檸檬醛對Malassezia furfur之抑制機制探討

The inhibitory activity of citral against Malassezia furfur

劉易慈(Yi-Tsz Liu)

指導教授 ： 賴雯玲

中山醫學大學/醫學科技學院/醫學檢驗暨生物技術學系碩士班/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00064

摘要

嗜脂性酵母菌Malassezia furfur (M. furfur)為正常人體皮膚共生的常在菌叢，也被視為機緣性病菌，與一些慢性發炎皮膚疾病之發生有關，例如頭皮屑、花斑癬、毛囊炎、脂漏性皮膚炎等。這些疾病的治療都相當困難，因此急需發展有效且安全的治療策略。本實驗室在先前的實驗中發現檸檬香茅精油對M. furfur具有抑制作用，因此，本研究之目的，即在探討檸檬香茅之主成分citral對病原菌M. furfur的之抑菌活性與作用機制，以及citral對M. furfur感染角質細胞之影響。結果顯示citral對M. furfur 的最低抑菌濃度與最低殺菌濃度，分別為200 μg/ml與300 μg/ml，同時也發現了citral能夠抑制此菌由yeast轉為mycelia的現象。在作用機制的部份我們分別透過Annexin V 、TUNEL assay以及CaspACE FITC-VAD-FMK染色來證實citral能夠促使M. furfur進行細胞凋亡(apoptosis)。而在感染的實驗中，我們使用人類角質細胞株(HaCat)做為M. furfur-infection平台，發現citral並無細胞毒性，且在sub-MIC的濃度下可以有效抑制M. furfur對HaCat細胞的貼附以及感染。透過Real-time PCR與ELISA，證實當M. furfur 感染角質細胞時，citral不但能夠抑制細胞TLR2與IL-6的表現，同時也能刺激HBD-2及TSLP的上升。綜合上述結果，顯示高濃度citral具有抗真菌之功能，而在低濃度下可調節細胞免疫反應，以增強對M. furfur的感染防禦，此也顯示citral對於防治M. furfur相關皮膚病具有潛力。

關鍵字

Malassezia furfur ； citral ； 細胞凋亡 ； 角質細胞 ； 免疫調控

並列摘要

The lipophilic yeast Malassezia furfur (M. furfur) is a member of the cutaneous commensal microbiota, associated with several chronic diseases such as dandruff pityriasis versicolor, folliculitis, seborrheic dermatitis, which are often difficult to therapy. The development of alternatively effective antifungal therapies is therefore of paramount importance. In this study, I investigated the antifungal effect of citral on M. furfur. The minimal inhibitory concentration of citral for M. furfur is 200 μg/ml, and the minimal fungicidal concentration is 300 μg/ml. Citral significantly reduced the proportions of yeast cells switched to mycelia growth. Cellular changes that accompany apoptosis were further analyzed using fluorescent annexin V probe, TUNEL assay and CaspACE FITC-VAD-FMK. Exposure of M. furfur to citral inducing phosphatidylserine externalization, DNA fragmentation and metacaspase activation, which are important markers of early and late stage apoptosis respectively. I observed that cital can reduce the infection of M. furfur to keratinocyte (HaCat) at sub-MIC concentration. Finally, I demonstrated citral inhibited the expression of IL-6 and TLR-2, and enhanced the expression of HBD-2 and TSLP in M. furfur-infected human keratinocyte. Taken together, these results showed citral has antifungal activity at high concentration and can decrease the infection of M. furfur through modulate the immune response of keratinocyte at lower concentration. Our results suggest that citral is a potential candidate for topical therapeutic application to ameliorate M. furfur-associated human skin diseases.

並列關鍵字

Malassezia furfur ； citral ； apoptosis ； human keratinocyte ； immunomodulation

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