苜蓿芽乙酸乙酯萃物對實驗性自體免疫腦脊髓炎的影響

Abstract

實驗性自體免疫腦脊髓炎 (EAE) 為多發性硬化症中常用的動物模式，主要由Th1及Th17主導，且伴隨神經發炎的症狀。因此本研究擬以細胞模式找到具有調節Th1/Th17及抗神經發炎的食材，再應用於實驗性自體免疫腦脊髓炎中，探討是否具有減緩病情的作用。細胞實驗先以非特異性抗原anti-CD3/28刺激正常小鼠初代脾臟細胞，經苜蓿芽乙酸乙酯萃物 (ASEA) 預處理，可顯著降低IL-2及IFN-γ分泌量，也有降低IL-17、IL-6的趨勢。將MOG35-55誘發EAE小鼠脾臟細胞以特異性抗原MOG35-55刺激，ASEA同樣可顯著降低IL-2及IFN-γ，卻顯著增加IL-17及IL-6。而以流式細胞儀分析，發現ASEA顯著增加MOG35-55刺激下的CD4+及CD4+IFN-γ+細胞比例。最後以ASEA預處理C8-B4神經微膠細胞株，可降低TNF-α、IL-6、IL-12/23 p40及MCP-1的分泌量，可能具有抗神經發炎的作用。 在MOG35-55誘發EAE小鼠動物模式中，於誘發後每天管餵ASEA可延緩EAE病程，並降低浸潤於脊髓中CD4+細胞比例及脾臟細胞自發性的IL-2分泌量，且兩者皆與病情呈顯著正相關 (分別為r= 0.778, p< 0.001; r= 0.527, p< 0.001)，可能因此具有延緩病程的作用；此外，短期餵食ASEA也可增加與病情呈負相關 (r= -0.358, p= 0.0295) 之脾臟調節型T細胞比例，並降低淋巴結細胞對MOG35-55刺激所分泌的IL-17與IL-6。這些結果顯示短期餵食ASEA，可能藉由降低脾臟細胞自發性分泌的IL-2與減少浸潤於脊髓中的CD4+細胞，而有延緩EAE病程發展的作用。

Experimental autoimmune encephalomyelitis (EAE), which is a common animal model of multiple sclerosis, is dominated by Th1 and Th17, accompanied by neuroinflammation. This study was first to investigate the capability of modulating Th1/Th17 and anti-neuroinflammation in food extract in vitro, and then we evaluated the effects of the food extract on EAE in vivo. First, the primary splenocytes from normal C57BL/6J female mice were pretreated with ASEA, followed by treatment with anti-CD3/28. The results showed that ASEA could significantly decrease Th1 (IL-2, IFN-γ) and Th17 (IL-17, IL-6) cytokines. Then, primary splenocytes and lymph node cells from MOG35-55-induced EAE C57BL/6J female mice were treated as before. Similarly, ASEA could significantly decrease Th1 cytokines, but significantly increase Th17 cytokines. Flow cytometric analysis of splenocytes showed ASEA significantly increased the percentage of MOG35-55-stimulated CD4+ cells and CD4+IFN-γ+ cells, but there was no difference in CD4+IL-17+ cells. Further, ASEA significantly reduced IL-6, IL-12/23p40 and MCP-1 secretions by C8-B4 microglial cell line, which suggested that ASEA had the potential for anti-neuroinflammation. Finally, female C57BL/6J mice were fed intragastrically with ASEA once a day (3.6 mg and 14.5 mg/kg BW, as the preASEA-L group and the preASEA-H group, respectively) for 59 days until sacrificed. The ASEA-H group was fed with 14.5 mg ASEA/kg BW for 17 days since the day of immunization. EAE clinical symptoms were delayed in the ASEA-H group. We examined the changes in the immunologic parameters, including Th1/Th17 population in CNS, spleen and lymph nodes, and ex vivo cytokines production. The preASEA-L group had lower Th1 population and ex vivo Th1 cytokine production in splenocytes. The infiltrating CD4+ cells population in spinal cord and spontaneous IL-2 secretion in the splenocytes of MOG35-55-induced EAE mice were higher than in normal mice; moreover, positive correlation existed between both parameters and area under curve (AUC) of EAE score (r= 0.778, p< 0.001 in CD4+ cells; r= 0.527, p< 0.001 in IL-2). We found that the ASEA-H group significantly reduced both important parameters, and it also had lower ex vivo MOG35-55-stimulated IL-17 and IL-6 production in lymph nodes. In addition, the ASEA-H group enhanced regulatory T cell (Treg) population, which had a negative correlation with sacrificed EAE score (r= -0.358, p= 0.0295). These results showed that mice fed with ASEA at immunization delayed the development of MOG35-55-induced EAE, accompanied with lower infiltrating CD4+ cells population in spinal cord and spontaneous IL-2 secretion in splenocytes, which suggests the potential of beneficial effects of ASEA for delaying EAE symptoms.