黃樟素抑制人類牙齦造纖維母細胞機轉的探討

從流行病學調查研究顯示，估計台灣嚼食檳榔的人口數約已達二百八十萬之多，檳榔嚼食者相較於非檳榔嚼食者其罹患口腔癌與口腔黏膜下纖維化的危險性皆會提高，然而嚼食檳榔對於牙周病之影響，卻缺乏充分之流行病學研究，目前雖有不少研究顯示，嚼食檳榔與牙周病有相關性，但以台灣特有檳榔嚼塊(紅灰檳榔)中荖花所含有的多酚化合物—黃樟素—對於人類牙齦造纖維母細胞之影響，目前仍缺如。因此本實驗以黃樟素作用於人類牙齦造纖維母細胞(Human gingival fibroblasts, HGF)來觀察其對於細胞在修復，再生上的影響，並進一步探討其可能機轉。首先以體外培養HGF在黃樟素30µM-960µM濃度下作用24小時，發現黃樟素對HGF存活率具有劑量-依賴效應(dose-dependent effect)(p<0.05)，當黃樟素濃度大於240µM時會抑制HGF的擴展與遷移。當黃樟素濃度愈高其細胞附著能力愈差(p<0.05)。當黃樟素作用HGF至24h時，phospho extracellular signal-regulated protein kinase 明顯被抑制的現象(p<0.05) 。隨著時間的增加在黃樟素作用下其Matrix metalloproteinases-2(MMP-2) 、tissue type plasminogen activator( t-PA) 都大幅的上升(p<0.05)。隨著黃樟素作用時間的增加cyclooxygenase-2 表現也不斷上升(p<0.05)。先加入Aa、 Pg 、IL-1-β、TNF-α來模擬牙周發炎下黃樟素對HGF的影響，發現MMP-2，t-PA的表現相對於控制組而言亦大量的增加(p<0.05)，顯示黃樟素具有破壞牙周組織的能力。從上述實驗得知，黃樟素可能具有抑制人類牙齦造纖維母細胞修復與再生的能力，乃藉由破壞MMP-Plasmin system，促使細胞與基質間交互作用破壞來抑制細胞修復，再生的能力。因此檳榔嚼食者對於牙周的破壞可能具有更高的感受性且對於牙周再生手術的反應較差。

關鍵字

黃樟素；牙周病；纖維蛋白溶解系統；基質金屬蛋白水解

並列摘要

The habit of areca (betel nut, Areca catechu) quid chewing is widespread in Taiwan, Southeast Asia, and India. Epidemiological studies have shown that areca quid chewing increases the risk of oral cancer and oral submucous fibrosis. In addition, areca quid chewers have a higher prevalence of periodontal diseases than non-chewers. In this study, the pathological effects of safrole, a major polyphenol compound in Piper betel leaf used when quid chewing, were investigated in cultured human gingival fibroblasts (HGFs). Little is known about the cytopathological effects of safrole on human gingival fibroblasts (HGF). Hence, we established 6 primary human gingival fibroblast strains for cell migration, attachment and spreading assays with the treatment of safrole. In addition, HGFs were challenged with safrole analyzed by 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyl-tetrazolium (MTT) colorimetric assay, Western blotting, gelatin zymography and casein zymography. Our results demonstrated that safrole was cytotoxic to HGFs in a dose-dependent manner (p<0.05). Safrole also inhibited cell attachment (p<0.05). A control culture exhibited a normal fibroblast monolayer of long spindle-shaped morphology. Safrole-treated HGFs showed a rounded appearance and detachment at higher concentrations. At concentrations higher than 20 µg/ml, safrole inhibited cell spreading and migration. Safrole was found to induced extracellular signal-regulated protein kinase phosphorylated (p-ERK) in a dose dependent manner.(p<0.05) Matrix metalloproteinases-2(MMP-2) and t-plasminogen activator(t-PA) expression was up-regulated by safrole treatment with time-dependent effect compare with control groups (p<0.05). Cyclooxygenase-2(COX-2) expression was increased by safrole (p<0.05). The addition of periodontal pathogens and proinflammation cytokines significant enhanced MMP-2 and t-PA expression (p<0.05) as compare with safrole alone. Taking together, these results indicate that safrole is a cytotoxic agent to HGFs. Areca quid chewers might be more susceptible to destruction of periodontium and less responsive to a regeneration procedures during periodontal therapy.

並列關鍵字

safrole ； periodontitis ； matrix metalloproteinase ； plasminogen/plasmin system, repair and regeneration

參考文獻

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黃樟素抑制人類牙齦造纖維母細胞機轉的探討

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