Curcumin抑制Phenytoin對於人類牙齦纖維母細胞誘發CTGF/CCN2表現之研究

Abstract

Phenytoin為治療癲癇的常用藥物之一，但是常會造成病人產生牙齦腫大(Gingival overgrowth，GO)的不良影響，對於病患的口腔衛生清潔、語言、吞嚥等生活品質會造成嚴重影響，經由手術方式雖然可以獲得症狀緩解，但是再發生率卻很高。轉形生長因子-β1 (Transforming growth factor-β1，TGF-β1) 在牙齦過度增生的疾病發展進程中扮演重要角色。結締組織生長因子(Connective tissue growth factor，CTGF/ CCN2)表現被報告和GO的疾病嚴重程度呈正相關。但是Phenytoin誘導GO的機制並不十分清楚。本研究希望藉由研究Phenytoin對於人類牙齦纖維母細胞誘導的作用進而找出未來有效治療牙齦過度增生的藥物。我們發現人類牙齦纖維母細胞(Human gingival fibroblast，HGF)在Phenytoin的刺激下，具有隨濃度及時間誘導CCN2表現的特性。使用TGF-β1中和抗體、ALK5抑制劑SB431542、Smad3抑制劑可以有效降低Phenytoin誘導的CCN2蛋白表現。顯示Phenytoin的成纖維化特性可能經由TGF-β1訊息路徑調控。ELISA分析發現Phenytoin可使細胞培養液中活化態的TGF-β1表現量增加。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)、薑黃素(Curcumin)和茶多酚(EGCG)對牙齦纖維母細胞做前處理，會明顯抑制由Phenytoin所誘發活化態的TGF-β1表現量，然而使用Apocynin (NOX2抑制劑)進行前處理則無明顯抑制，表示TGF-β的活化可能受到過氧化物(Reactive oxygen species，ROS)的調控，ROS的來源可能是NOX4。進一步確認薑黃素的抑制效果隨濃度增加而增加，具有濃度依賴性。薑黃素可能成為預防或治療GO的潛力藥物。

Gingival overgrowth (GO) is a common complication of the usage of phenytoin. It causes severe adverse effects in life quality of patient in maintenance of oral hygiene, language and swallowing. Clinically, periodontal therapy offers removal of the inflammatory component of the GO through scaling and gingival curettage, followed by surgical interventions. However, the high recurrence rate of drug-induced gingival overgrowth requires gingivectomy must be repeated periodically. Transforming growth factor β (TGFβ) is a key regulator associated with the pathogenesis of GO. Connective tissue growth factor (CTGF/CCN2) is positively correlated with severity of GO. In this study, we found phenytoin induced CCN2 production in human gingival fibroblasts (HGF) in a time- and dose-dependent manner. Pretreatment with TGF-β1 neutralizing antibody, ALK5 inhibitor SB431542 and Smad3 inhibitor significantly reduced phenytoin-induced CCN2 protein production. Furthermore, we found phenytoin increased activated TGF-β1 levels in HGFs. Pretreatment of N-acetyl-cystenine(ROS inhibitor), Diphenylene iodonium (NOX inhibitor), Plumbagin (NOX4 inhibitor), cucurmin and EGCG on HGF significantly inhibits production of active form TGF-β1 induce by phenytoin, but not Apocynin (NOX2 inhibitor). These results suggested that the increased activated TGF-β levels is regulated by ROS and ROS came from NOX4. Furthermore, curcumin significantly inhibited phenytoin-induced CCN2 protein production in HGFs. Curcumin could be a potential medicine for prevention and therapy of GO.