山苦瓜與共軛次亞麻油酸對氣喘模式小鼠發炎與過敏免疫反應的影響

Effects of bitter gourd and 9c, 11t, 13t-conjugated linolenic acids on airway inflammation and immune responses in murine allergic asthma model

呂學耘(Hsueh-Yun Lu)

指導教授 ： 林璧鳳

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00663

摘要

已有研究指出山苦瓜的降血糖功效與其乙酸乙酯萃物含共軛次亞麻油酸 (9c, 11t, 13t-conjugated linolenic acid, CLN)活性成份，但對於免疫調節的影響，尚未有研究發表。本研究首先以細胞培養探討花蓮四號山苦瓜正己烷(BGP-Hex)、乙酸乙酯(BGP-EA)、酒精(BGP-EtOH)與水萃物(BGP-H2O)和CLN，對巨噬細胞株RAW264.7、小鼠T細胞株EL4、小鼠初代細胞分泌細胞激素的作用。結果發現BGP-EA、BGP-Hex和BGP-EtOH能顯著的降低RAW264.7的TNFα及IL-6分泌，也顯著的抑制初代腹腔細胞IL-6的分泌。CLN能降低初代腹腔細胞TNFα及IL-6的分泌，也能顯著減少小鼠初代脾臟細胞IFNγ與IL-4。在體外實驗初步觀察到山苦瓜萃物與CLN具有抗發炎的潛力及調節免疫的效果。本研究接續探討山苦瓜與所含的CLN對氣喘模式小鼠發炎與過敏免疫反應的影響，將 BALB/c 小鼠8週大時以卵蛋白（ovalbumin, OVA）加佐劑氫氧化鋁，隔週腹腔致敏以誘發過敏免疫傾向。三次致敏之後，將以小鼠分為五組，分別為OVA-oil控制組 、飼料含5%山苦瓜凍乾粉的OVA-BGP組、每天管餵35 mg共軛次亞麻油酸OVA-CLN組、每天管餵35 mg共軛亞麻油酸 (conjugated linoleic acid, CLA) 的OVA-CLA組、和固醇類藥物prednisolone正對照OVA-Pred組 ，另有一組為未致敏的PBS-oil組。餵食期間以5%霧化之OVA給予致敏小鼠呼吸道刺激，誘發過敏性呼吸道發炎，而PBS-oil組給予霧化PBS。小鼠餵食13天後，以methacholine (Mch)刺激呼吸道，測定呼吸道過度反應(airway hyperresponsiveness, AHR)時的呼吸阻力。小鼠餵食16天後犧牲，採血液、肺氣管沖洗液、分離脾臟及腹腔細胞、取肺組織進行切片觀察與抽取RNA分析基因表現。結果顯示，OVA-BGP組與OVA-CLN組在Mch各濃度下呼吸道阻力係數Penh值顯著低於OVA-oil控制組，OVA-CLA組在大於25 mg/mL Mch刺激下也顯著較低，表示山苦瓜、CLN和CLA可減緩過敏性氣喘。與OVA-oil控制組相較， OVA-BGP組顯著減少肺氣管沖洗液中的總細胞、嗜伊紅性白血球及嗜中性白血球的數目、也降低Th2細胞激素IL-4、IL-5、IL-13與促發炎介質IL-6、PGE2和eotaxin含量，肺組織中的COX-2 mRNA表現也顯著較低，且顯著的回復肺組織中的PPARα基因表現量，顯示山苦瓜抑制發炎反應與Th2免疫反應。並且，OVA-BGP組的脾臟細胞在ConA刺激之下，IFN-γ分泌量顯著高於OVA-oil組，在特異性OVA刺激下的脾臟細胞IL-13分泌量顯著較低，血清總IgE生成量也顯著較低，顯示山苦瓜對調節傾免疫向Th1反應。 OVA-CLN組則是顯著降低肺氣管沖洗液中嗜伊紅性白血球的數目和IL-5含量，回復肺組織PPARγ mRNA的表現量，顯示CLN的抗發炎作用。而OVA-CLA組顯著降低脾臟細胞在ConA刺激之下IL-13的分泌，肺組織IL-12與PPARα mRNA表現量也顯著較高，故也測得顯著低的血清總IgE生成量，顯示CLA具調節免疫傾向Th1的作用。因此，由誘發致敏小鼠產生過敏性氣喘的動物模式顯示，山苦瓜具抑制過敏的免疫調節與抗發炎作用，所含成份的CLN則以降低呼吸道發炎作用較顯著，山苦瓜能減少過敏性抗體IgE的生成，應有助於減緩過敏免疫反應，值得推廣。

