效果較佳。松杉靈芝多醣與三萜類均不影響IL-4 promoter 下游的報導基因表現
(luciferase activity)，但三萜類可以顯著的抑制EL4 細胞分泌IL-4 與IL-5。本實驗另採用
含NF-κB 接合位的質體，探究NF-κB 轉錄活性與IL-4 生成之相關性，結果顯示，松杉
靈芝三萜類可以顯著的抑制NF-κB 下游報導基因表現，若在PPARγ拮抗劑與三萜類區分
物共同培養下， IL-4 分泌受抑制的情形，則有回復的現象。由此得知，對於過敏性免疫
反應而言，三萜類是松杉靈芝中較有效抑制Th2 免疫反應之區分物。
為探討松杉靈芝三萜類對體內T 細胞調節的影響，以及是否能夠減緩氣喘性過敏免
疫反應。本實驗接著以OVA 進行腹腔注射與吸入性致敏，誘發過敏性氣喘小鼠研究模
式進行動物實驗，並以固醇類藥物prednisolone 為對照組。結果顯示，松杉靈芝三萜類
補充可以顯著減緩呼吸道過度反應 (AHR)，減少免疫細胞在肺沖洗液及呼吸道週邊組織的聚集、降低肺沖洗液中IL-5、eotaxin、IL-4 與IL-13 含量，以及減緩氣喘之局部性發
炎的作用，與固醇類藥物類似。同時三萜類對血清特異性IgG1 抗體生成，與脾臟細胞分
泌IL-4 及IL-5 也有抑制作用；但對血清IgG2a，及脾臟細胞生成Th1 細胞激素，則無顯
著影響。由以上結果得知，松杉靈芝三萜類可以顯著減緩氣喘小鼠呼吸道過度反應與發
炎反應，亦可以顯著的抑制Th2 免疫反應，但不影響Th1 免疫反應。
綜合本研究之結果得知，由RBL-2H3 肥大細胞、OVA 特異性T 細胞、EL4 T 細胞
株至動物實驗，松杉靈芝尤其是三萜類可有效的抑制組織胺釋放、呼吸道發炎介質生成、
減緩氣喘小鼠呼吸道過度反應、抑制Th2 細胞激素分泌，與OVA 特異性IgG1 生成，但
不影響Th1 免疫反應，顯示松杉靈芝三萜類可以主要抑制氣喘的過敏免疫反應。推論松
杉靈芝三萜類可透過PPARγ活化，抑制NF-κB 之轉錄活性與Th2 免疫反應，具有開發為
較具安全性的氣喘用藥之潛能。

關鍵字

氣喘 ； 過敏免疫反應 ； 松杉靈芝 ； 三

並列摘要

The purpose of this study was to establish both in vitro and in vivo systems to study and explore the possible functional foods for the allergic diseases. It has been well documented that fungi might exert immunomodulatory effect, and thus the Agaricus blazei, Poria cocos, Ganoderma tsugae and Antrodia camphorate were tested for allergic immunomodulatory effect, by using RBL-2H3 mast cell line, primary splenocyte form BALB/c; and OVA-specific CD4+ T cells from T cell receptor (TCR) transgenic DO11.10 mice (nanaïve OVA-specific T
cells). The results showed that ionomycin-stimulated histamine production form RBL-2H3
cells did not affect by Agaricus blazei, Poria cocos and Ganoderma tsugae, but increased by
Antrodia camphorate. OVA-stimulated IL-2 secretion form Th1 cells was increased, but IL-4
and IL-5 secretion from Th2 cells was decreased by all the fungi samples. Furthermore, fungi
treatment increased the IL-2 level and decreased Th2 cytokines production during naïve
OVA-specific T cell differentiation in vitro. To further investigate the effect of fungi on airway
inflammation, the allergen-sensitized mice challenged with aerosol allergen supplemented
with Agaricus blazei, Poria cocos or Ganoderma tsugae. The data showed that the percentage
of eosinophils, IL-5 and total protein content in bronchoalvelor lavage fluid (BALF), and IL-4,
IL-5 levels produced by splenocytes were significantly decreased in mice fed with Agaricus
blazei, Poria cocos and Ganoderma tsugae.
Furthermore, two major fractions of Ganoderma tsugae, triterpenoids (Gt-TRE) and
polysaccharides (Gt-PS) were tested for their contribution for regulation of histamine and
cytokines productions using several different cell models described above. The results showed
that ionomycin-stimulated histamine secretion from RBL-2H3 cell was significantly
suppressed by Gt-TRE, but not Gt-PS fraction. PMA/ionomycin-stimulated IL-4 secreted from
a murine T cell line EL4 was also significantly decreased by Gt-TRE only. To further
investigate whether Gt-PS and Gt-TRE affect CD4+ T cell polarization, naïve OVA-specific T
cells were cultured with Gt-PS or Gt-TRE. The results showed that Gt-PS enhanced Th1
cytokine IL-2 but suppressed IFN-γ significantly. Gt-TRE had no effect on Th1 cytokines but
dose-dependently suppressed Th2 cytokines more significantly than that of Gt-PS. To evaluate
the effects of Ganoderma tsugae on IL-4 production, EL4 cell was transfected a plasmid
containing reporter gene luciferase with IL-4 promoter. The results shown that luciferase activity did not affect by Gt-PS and Gt-TRE treatment, but IL-4 and IL-5 production were
decreased by Gt-TRE. In addition, both EL4 cell and RAW264.7 macrophage cell line
transfected a plasmid containing reporter gene luciferase with NF-κB binding sites promoter
were cultured with Gt-PS or Gt-TRE. The results showed that the luciferase activity was
significantly increased by Gt-PS but significantly decreased by Gt-TRE, suggesting that these
two fractions may exert different effect on NF-κB related cytokine expression.
An in vivo study was meant to investigate if triterpenoid extracts have anti-inflammatory
effects on airway responses and regulatory effects on Th2 responses. BALB/c mice sensitized
intra-peritoneally and challenged with OVA were treated with either TRE or prednisolone for 2
weeks. The effects of TRE on bronchial AHR, airway inflammation, serum antigen-specific
antibody levels, and cytokine secretions from splenocytes were evaluated. TRE treatment
significantly decreased AHR and reduced the total infiltrating leukocytes and eosinophils in
BALF when compared to the control group. Furthermore, TRE decreased the production of
inflammatory mediators, such as IL-4, IL-5, and eotaxin in BALF, as well as secretions from
splenocytes of OVA-sensitized mice. TRE treatment also significantly decreased OVA-specific
IgG1 production, but not IgG2a production. These data suggest that triterpenoids is the fraction
of Ganoderma tsugae that alleviate bronchoalveolar inflammation in allergen-induced
asthmatic animal model through the biological activity for suppression of Th2 responses.
Overall, these data suggest that triterpenoid-rich extracts of Ganoderma tsugae attenuated
histamine release, airway inflammation, airway hyperresponsiveness, Th2 cytokines secretion,
and serum OVA-specific IgG1 production, but not affect Th1 responses. The regulatory
mechanism may exert the NF-κB related Th2 cytokine expression through the PPARγ
activation. Triterpenoid-rich extracts of Ganoderma tsugae exert anti-inflammatory effects on
airway responses and attenuates Th2 responses without the overall immuno-suppression
effects in allergic murine models of asthma.

並列關鍵字

Asthma ； Allergic immune responses ； Ganoderma tsugae ； Triterpenoids

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