山苦瓜乙酸乙酯萃物與奇異果對氣喘模式小鼠發炎與過敏免疫反應的影響

Effect of ethyl acetate extract of bitter gourd and kiwifruits on airway inflammation and immune responses in murine allergic asthma mode

許永瀚(Yung-Han Hsu)

指導教授 ： 林璧鳳

國立臺灣大學/生命科學院/生化科技學系/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10590

摘要

在亞洲地區，山苦瓜為傳統用來治療糖尿病的藥用植物。有研究指出山苦瓜乙酸乙酯萃物含有PPARα/γ ligand活性物質，並具有抗發炎的功效。另外，奇異果是富含抗氧化物質的水果，有些特定品種奇異果在東方國家被用作治療類風濕性關節炎、腦中風等疾病，然而市售常見的紐西蘭綠色奇異果對免疫調節的影響，鮮少被探討。過敏性氣喘為慢性呼吸道發炎疾病，屬於最常見的第一型過度免疫反應(Type I hypersensitivity)，伴隨著嗜伊紅性白血球浸潤於肺部、血液中IgE濃度升高、Th1/Th2免疫平衡失調並且呼吸道過度反應，為目前世界上最普遍的過敏疾病。故本研究探討經由攝食山苦瓜乙酸乙酯萃物與奇異果能否改善過敏氣喘小鼠體內過敏反應與呼吸道發炎現象。首先以in vitro細胞培養探討花蓮四號山苦瓜乙酸乙酯萃物(BGP-EA)和紐西蘭綠色奇異果乙酸乙酯萃物(kiwi-EA)，對巨噬細胞株RAW264.7、小鼠初代細胞分泌細胞激素的影響。結果顯示，BGP-EA顯著降低LPS活化RAW264.7的TNF-α與IL-6的分泌量，也能顯著的抑制初代腹腔細胞TNF-α和IL-6分泌、樹突細胞分泌IL-12和TNF-α以及初代脾臟細胞分泌IFN-γ。Kiwi-EA則是能顯著減少小鼠初代腹腔細胞分泌TNF-α與IL-6，和初代脾臟細胞分泌IFN-γ因此由體外實驗顯示BGP-EA和kiwi-EA具有抗發炎及免疫調節的功效，進一步探討對過敏性氣喘小鼠的免疫調節，方法為BALB/c小鼠8週齡時以卵蛋白(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，將小鼠分為八組，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BGP-EA的低劑量OVA-L組(0.15 mg/day)、中劑量OVA-M組(0.6 mg/day)和高劑量OVA-H組(2.4 mg/day)、飼料含20%奇異果凍乾粉的OVA-kiwi組和固醇類predisolone的藥物正對照組OVA-Pred組，另有一組為未致敏的負對照組PBS-oil組。餵食為期5週後，抽血測OVA-IgE抗體以methacholine刺激呼吸道，測定呼吸道過度反應時的呼吸阻力。氣喘小鼠於21週齡時犧牲，採取血液、肺氣管沖洗液、分離脾臟細胞、取肺組織抽取mRNA分析基因表現。結果顯示，OVA-L組與OVA-H組在Mch 50 mg/mL濃度下，Penh值顯著低於OVA-oil組。另外，與OVA-oil控制組相比，管餵BGP-EA的小鼠低、中、高劑量，都顯著減少肺氣管沖洗液中的嗜伊紅性白血球的數目，增加肺組織中的PPAR-γ mRNA表現、顯著降低IL-12p35表現和增加COX2基因表現，並且降低血清總IgE濃度，其中氣喘小鼠OVA-H組血清OVA特異性IgE觀察到顯著下降。小鼠脾臟細胞方面，在ConA刺激下，OVA-L組與OVA-M組IL-4分泌顯著降低，低、中、高劑量組IL-13分泌量都顯著下降。另外，OVA-H組的IFN-γ分泌顯著較低，而OVA-L組小鼠的IL-17分泌顯著下降。特異性OVA刺激之下，IL-2在OVA-L和OVA-M組有下降的趨勢，而IL-4分泌量在低、中、高劑量組都顯著下降，OVA-M組和OVA-H組則顯著減少IL-13分泌。OVA-kiwi組則是肺嗜伊紅性白血球數目顯著降低，肺組織PPAR-α、IL-12p35、iNOS、TSLP表現較低，而血清總IgE含量也下降。脾臟細胞在ConA刺激下，OVA-kiwi組小鼠的IFN-γ、IL-17、IL-4、IL-13分泌量都顯著下降。OVA特異性刺激下，餵食奇異果小鼠顯著抑制IL-4與IL-13分泌，且IL-10分泌量顯著增加。因此，從OVA致敏氣喘模式小鼠顯示，山苦瓜乙酸乙酯萃物可減緩全身Th2免疫反應，降低氣喘發炎情形。而攝取紐西蘭奇異果可以活化脾臟細胞，增加調節型T細胞分泌IL-10的能力，調節身體過度免疫反應。

