薑黃素抑制環孢素經由活性氧誘導牙齦上皮細胞活化潛伏性TGF-β

Abstract

實驗目的和背景：轉型生長因子-β (Transforming growth factor-β (TGF-β) ) 在牙齦腫大 (gingival overgrowth, GO)的致病機轉中扮演重要角色。先前研究發現環孢素(Cyclosporin-A, CsA) 在牙齦纖維母細胞可經由TGF-β訊息傳遞路徑引發GO。TGF-β誘導上皮-間質轉換(epithelial to mesenchymal transition (EMT))在GO的致病機轉中亦扮演重要角色。然而CsA於人類牙齦上皮細胞的作用並未完全明瞭。本研究的實驗目的探討環孢素於人類牙齦上皮細胞經由TGF-β訊息傳遞路徑引發GO機轉。 實驗方法：利用西方墨點法檢測環孢素所引發的上皮-間質轉換(epithelial to mesenchymal transition (EMT)) 之表現及活化態TGF-β Enzyme Linked Immunosorbent Assay (ELISA) 檢測環孢素CsA及前處理各種不同抑制劑對於人類牙齦上皮細胞的作用。 實驗結果：本研究發現人類牙齦上皮OECM-1和Ca9-22細胞株在處理環孢素48小時後，有上皮鈣黏蛋白(E-cadherin)表現量下降，slug表現量上升的EMT現象。以TGF-β1的中和抗體前處理後可抑制環孢素所誘導的EMT表現。前處理抗氧化劑N-acetyl-cystenine (ROS抑制劑)、Diphenylene iodonium (NOX抑制劑)、Plumbagin (NOX4抑制劑)、Apocynine (NOX2抑制劑)、薑黃素(Curcumin) 和Lovastatin可以抑制環孢素誘導的活化態TGF-β產生及過氧化物(ROS)生成。另外我們也發現薑黃素劑量依賴性地抑制OECM-1和Ca9-22細胞株中環孢素所誘導的過氧化物生成及活化態TGF-β1的釋放。 結論：環孢素可透過過氧化物誘導活化態TGF-β1的釋放於人類牙齦表皮細胞，薑黃素可以顯著抑制環孢素所誘導的過氧化物生成及活化態TGF-β1的釋放，冀望未來薑黃素可以成為治療牙齦過度增生的潛力藥物。

Objectives: Transforming growth factor-β (TGF-β) is a key regulator associated with pathogenesis of gingival overgrowth. Cyclosporin-A (CsA) has been shown to induce gingival overgrowth through TGF-β signaling pathway. However, the mechanism(s) of CsA-induced TGF-β1 activation in human gingival epithelium (hGE) cells is still unknown. The aims of this study were to investigate the mechanisms of CsA-induced TGF-β1 activation in hGE cells. Materials and Methods: Effects of CsA on EMT and activated-TGF-β1 levels in hGE cells (OECM-1 and CA9-22) were assessed by Western blot analysis and enzyme-linked immunosorbent assay. Results: The expression of E-cadherin decreased and slug increased after 48 hours treatment with CsA in both OECM-1 and CA9-22 cells. Pretreatment with TGF-β1 neutralizing antibody in OECM-1 and CA9-22 cells inhibited the effects of CsA on OECM-1 and CA9-22 cells. Pretreatment with N-acetyl-cystenine (reactive oxygen species (ROS) inhibitor), Diphenylene iodonium (NOX inhibitor), Plumbagin (NOX4 inhibitor), Apocynine (NOX2 inhibitor), Curcumin and Lovastatin in OECM-1 and CA9-22 cells significantly inhibited CsA-induced TGF-β1 activation. Moreover, curcumin dose-dependently inhibited CsA-induced latent TGF-β1 activation in OECM-1 and CA9-22 cells. Conclusion: CsA induced latent TGF-β1 activation via ROS generation in hGE cells. Curcumin significantly inhibited CsA-induced ROS generation and latent TGF-β1 activation in hGE cells and could be a potential therapy of gingival overgrowth.