- 學位論文
洛神葉萃取物抑制紫外線B誘發人類真皮層纖維母細胞基質金屬蛋白酶表現之影響
The inhibitory effects of Hibiscus sabdariffa leaf extracts on UVB-induced expressions of matrix metalloproteinases in human dermal fibroblast cells
摘要
紫外線 (Ultraviolet; UV)是導致皮膚光老化 (photoaging)的主要原因。長期UV暴露下，會導致皮膚膠原蛋白降解 (collagen degradation)、皺紋生成 (wrinkles generation)及皮膚免疫力下降，更可能造成皮膚癌。近來研究指出此光老化過程中，UVB會刺激纖維母細胞 (fibroblast)分泌基質金屬蛋白酶 (matrix metalloproteinases; MMPs)造成過度膠原蛋白的降解。因此，如何抑制膠原蛋白降解及減少皺紋的生成成為重要議題，也是抗UV致光老化重要的機制之一。洛神葉富含多酚 (polyphenol)化合物，包括鞣花酸 (ellagic acid; EA)及兒茶素 (catechin)。過去研究顯示，洛神葉具有抗癌、降血脂及抗氧化之效能，但在抗UVB之活性仍是未知。因此本實驗以洛神葉水萃取物 (Hibiscus sabdariffa leaf water extract; HLE)和其多酚萃取物 (Hibiscus sabdariffa leaf polyphenolic extract; HLP)為實驗材料，利用UVB照射裸鼠模式及人類真皮層纖維母WS-1細胞 (human dermal fibroblast WS-1 cells)，分析HLE及HLP抗UVB作用及其抗光老化機轉。 在動物實驗中，藉由西方墨點法及免疫組織染色法分析，經UVB照射裸鼠皮膚下，預處理HLE、HLP和其主要功能性成份EA皆能透過抑制MMP-1/8/13 (collagenases)及MMP-2/9 (gelatinases)的表現來增加膠原蛋白的含量。此外，在細胞實驗中，經由西方墨點法及real-time PCR分析證實，UVB照射同樣能誘發WS-1細胞中MMP-1/8/13 (collagenases)及MMP-2/9 (gelatinases)的表現，而預處理HLE下，再照射UVB後發現，HLE在0.2 mg/ml劑量下能顯著的抑制細胞內MMP-1/2/8/9/13的表現，並增加前膠原蛋白 (pro-collagen)的含量。接著分析細胞核中調控MMPs轉錄因子之核內表現及與DNA結合能力，結果發現HLE能抑制核內c-Fos及c-Jun的表現。最後透過EMSA証實HLE透過減少此轉錄因子AP-1 (c-Jun/c-Fos二元體)與DNA結合的表現達到抑制MMPs的效果。此外也發現，HLE可能是透過抑制JNK磷酸化作用來抑制轉錄因子c-Fos及c-Jun的表現。綜合以上結果顯示在WS-1細胞株中，HLE能抑制UVB所誘發的MMP-1/2/8/9/13蛋白表現是藉由抑制p-JNK表現來降低轉錄因子AP-1的活性，以減少MMPs表現，達到增加pro-collagen的含量。因此，未來洛神葉萃取物可能有潛力作為抗UVB致皮膚光老化作用之試劑。
並列摘要
Ultraviolet (UV) radiation is one of the main factors of skin photoaging. Long-term exposure to the UV leads to skin collagen breakdown, and generate wrinkles in the skin. Recently, it has been known that excessive matrix degradation was performed by UVB-induced matrix metalloproteinases (MMPs) secreted by fibroblasts cells during photoaging. Hibiscus sabdariffa leaf, the edible part of H. sabdariffa Linne (Malvaceae), is usually ignored and discarded. Previous studies have indicated H. sabdariffa leaf extract has antioxidant and anticancer activities. However, few studies have investigated the anti-UVB activity of H. sabdariffa leaf. In this study, we evaluated the effect of H. sabdariffa leaf water extract (HLE) and its polyphenolic extract (HLP) on MMPs expressions in hairless mice and human dermal fibroblast WS-1 cells exposed to UVB irradiation. In vivo study, Western blotting and immunohistochemistry demonstrated HLE、HLP and ellagic acid (EA) inhibited MMP-1/2/8/9/13 production and increased collagen synthesis in the dorsal skin of hairless mice exposed to UVB, while HLE is more potential. In addition, our in vitro study showed that non-cytotoxic doses of HLE and their main component EA rescued the UVB-regulated the expressions of MMP-1/2/8/9/13 and pro-collagen. The HLE could abolish UVB-induced MMPs expressions appeared be a consequence of activator protein-1 (AP-1) inactivation, because its DNA binding activity was suppressed by HLE. Further, HLE also suppressed the protein levels of p-JNK causing the consequence disturbed activation of AP-1 in UVB-induced WS-1 cell. These findings suggest that HLE inhibited the UVB-induced MMP-1/2/8/9/13 expressions by suppressing AP-1 activation in WS-1 cells. In summary, H. sabdariffa leaf extracts might be used as potential agents for treatment of UV-induced skin photoaging.