口腔黏膜下纖維化症(Oral submucous fibrosis，簡稱OSF)是一種隱襲性及慢性發炎的口腔黏膜病變，主要特徵是張口受限、咀嚼吞嚥因難，流行病學研究顯示口腔癌和OSF的致病原因和咀嚼檳榔最有關係，檳榔子內夾荖花、外包荖葉、熟石灰及特殊的香料是台灣常見的吃法，荖花中以多酚類的黃樟素含量最多，黃樟素具有基因毒性及致癌性，會形成safrole-DNA adducts。文獻指出，檳榔萃取物會活化NF-kB訊息傳遞路徑，但是黃樟素和NF-kB之間的關係及其訊息傳遞路徑的機轉仍不明瞭。因此，本實驗收集了17個OSF及3個正常的口腔頰黏膜組織用免疫組織化學染色(Immunohistochemistry，IHC)來看NF-kB在口腔黏膜下纖維化症的表現，另外培養三株健康初代培養頰黏膜造纖維母細胞，運用細胞毒性試驗及西方點墨法(Western blotting)來探討黃樟素的毒性及其是否會對頰黏膜造纖維母細胞誘發NF-kB的表現，另外本實驗加入N-Acetyl-L-cysteine (NAC)、PD98059 (ERK抑制劑)、NS-398 (COX-2抑制劑)、Dexamethasone (使IkB上升)及cyclosporin A (NF-kB抑制劑，拮抗proteasome 26s)來觀察黃樟素誘發NF-kB活化可能的調控機轉。實驗結果顯示，在正常的口腔頰黏膜組織中，NF-kB大部份表現在上皮細胞的基底層、血管的內皮細胞及少數的發炎細胞，而在OSF病理切片中，NF-kB在結締組織造纖維母細胞有強烈的表現而且比正常的口腔頰黏膜組織還多；此外本實驗發現，黃樟素(12~768 μM)對細胞的毒性是隨著濃度的增加而增加(p<0.05)，以西方點墨法發現，隨著黃樟素濃度的增加及時間的增加，NF-kB的表現有明顯上升的趨勢(p<0.05)，另外在黃樟素無毒性的劑量下，本實驗發現NAC、PD98059、NS-398、Dexamethasone及cyclosporin A都可以抑制黃樟素對造纖維母細胞誘發NF-kB的表現(p<0.05)。實驗結果顯示，咀嚼內含荖花的檳榔誘發NF-kB，這可能是造成OSF致病原因之一，此外，黃樟素在造纖維母細胞中誘發NF-kB可能是藉由ERK及COX-2傳導路徑來調控的。
Oral submucous fibrosis (OSF) is an insidious and chronic inflammatory disease characterized by restriction of mouth opening and difficulty in mastication and swallowing. Epidemiological studies have reported that betel quid (BQ) chewing is highly associated with oral cancer and OSF. In Taiwan, chewing BQ containing piper betel influorescence (PBI) and piper betel leaf (PBL) is very popular. Safrole is the major component of PBI. Safrole is known to be genetic toxicity and carcinogenesis, and forms safrole-DNA adducts. Previous study has shown that areca nut extract activates NF-kB signal transduction pathway. However little is known about the signal transduction between safrole and NF-kB. Hence, 17 OSF specimens and 3 normal buccal mucosa were examined the expression of NF-kB by immunohistochemistry (IHC). 3 primary human buccal mucosal fibroblasts were established and challenged with safrole analyzed by MTT colorimetric assay and western blotting. Furthermore, N-Acetyl-L-cysteine (NAC), PD98059 (ERK inhibitor), NS-398 (COX-2 inhibitor), Dexamethasone (up-regulation of IkB), Cyclosporin A (antagonist of proteasome 26s) were added to find the possible mechanism. NF-kB expression is almost limited to the basal layer of epithelial cells, endothelial cells of vessel and a few inflammatory cells in normal buccal mucosa specimens. In the OSF specimens, NF-kB is strongly expressed in the fibroblasts of connective tissue more than normal buccal mucosa specimens. Our studies indicated that cytotoxicity of safrole (12~768 μM) was in a dose-dependent manner (p<0.05). In addition, the expression of NF-kB was in a dose- and time- dependent manner by western blotting assay (p<0.05). Beside, pretreatment of NAC, PD98059, NS-398, Dexamethasone and Cyclosporin A all markedly inhibited the NF-kB expression induced by safrole at the non-toxic concentration (p<0.05). The result suggests that chewing betel quid containing PBI may activate NF-kB expression that may be one of pathogenesis of OSF. NF-kB expression induced by safrole in fibroblasts may be mediated by ERK activation and COX-2 signal transduction pathway.
