以誘發小鼠關節炎模式探討攝食山藥對類風濕性關節炎的影響

Abstract

本研究主要探討山藥對發炎相關疾病的影響，首先以 RAW264.7細胞培養in vitro 添加方式，探討台農二號山藥 (Dioscorea alata L. cv. Tainung No.2) 乙酸乙酯萃取物 (DAEA) 對巨噬細胞株RAW246.7及C57BL/6小鼠初代細胞免疫調節的作用。結果顯示山藥EA萃物能顯著降低活化巨噬細胞IL-6的分泌，也顯著降低小鼠初代活化脾臟細胞IFN-γ的分泌，對於小鼠活化初代腹腔細胞分泌IL-6的能力有降低的現象。顯示體外試驗山藥EA萃物具有抗發炎的潛力。接著探討攝食山藥EA萃物對於以膠原蛋白誘發C57BL/6關節炎小鼠之影響。C57BL/6小鼠八週大時，以第二型膠原蛋白 (type II collagen，CII) 伴隨含結核桿菌的complete Freund佐劑，誘發關節炎，13週大時以CII特異性抗體IgG2a、體重及score，將小鼠分為四組，分別餵食chow diet飼料的控制組、PBS組，以及分別每天以管餵的方式補充100 μg或200 μg的DAEA-L組及DAEA-H組。餵食17天後，DAEA-H組的score較控制組有降低的現象，犧牲小鼠，並採集血液、培養脾臟及腹腔細胞。結果顯示，在ConA或LPS刺激下，均顯著促進脾臟細胞分泌IFN-γ。但因以膠原蛋白誘發C57BL/6成關節炎小鼠的成功機率不高，因此改而使用六週大的BALB/c小鼠，以抗體誘發成關節炎小鼠，依體重及小鼠足部腫脹程度計分總值(總score) 分為五組，分別為餵食AIN-76飼料的控制組、PBS組、AIN-76飼料含0.9%熟山藥粉的DA-cook組、每天分別以管餵的方式補充100 μg或200 μg的 DAEA-L組及DAEA-H組。餵食11天後，結果顯示DA-cook組與DAEA-H組的score顯著較控制組低，而DAEA-L也有降低的現象，隨即犧牲小鼠，並採集血液、培養脾臟與腹腔細胞及取小鼠四肢。結果顯示在LPS刺激下，DAEA-L均顯著抑制脾臟細胞TNF-α、IL-6與IL-1β等促發炎細胞激素的分泌；DAEA-L可顯著降低ConA刺激之脾臟細胞IFN-γ的分泌；在CII刺激下，顯著降低IL-12p40的分泌。此外，在小鼠後腳的組織化學切片染色的結果顯示，DAEA-L組與DAEA-H組有較控制組明顯改善細胞聚集與關節軟骨破壞的情形，而DA-cook組也略有改善的現象。由以上結果可知，台農二號山藥萃物能夠降低關節炎小鼠Th1及促發炎細胞激素的分泌、減少關節處的損傷，可能有助於減緩類風濕性關節炎的症狀。

The purpose of this study is to evaluate whether the yam may possess an immunomodulation effect on the inflammatory disease such as rheumatoid arthritis. First, the effect of ethyl acetate extract of Taiwanese Yam (Dioscorea altata L. cv. Tainung No.2) (DAEA) on in vitro cytokines secretion by RAW264.7 cell, primary macrophages or splenocytes from C57BL/6 mice was investigated. The results showed that DAEA significantly inhibited IL-6 secretion in LPS-stimulated RAW264.7 cell and IFN-γ secretion from ConA-stimulated splenocyte, and tended to decrease IL-6 secretion in LPS-stimulated primary macrophages, suggesting an anti-inflammatory effect. Therefore, in vivo anti-inflammatory effect of DAEA on rheumatoid arthritis was evaluated using a collagen-induced arthritis (CIA) murine model. Female C57BL/6 mice were immunized with type II collagen (CII) emulsion with CFA containing 5 mg/mL of M. Tuberculosis subcutaneously. On day 21, mice were boosted with the same dose of CII and CFA, and then divided into four groups by titer of CII-specific IgG2a, body weight and the swelling of joints (toal score). Four groups of mice were fed chow diet (control and PBS), tube-feeding of 100 (DAEA-L) or 200 μg (DAEA-H) EA extracts of yam per day. After 17 days feeding, the total score of DAEA-H group tended to decrease compared to the control group. Then, mice were sacrificed for further in vivo analysis. Compared to the control group, DAEA-H significantly increased IFN-γ secretion under ConA or LPS-stimulated splenocytes. To obtain better induction of arthritis in murine model, second in vivo experiment was conducted by using a type II collagen antibody-induced arthritis (CAIA) model. Female BABL/c mice were immunized with type II collagen antobody and then divided into five groups by weight and score. Five groups of mice were either fed an AIN-76 diet (control and PBS), AIN-76 diet containing 0.9% cooked yam (DA-cook), tube-feeding of 100 (DAEA-L) or 200 μg (DAEA-H) EA extracts of yam per day. After 11 days’ feeding, the total score of DA-cook and DAEA-H group was significantly lower than that of the control group. Total score of the DAEA-L group tended to be lower than the control group. Mice were sacrificed for further in vivo analysis. The results showed that DAEA-L significantly inhibited TNF-α、IL-6 and IL-1β secretion in LPS-stimulated splenocytes. DAEA-L decreased IFN-γ secretion by ConA-stimulated splenocytes and IL-12p40 on CII-stimulated splenocytes. Furthermore, histology also showed the group of DAEA-L and DAEA-H had eased immune cells recruitment and bone erosion. In conclusion, these results suggest that the EA extract of yam, Dioscorea alata L. cv. Tainung No.2 used in this study, may alleviate arthritis trough suppression of Th1 cytokines and pro-inflammatory mediators, which may be beneficial for rheumatoid arthritis.