黃耆發酵產物對腸黏膜免疫調節作用之研究

Abstract

腸道黏膜佔有人體 70 % 的淋巴組織，是身體最頻繁與外界抗原接觸的場所，因此調節腸黏膜的免疫反應與耐受性，是促進全身性免疫平衡的重要標的。在衛生福利部認可的藥食同源中草藥中，黃耆（Radix Astragali）在免疫調節方面擁有豐碩的研究成果。此外，利用微生物進行中草藥的加工炮製，可望透過微生物對其成分進行生物轉換，提升生理活性。本實驗利用Bifidobacterium infantis （BCRC14602）、B. adolescentis（BCRC14606）、B. bifidum（BCRC14615）、B. longum（BCRC14634）及B. animalis subsp. Lactis（BB-12）進行黃耆的發酵，得到黃耆發酵產物（Fermented Radix Astragali, FRA），並與未發酵之黃耆（Non-fermented Radix Astragali, NRA）進行比較，探討是否能透過發酵的方式，提升黃耆的腸道黏膜免疫調節活性。實驗結果發現，在非特異性免疫方面，經BCRC 14615、14634與BB-12發酵的黃耆與NFR相比，可顯著促進RAW 264.7的NF-κB轉錄活性，且FRA對LPS誘導之NO生成具有更佳的抑制作用。接著以分化的Caco-2細胞模擬類小腸上皮細胞，發現在給予BCRC 14606與BB-12發酵的黃耆後，可在不影響細胞生長的前提下，顯著提升跨上皮細胞電阻（TEER），促進Caco-2細胞間的緊密連結，以強化腸道上皮的屏障功能。另一方面，以NRA及BCRC 14606、BB-12發酵黃耆處理THP-1誘導的未成熟樹突細胞，可抑制其表面MHC II與共同刺激分子（CD40、CD80、CD86）的表現，限制樹突細胞的成熟及活化適應性免疫反應的能力。在Caco-2與THP-1誘導的未成熟樹突細胞所建構的共培養模式中，極化上皮細胞的存在使得樹突細胞具有較高的耐受性，讓共培養系統更能模擬實際腸黏膜免疫環境。在共培養系統下，NRA與BCRC 14606、BB-12發酵黃耆可誘導正常生理狀態下的樹突細胞產生免疫耐受性，並抑制LPS活化的MHC II、共同刺激分子及促發炎細胞激素（IL-1β與IL-6）的表達，使樹突細胞保持在未成熟狀態，藉此阻止適應性免疫反應的發生，恢復腸黏膜的免疫平衡狀態。最後，透過NRA與FRA中的活性物質分析，推測Bifidobacterium spp.可對黃耆中極性較高的成分進行生物轉換作用，提升極性較低之成分（例如黃耆皂苷AS I – IV）的含量，因而提升黃耆的腸黏膜免疫調節活性。綜合以上實驗結果，本實驗所製備出的黃耆發酵產物有作為腸黏膜免疫調節膳食補充劑的潛力。未來可針對黃耆發酵前後的活性成分變化作進一步的分析，以釐清黃耆發酵產物中具有腸黏膜免疫調節活性的關鍵成分。

Gut-associated lymphoid tissue (GALT) represents approximately 70 % of the entire immune system, and it is the main route of contact with the external antigens. Therefore, regulation of immune response and tolerance of gut mucosal immune system is essential for promoting systemic immune balance. Radix Astragali is an important traditional Chinese medicine widely used in regulating immune system, and it is also approved by the Ministry of Health and Welfare to be used as a food ingredient due to its safety. Since fermentation is often used for processing Chinese herbal medicine to enhance its bioactivity. In this study, we fermented Radix Astragali by using Bifidobacterium infantis (BCRC14602), B. adolescentis (BCRC14606), B. bifidum (BCRC14615), B. longum (BCRC14634) and B. animalis subsp. Lactis (BB-12) to get fermented Radix Astragali (FRA) in comparison with non-fermented Radix Astragali (NRA). The purpose of this study was to investigate whether the gut mucosal immunomodulatory activity of Radix Astragali can be improved by fermentation. In respect of non-specific immunity, the expression of NF-κB in RAW 264.7 was significantly enhanced by BCRC 14615, 14634 and BB-12 FRA compared with NRA, and FRA had a better inhibitory effect on LPS-induced NO production. Then we used differentiated Caco-2 cells to mimic small intestinal epithelial cells. Results showed that BCRC 14606 and BB-12 FRA could significantly increase transepithelial electrical resistance (TEER) without influening cell growth. It appeared that BCRC 14606 and BB-12 FRA can promote tight junction between Caco-2 cells thus enhance the intestinal barrier function. On the other hand, we found that NRA, BCRC 14606 FRA and BB-12 FRA could inhibit the expression of MHC II and co-stimulatory molecules (CD40, CD80, CD86) on THP-1 induced immature dendritic cells, which in term limit the maturation of dendritic cells and prevent the activation of adaptive immune responses. We further constructed an in vitro co-culture model using differentiated Caco-2 cells and THP-1 induced immature dendritic cells. We found that the dendritic cells in the environment of polarized epithelial cells had a higher tolerance, thus the co-culture model is more similar to human gut mucosal immune environment. In this co-culture model, NRA, BCRC 14606 FRA and BB-12 FRA could induce dendritic cells to yield immune tolerance in normal physiological state and inhibit the expression of MHC II, co-stimulatory molecules and pro-inflammatory cytokines (IL-1β and IL-6), which keeps dendritic cells in immature state and prevents the activation of adaptive immune responses. As a result, the fermentation samples we used can restore the immune homeostasis of gut mucosa. Finally, by the analyses of the active compontents of NRA and FRA, we found that the Bifidobacterium spp. could transform the high polarity compounds in Radix Astragali and increase the content of low polarity compounds such as astragalus saponins (asrtragaloside I-IV), and thus enhanced the gut mucosal immunoregulatory activity of Radix Astragali. In conclusion, our Radix Astragali fermentation products may have the potential to be a dietary supplement for gut mucosal immunoregulation. In the future, we should conduct more analyses and compared the changes in the active ingredients before and after fermentation, thus we may be able to identify the key compounds in fermented Radix Astragali, which have gut mucosal immunomodulatory activity.