苜蓿芽乙酸乙酯萃取物改善 MRL-lpr/lpr 自體免疫鼠病程發展之探討

Abstract

本研究係探討具有抗發炎能力與調節 T helper (Th)1 和 Th2 免疫反應，以及雌激素受器調節劑 (selective estrogen receptor modulator, SERM) 的植物食材樣品，對於自體免疫傾向鼠病情的影響。首先以 BALB/c 正常小鼠的初代腹腔細胞，篩選具有降低 lipopolysaccharide (LPS) 刺激下 interleukin(IL)-1β、IL-6 與 tumor necrosis factor (TNF)-α 等促發炎細胞激素分泌量之抗發炎潛力樣品，再以 LPS 誘發的急性發炎模式，檢測這些樣品 in vivo 是否降低促發炎細胞激素的分泌量，因而改善發炎反應。篩選結果顯示，經發酵三天的黑豆乙酸乙酯萃取物 (fermented black soybean methanol extracts, F3BME) 與苜蓿芽乙酸乙酯萃物 (alfalfa sprouts ethyl acetate extracts, ASEA) 明顯抑制促發炎細胞激素的分泌量，並且增加 LPS 致急性發炎小鼠的存活率。由於 18 週齡相較 9 週齡的 MRL-lpr/lpr 小鼠之脾臟細胞，在 Con A 刺激下 interferon (IFN)-γ 與 IL-10 的分泌量明顯增加，表示這些細胞激素與病程的發展有關，接續以 16~18 週齡 MRL-lpr/lpr 小鼠的初代細胞，檢測篩選樣品對於細胞激素的影響，樣品包括 F3BME、ASEA、以及山藥乙酸乙酯萃物 (Dioscorea alata L. ethyl acetate extracts, DAEA)。結果表示 50 mg/ml ASEA 或 40 mg/ml DEAE 顯著降低脾臟細胞在 Con A 刺激下 IFN-γ、IL-4 與 IL-10 的分泌量，40 mg/ml F3BME 則只有降低 IFN-γ 的分泌量。同時， ASEA 與 F3BME 可以抑制腹腔細胞在 LPS 刺激下促發炎細胞激素的分泌量， DEAE 則只降低 IL-6 的分泌量。在 CHO-K1 細胞的 estrogen receptor (ER) 轉錄活性分析中， 50 mg/ml ASEA 活化 ERβ 的能力高於活化 ERα 的能力，表示有 SERM 的能力；同時， ASEA 可以抑制雌激素促進 MCF-7 細胞增生的作用，具有拮抗雌激素之作用。綜合以上結果， ASEA 可能有助於自體免疫病情的改善，因此以 ASEA 介入 MRL-lpr/lpr 小鼠的飲食中，測試能否緩和小鼠的病程發展。實驗一將 12 週齡 MRL-lpr/lpr 小鼠依據體重、尿蛋白與血清抗雙股 DNA IgG 抗體分成五組。分別為控制組 (Control) 與苜宿芽粉組 (AS)、苜蓿芽乙酸乙酯萃物組 (ASEA, 25 mg/kg BW)、香豆素 coumestrol 組 (CUM, 0.075 mg/kg BW)、與 tamoxifen 組 (TAM, 0.375 mg/kg BW)， 各組管餵葵花油 (Control 與 AS 組) 或含有待測樣品的葵花油，每天管餵量為 50 ml ，每週共管餵 6 天，各組餵食的飼料皆為 AIN-76 配方，AS 組的飼料額外添加 5.5% 的苜蓿芽粉。結果顯示，ASEA 組與 TAM 組可以降低血清 anti-dsDNA IgG2a/IgG1 比率，抑制血清 IL-12p40 的含量，降低尿蛋白的發生率，而且提高小鼠的存活率。AS 組與 CUM 組並未影響 MRL-lpr/lpr 小鼠的病情發展。因此實驗二再次將 12 週齡 MRL-lpr/lpr 小鼠分成三組，分別是 Control組、 ASEA 組與 TAM 組，飼料、管餵劑量與時程與上述實驗相同。在管餵補充六週後，犧牲小鼠進行分析，結果顯示，ASEA 組與 TAM 組顯著降低血清 IL-12p40 與 IL-6 的生成，同時延後尿蛋白的發生，與實驗一結果一致。在免疫細胞分析方面，ASEA 組相較於 Control 組，脾臟細胞在 Con A 刺激下 IFN-γ、IL-4 與 IL-6 的分泌量顯著降低，腹腔細胞在 LPS 刺激下促發炎細胞激素的分泌量有略低的趨勢，而且脾臟細胞中的 CD3+CD69+ T 細胞族群明顯減少。腎臟切片圖顯示 ASEA 組的間質細胞的增生與絲球體腫大程度較 Control 組輕微。為了解 ASEA 中的生物活性分子，遂進行分離與純化，將 ASEA 以開放式矽膠管柱層析與製備式 HPLC 的分離，經過抗發炎與 SERM 活性鑑定，取得具抗發炎能力的 loliolide 2.5 mg 與 4-hydroxy-6-nonadecyl-2H-pyran-2-one 1.9 mg，以及具抗發炎和 SERM 活性的 liquiritigenin 2.1 mg 與isoliquiritigenin 5.2 mg。本研究中檢測抗發炎能力與 SERM 能力、以及抑制自體免疫傾向小鼠細胞分泌 IFN-γ 和 Th2 細胞激素的能力，篩選出苜蓿芽乙酸乙酯萃物 ASEA，在 in vivo 中明顯改善 MRL-lpr/lpr 鼠體內的發炎狀況，而有助於延緩病情的發展。自分離純化 ASEA 結果可知，loliolide、4-hydroxy-6-nonadecyl-2H-pyran-2-one、liquiritigenin、與 isoliquiritigenin 可能為延緩病情的主要化合物。 關鍵字: 自體免疫、發炎、MRL-lpr/lpr、苜蓿芽乙酸乙酯萃取物、促發炎細胞激素、雌激素受器調節劑

