探討4-苯基丁酸鈉鹽對卵清蛋白誘發的過敏性氣喘 小鼠動物模式之抗過敏效果與其作用機制

The anti-allergic effect and the underlying mechanism of sodium 4-phenylbutyrate in ovalbumin-induced asthmatic mice

盧珈伶(Jia-Ling Lu)

指導教授 ： 李岳倫

臺北醫學大學/醫學院/醫學科學研究所/碩士(2014年)

摘要

過敏性氣喘是一種呼吸道慢性發炎的疾病，是由於第二型輔助型T細胞過度反應造成，其主要特徵為嗜酸性白血球增生與聚集於肺部、以及支氣管平滑肌收縮、黏液大量分泌等。本研究主旨在於探討短鏈脂肪酸類對於氣喘的治療效果與免疫調節機制，而我們則以消化道常見的短鏈脂肪酸作為材料來源。我們先以小鼠骨髓衍生性樹突細胞以及CD4+ T細胞作為研究平台，觀察短鏈脂肪酸對免疫細胞之活化與功能的影響。細胞實驗結果顯示：短鏈脂肪酸中的NaB與NaPB能抑制LPS刺激的樹突細胞分泌前發炎細胞激素TNF-α、IL-12與IL-10；而進一步分析NaB與NaPB對樹突細胞表面分子表現的影響，則發現MHC class II、CD80、CD86、CD40表現量皆有下降的趨勢，由此我們推測NaB與NaPB可能藉由抑制樹突細胞的活化與成熟，進而調控CD4+ T細胞的活化與分化。因此我們進一步將經LPS合併NaB或NaPB作用後的樹突細胞經放射線照射後，與CD4+ T細胞共同培養，發現給予NaPB刺激的樹突細胞進而能抑制CD4+ T細胞的增殖。另外將NaB與NaPB直接作用於anti-CD3/anti-CD28抗體活化的CD4+ T細胞上，同樣發現NaPB能顯著抑制CD4+ T細胞的增殖與細胞激素的分泌。故由以上結果顯示：NaPB能同時調控樹突細胞和T細胞的活化。此外，我們進一步使用對卵清蛋白(ovalbumin, OVA)具抗原專一性的DO11.10小鼠，同時給予DO11.10小鼠脾臟細胞體外OVA和NaPB合併刺激後，測其細胞激素的表現。結果顯示：NaPB能顯著抑制Th2細胞分泌IL-5，IL-13，同時提升IFN-γ和IL-10這兩種細胞激素的產量。綜合以上細胞實驗結果，我們推測NaPB可能具有抑制過敏性發炎反應的功能。因此，我們將NaPB應用於預防氣喘發病的小鼠動物模式上，動物實驗結果顯示：NaPB能降低小鼠血清中OVA專一性抗體IgE和IgG1的產量，且能明顯減低氣喘小鼠的呼吸道阻力，並抑制嗜酸性白血球聚集至肺部，減少Th2細胞激素IL-5、IL-13的分泌量，同時提升IL-10和IFN-γ的產量；此外，NaPB亦能促進小鼠體內Foxp3+CD4+的調節型T細胞的生成。綜合以上實驗結果，我們認為NaPB對過敏性氣喘有預防效果，有潛力發展為改善過敏體質之藥物。

關鍵字

過敏性氣喘 ； 短鏈脂肪酸 ； 4-苯基丁酸

並列摘要

Atopic asthma is a chronic inflammatory disease of the airways induced by type 2 T helper cell (Th2) immune response. It is characterized by airway eosinophilia, goblet cell hyperplasia with mucus hypersecretion and hyper-responsiveness in response to allergens. Short-chain fatty acids (SCFAs) have anti-inflammatory and immune-suppressive properties and are considered good candidates to inhibit allergen-induced disorders. In this study, we aim to investigate the anti-allergic ability and regulatory mechanisms of SCFAs on asthma. Firstly, we assessed the immunomodulatory effects of these SCFAs on the activation and function of bone marrow-derived dendritic cells (DCs) and CD4+ T cells in vitro. Our data showed that SCFAs, sodium butyrate (NaB) and sodium phenylbutyrate (NaPB), inhibited the secretion of TNF-α, IL-10 and IL-12 in LPS-stimulated DCs. Besides, the expression of surface makers on DCs was also reduced upon NaB or NaPB treatment. Thus, we proposed that NaB and NaPB might impair DCs’ maturation and activation and further affect T cell response. By co-culturing irradiated DCs with CD4+ T cells, we found that NaPB-treated DCs could inhibit the proliferation of CD4+ T cells. Additionally, we found that NaPB could directly suppress anti-CD3/CD28 antibodies-activated CD4+ T cell proliferation and cytokine production. On the other hand, DO11.10 splenocytes were cultured with NaPB in the presence of OVA and the effects on cytokine release were assessed. NaPB treatment suppressed IL-5 and IL-13 release, but had positive effect on IL-10 and IFN-γ production. Base on the above results, we thought that NaPB may have the ability to modulate allergic-specific immune responses in vivo. Finally, a murine model of OVA-induced asthma was used to investigate the preventative effect of anti-allergic effect of NaPB in these mice. Our data showed that NaPB-treated mice expressed lower levels of serum IgE and IgG1 than control mice. Besides, the development of airway hyper-responsiveness and the infiltration of eosinophils in lungs were alleviated by NaPB treatment. Notably, the secretion of Th2 cytokines was decreased but the production of IL-10 and IFN-γ was markedly increased in NaPB-treated mice. NaPB also enhanced the generation of Foxp3+CD4+ regulatory T cells inthese mice. Taken together, these results indicate that NaPB acts as anti-allergic regulator on immune effector cells and exerts preventative effects on OVA-induced asthma in mice.a In the future, we hope that NaPB can be developed as a novel agent to treat allergic diseases.

並列關鍵字

allergic asthma ； short-chain fatty acids ； 4-phenylbutyrate

參考文獻

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