關鍵字

氣喘 ； 山苦瓜 ； 共軛次亞麻油酸 ； IgE ； 呼吸道發炎 ； 共軛亞麻油酸

並列摘要

Studies show bitter gourd (Momordica charantia) exerts hypoglycemic effect and 9c, 11t, 13t-conjugated linolenic acid (CLN) from its ethyl acetate extract is the bioactive compounds. Only few reports on immunomodulatory effect of bitter guard have been published. First, the effect of hexane extract (BGP-Hex), ethyl acetate extract (BGP-EA), ethanol extract (BGP-EtOH), water extract (BGP-H2O) and CLN on cytokines secretion by RAW264.7 cell, EL4 cell and primary peritoneal cells or splenocytes from BALB/c mice was investigated. The results showed that BGP-EA, BGP-Hex and BGP-EtOH significantly inhibited TNFα and interleukin (IL)-6 secretion in LPS-stimulated RAW264.7. Furthermore, BGP-EA and BGP-EtOH significantly reduced IL-6 secretion and CLN could decrease the TNFα and IL-6 production in LPS-stimulated primary peritoneal cells. Treatment with CLN inhibited IFNγ and IL-4 secretion in ConA-stimulated splenocytes. Therefore, bitter gourd and CLN may have an anti-inflammatory and immunoregulatory effect. Subsequently, we investigated effect of bitter gourd and 9c, 11t, 13t-CLN may on airway inflammation and immune response using a murine model of allergic asthma. Eight-week-old BALB/c mice sensitized intraperitoneally and challenged with ovalbumin (OVA) were fed a diet either supplemented with 5 % (wt/diet) bitter gourd powder (OVA-BGP group), or tube-feeding 35 mg/day CLN (OVA-CLN group), 35 mg/day 9c, 11t-conjugated linoleic acid (CLA) (OVA-CLA group), or oil as control (OVA-oil group) for 16 days. The OVA-sensitized mice treated with prednisolone were the positive control (OVA-Pred group). Mice without sensitization were the negative control (PBS-oil group). The hyperresponsiveness (AHR) challenged by methacholine (Mch), airway inflammation, serum antibody levels, cytokine secretions from splenocytes and lung mRNA expression were measured. BGP, CLN and CLA supplementation significantly suppressed the AHR, suggesting the alleviative effects on allergy asthma. Compared to the OVA-oil group, the OVA-BGP group had significantly lower total cells, eosinophil and neutrophil infiltration and Th2 cytokines IL-4, IL-5 and IL-13, and inflammatory mediators IL-6, PGE2 and eotaxin in bronchoalveolar lavage fluid (BALF), and pulmonary COX-2 mRNA expression, suggesting anti-inflammatory effect of BGP. The OVA-BGP group also had lower OVA-specific IL-13 secretion and IFN-γ secretion from OVA- or ConA-stimulated splenocytes, and lower serum total immunoglobulin (Ig) E level, suggesting immunomodulatory effect of BGP. The OVA-CLN group showed significantly lower eosinophil infiltration and IL-5 in BALF, restored PPARγ mRNA expression that was down-regulated by sensitization, suggesting anti-inflammatory effect of CLN. The CLA group had significantly lower IL-13 secretion from Con-A stimulated splenocytes, higher pulmonary IL-12 and PPARα mRNA expression, lower total IgE level in serum, suggesting the immunoregulatory effect of CLA toward Th1 responses. In conclusion, Bitter gourd possessed both anti-inflammatory and immunoregulatory effects, while CLN exerted mainly reduced airway inflammation in a murine model of allergic asthma. Our study suggests that bitter guard suppressing allergic immune responses including serum IgE level may be beneficial for alleviation of allergic responses and worthy of dietary promotion.

並列關鍵字

asthma ； bitter gourd ； conjugated linolenic acid ； IgE ； airway inflammation ； conjugated linoleic acid

參考文獻

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