四君子湯及其單方對小白鼠免疫功能之調節

The Regulatory Effect of Si-Jun-Zi-Tang and Its Four Ingredients on The Murine Immune Function

劉倩君(Liu Chien Chiung)

指導教授 ： 曾哲明 ； 李銘亮

國立臺灣師範大學/理學院/生物學系/博士(2002年)

摘要

四君子湯為東方國家使用千年以上的補氣中藥，由人蔘、白朮、茯苓、甘草四種中藥材組成。本實驗室於1996年證實四君子湯具有免疫調節功能，因此本研究目的在透過一系列實驗，更完整探討四君子湯及單方調節免疫反應的機轉。
實驗藥材採用四君子湯及其單方之50%酒精萃取物，第一階段實驗老鼠連續三天腹腔注射藥劑，結果顯示，四君子湯及其單方都可顯著促進脾臟細胞分泌IgG及IgA，但是四君子湯及白朮、茯苓並未促進IgM的分泌。第二階段以相同方式給藥，探討四種單方對於脾臟細胞製造Th-1型（IL-2、IFN-γ）和Th-2型細胞激素 (IL-10、IL-4)的影響，結果發現四種單方對Th-1及Th-2型細胞激素的製造，皆有促進作用，影響層面包括基因表現層次及蛋白質層次，佐以IgG亞型（IgG1, IgG2a, IgG2b）分泌量改變的數據，確認四種單方藉由IFN-γ(Th-1型)及IL-4 (Th-2 型)的分泌，影響B淋巴球的抗體類型轉換（class-switching）。第三階段主要探討人蔘途徑及時間長短對其免疫調節功能所造成的影響，實驗結果發現，經由口服途徑，除了對IgA仍保持促進作用外，IgG及IgM皆呈現抑制效應，推論經由口服途徑可以增加黏膜性免疫反應，且口服人蔘會促進Th-1型細胞激素製造，但是對IL-4製造卻無顯著影響，對IL-10則有促進作用，佐以IgG亞型（IgG1, IgG2a, IgG2b）分泌量改變的數據，確認人蔘藉由IFN-γ，使 B淋巴球由IgM製造者類型轉換成IgG2a製造者，同時抑制IgM轉型為IgG1及IgG2b。口服人蔘會促進NK細胞活性，增加細胞毒殺作用，整體效應應該趨向於細胞性免疫反應的調節，但短期口服人蔘卻抑制CD3+T淋巴球的數量，伴隨抑制了CD4+ 及CD8+細胞的量，造成的原因須待進一步探討。
口服時間長短也會影響免疫調節機制，長期服用後，失去對IgA分泌的促進作用，只有IL-10維持促進效果，推測IL-10在長期口服中扮演細胞激素及抗體分泌的負面協調角色。長期口服對NK細胞活性無影響，CD3+T細胞及CD8+T細胞的比例降低，但CD4+T細胞比例與控制組無顯著差異。最後探討長期口服人蔘對抗雞卵蛋白 (ovalbumin;OVA)專一性免疫反應的調節，老鼠餵食人蔘三十天中，第一天免疫注射OVA，第十四天追加注射，第三十天測定抗OVA抗體效價，發現長期口服人蔘能增進專一性抗OVA抗體反應。總結本研究之結果，可確定以50﹪乙醇萃取之人蔘萃取物，劑量如在0.4至4 g/kg/day之間，則短期口服人蔘萃取物，有利於黏膜免疫反應及細胞性免疫反應，尤其是與NK細胞有關的防禦機轉；而長期口服人蔘對整體免疫功能無幫助，不過透過某些未知的機制，能增進專一性抗體反應。

關鍵字

四君子湯 ； 免疫 ； 細胞激素 ； 抗體

並列摘要

Si-Jun-Zi-Tang is a general tonic medicine that has been used in Asian countries for more than a thousand years. It is composed of four major ingredients including Ginseng (Panax ginseng C.A Meyer), Bai-zhu (Atraclylodes macrocephala Koidz), Licorice (Glycyrrhiza uralensis Fisch) and Fu-ling (Poria cocos (Schw.) Wolf). In 1996, our research team had demonstrated that Si-Jun-Zi-Tang was a potential immunoregulatory medicine. Therefore, the research was taken through a series of carefully designed experiments to investigate closely into the mechanism by which the Si-Jun-Zi-Tang modulated the immune function. The reagents used in this study were prepared by boiling the Si-Jun-Zi-Tang and its four major ingredients separately in 50% ethanol. In the primary stage of the experiment, the mice were injected intraperitoneally (i.p.) with the extracts for three consecutive days. Results indicated that injecting the drug all significantly enhanced both IgG and IgA secretion by spleen cells, but IgM secretion was not augmented significantly by Si-Jun-Zi-Tang, Bai-zhu and Fu-ling. In the secondary stage, assayed for production of Th-1 type (IL-2, IFN-g) and Th-2 type (IL-4 and IL-10) cytokines by spleen cells cultured in vitro. Results indicated that treatment with drug all significantly enhanced both cytokine mRNA expression and cytokine secretion by spleen cells. This observation was further supported by showing that the IgG1, IgG2b (mediated by IL-4) and IgG2a (mediated by IFN-g) secretion by spleen cells were increased significantly after the drug treatments. The result also implied that antibodies production by the B-lymphocytes might undergo class-switch. In the third stage, the study was focused on the possible effects of the route and the duration of drug treatment on the Ginseng-mediated immunoregulation. Oral administration of Ginseng extract only enhanced the secretion of IgA, but showed a significantly suppressive effect on IgG and IgM secretion, suggesting that Ginseng might directly stimulate the mucosal-associated lymphoid tissue in gut and augmented IgA production though oral administration. Oral administration of Ginseng extract significantly enhanced Th-1 type cytokine production and IL-10 production. This observation was further supported by showing that the IgG2a secretion by spleen cells, which was induced by IFN-g, were increased significantly after the drug treatments; but IgG1 and IgG2b secretion, which was induced by IL-4, were suppressed. In addition, oral administration of Ginseng significantly increased the cytotoxic activity of natural kill cells (NK cells), suggesting that Ginseng can also induced an innate cell-mediated immune response. However, oral administration of Ginseng extract significantly reduced the percentage composition of CD3+ T-lymphocytes,CD4+8- and CD4-8+ subpopulations. The Ginseng-mediated immunoregulation was also affected by the duration of drug treatment. Long-term (30 days) oral treatment of Ginseng almost failed to modulate both immunoglobulin and cytokine productions, except that the production of IL-10 was significantly induced. Since, the major biological activity of IL-10 is to inhibit the synthesis of lymphokines and monokines. Therefore, long-term oral treatment of Ginseng extract might down-regulate the immune system by increasing IL-10 production. Finally, the effect of long-term oral treatment of Ginseng extract on the ovalbumin (OVA)-induced specific antibody response was studied. During the 30 days of treatment, the mice were primary immunized with OVA at day one and were boosted at day 14. The anti-OVA antibody titer of the mice treated with Ginseng was significantly higher than that of the control group. In conclusion, for the 50% ethanol extract of Ginseng, the dosage ranged from 0.4 g/kg/day to 4 g/kg/day, the short-term oral administration will up-regulate both MALT response and cell-mediated immune response, especially the cytotoxicity of NK cells. However, long-term oral administration will not be able to augment immune response but may have advantage in the antigen-specific antibody response.

並列關鍵字

Si-Jun-Zi-Tang ； immune ； cytokine ； antibody

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