The purpose of this study was to investigate the effect of plant food components sample possessing the abilities of anti-inflammation, Th1/Th2 immune regulation, and selective estrogen receptor modulator (SERM) on the autoimmune-prone disease in MRL-lpr/lpr female mice. First, in primary cell culture test, potential plant food components which could inhibit LPS-stimulated pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α, secretion from peritoneal macrophages of BALB/c mice were screened out. These potential food components were subsequently tested (per oral) to reduce serum levels of pro-inflammatory cytokines and attenuate inflammatory conditions in a murine model of LPS-induced acute inflammation. The results showed fermented 3-day blacksoybean methanol extract (F3BME) and alfalfa sprouts ethyl acetate extract (ASEA) could inhibit pro-inflammatioy cytokines secretion in vitro and in vivo. Due to significantly higher IFN-γ and IL-10 secretion from Con A-stimulated splenocytes at age 18 weeks compared to those at age of 9 weeks in MRL-lpr/lpr mice, we cultured primary cells of MRL-lpr/lpr mice at age of 16~18 weeks and tested anti-inflammatory and Th1/Th2 immune-regulatiory abilities of potential food components. The results showed 50 mg/ml ASEA and 40 mg/ml DAEA could significantly reduce IFN-γ, IL-4, and IL-10 secretions from Con A-stimulated splenocytes of MRL-lpr/lpr mice, and 40 mg/ml F3BME just inhibited IFN-γ secretion from these cells. ASEA and F3BME could evidently inhibit LPS-stimulated pro-inflammatory cytokine secretions peritoneal exudates cells (PEC) of MRL-lpr/lpr mice, and DAEA only inhibited IL-6 secretion from these cells. Moreover, estrogen receptor (ER)β rather than ERα is activated by 50 mg/ml ASEA in CHO-K1 ER transactivation assay. This revealed ASEA likely be a SERM. ASEA also inhibited MCF-7 cell proliferation under 1 nM 17β-estradiol treatment. These data displayed ASEA have the potential to ameliorate inflammatory status and estrogen-related disease course in MRL-lpr/lpr mice. twelve-week-old MRL-lpr/lpr mice depend on their body weight, urine protein levels and sera levels of anti-dsDNA IgG were divided into five groups, Control group (50 ml sunflower oil (SO)/day), AS group (50 ml SO/day), ASEA group (25 mg/kg BW), CUM group (coumestrol, 0.075 mg/kg BW), TAM group (tamoxifen citrate, 0.375 mg/kg BW). The mice in each group were tube-fed either SO or tested samples in SO (50 ml/day) six days per week. Each group was fed AIN-76 diet, but the AS group was fed AIN-76 diet plus containing 5.5% alfalfa sprouts dry powder. The results exhibited that ratio of sera IgG2a/IgG1, sera levels of IL-12p40 and appearance of proteinuria were reduced, and life span was extended in the ASEA and the TAM groups. The disease progress of mice was not affected in the AS and the CUM groups. Subsequently, to clarify ASEA how to improve the disease development of MRL-lpr/lpr mice, we sacrifice mice after supplementation of tested sample for six weeks. The results illustrated sera levels of IL-12p40 and IL-6 and appearance of proteinuria were reduced in the ASEA and the TAM groups. Con A-stimulated IFN-γ, IL-4 and IL-6 secretion from splenocytes were evidently attenuated in the ASEA group compared to the control. These data are in accordance with in vitro results. CD3+CD69+ T cell population of splenocytes was remarkable decreased in the ASEA group. The ASEA group also had less severe glomerulonephritis. To clarify what bioactive compounds in ASEA, this study purified ASEA (71 g) by silica gel chromatography and prepared HPLC separation. By using anti-inflammatory test and CHO-K1 ER transactivation assay, the candidate compounds were verified and identified as loliolide, 4-hydroxy-6-nonadecyl-2H-pyran-2-one, liquiritigenin, and isoliquiritigenin. In summary, this study demonstrated that ASEA having the abilities of anti-inflammation, Th1 and Th2 immune regulation and SERM could strikingly reduce the inflammatory progress and ameliorate disease course in autoimmune-prone MRL-lpr/lpr mice. Some purified compounds in ASEA such as loliolide, 4-hydroxy-6-nonadecyl-2H-pyran-2-one, liquiritigenin, and isoliquiritigenin are supposed to the contribution of disease amelioration.

Key words: autoimmune, inflammation, MRL-lpr/lpr, ASEA, pro-inflammatory cytokine, SERM