關鍵字

氣喘 ； 山苦瓜乙酸乙酯萃物 ； 紐西蘭綠色奇異果 ； IgE ； 呼吸道過度反應

並列摘要

Backbround:Bitter gourd (Momordica charantia L.) is common vegetable in Asia that has been used in traditional medicine for the treatment of Diabetes. It is reported that the ethyl acetate (EA) extract of bitter gourd activated peroxisome proliferator receptors (PPARs) α and γ and shown anti-inflammatory effect. Additionally, kiwi fruits are one of the foods found to have abundant anti-oxidant substance. In some oriental coutries, there are some species of kiwi fruit used in traditional medicine to treat rheumatic arthritis and stroke. Just only few reports on immune-modulating activities of common commercially available breed of kiwi, Zespri green kiwi (Actinidia deliciosa) have been published. Asthma is a common disease of type I hypersensitivity characterized by chronic allergic airway inflammation with eosinophils infiltration in lung, elevated IgE contents in serum, Th1/Th2 imbalance, and airway hyperresponsiveness (AHR). Allergic asthma is one of the most prevalent chronic airway inflammatory diseases in the world. Objective: In this study, we examined the effects of BGP-EA and kiwi fruit on the development of pulmonary inflammation in murine model of asthma. Results: First, the effect of ethyl acetate extract of bitter gourd (BGP-EA) and kiwi fruit (kiwi-EA) on cytokine secretion by RAW264.7 cell, primary peritoneal cells or splenocytes from BALB/c mice was investigated. The results showed that BGP-EA significantly inhibited TNF-α and interleukin-6 (IL-6) production in LPS-stimulated RAW264.7. Furthemore, BGP-EA and kiwi-EA significantly reduced TNF-α and interleukin-6 (IL-6) secretion in LPS-stimulated primary peritoneal cells. Treatment with BGP-EA and kiwi-EA inhibited IFN-γ secretion in ConA-stimulated splenocytes. Therefore, bitter gourd and kiwi fruit may have anti-inflammatory and immunoregulatory effect. Subsequently, we investigated effect of bitter gourd and kiwi on airway inflammation and immune response by ovalbumin (OVA)-induced asthmatic murine model. After three times OVA sensitized intraperitoneally, eight-week-old BALB/c mice were separated eight groups including of OVA-oil, OVA-BGP (5% BGP), OVA-L (0.15 mg/day BGP-EA), OVA-M (0.6 mg/day BGP-EA), OVA-H (2.4 mg/day BGP-EA), OVA-kiwi (20% kiwi powder), OVA-Pred and PBS-oil group. Feeding five weeks later, OVA-L and OVA-H significantly suppressed the AHR. Compared to the OVA-oil group, three groups with BGP-EA feeding had significantly lower eosinophil infiltration in bronchoalveolar lavage fluid (BALF). On pulmonary mRNA expression, oral administration of BGP-EA significantly downregulate PPAR-γ and IL-12p35 mRNA and induce COX-2 mRNA. By the way, BGP-EA can lower total IgE level in serum, including of decreased OVA-IgE in OVA-H group serum. The BGP-EA feeding groups also had lower OVA-specific IL-4 and IL-13 secretion from OVA- or ConA-stimulated splenocytes. The OVA-kiwi group had profound inhibitory effects on the accumulation of eosinophils into airways. Taking a view to lung mRNA expression, OVA-kiwi had a lower PPAR-α, IL12p35, iNOS, TSLP level. In addition, OVA-kiwi also had lower total IgE level in serum. On T cell secreting cytokine, OVA-kiwi significantly lower OVA-specific IFN-γ, IL-4, IL-13 production and increasing IL-10 secretion instead. Conclusion: BGP-EA has an inhibitory effect on airway inflammation and hyperresponsiveness and plays a crucial role as an immunomodulator which possess anti-inflammatory and anti-asthmatic proterty by modulating the relationship between Th1/Th2 balance. On the other side, kiwi fruit can enhance the expression of Tregs in spleen toward allergen and inhibit over-activated immune responses.

並列關鍵字

asthma ； ethyl acetate extract of bitter gourd ； Zespri green kiwi fruit ； IgE ； airway hyperresponsiveness

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