薑黃素可抑制環孢素經由TGF-β誘導牙齦上皮細胞的LOXL2表現

Abstract

環孢素A（Cyclosporin A）為一種免疫抑制劑，常用於器官移植的患者，服用此藥物的患者大約有七成會出現副作用牙齦過度增生（Gingival overgrowth，GO），進而導致患者美觀、功能以及生活上受影響。近期研究發現GO的組織中在結締組織及上皮有大量表現的離氨基氧化酶樣蛋白2（Lysyl Oxidase-like protein 2, LOXL2），且LOXL2也因為可以造成上皮間質轉化（Epithelial-mesenchymal transition, EMT）而被認為可能是器官纖維化的治療標的。先前實驗室以抑制劑研究，發現環孢素在人類牙齦上皮細胞Ca9-22中可能經由TGF-β1及LOXL2誘導EMT的產生。本研究確認以TGF-β1處理人類牙齦上皮細胞Ca9-22及另一株牙齦上皮細胞OECM-1可誘導LOXL2的表現。表皮生長因子（EGFR）抑制劑AG1478、NF-kB抑制劑BAY 11-7082、PI3K抑制劑LY294002能夠明顯降低TGF-β1誘導Ca9-22細胞中LOXL2的表現。顯示TGF-β非典型路徑可經由EGFR、NF-kB 、PI3K 訊息傳導路徑誘導LOXL2的表現。LOXL2 siRNA能明顯降低TGF-β1誘導Ca9-22的EMT標誌蛋白N-Cadherin、Vimentin、Snail表現，確認LOXL2在TGF-β誘導Ca9-22的EMT扮演重要的角色。活性氧化物（Reactive oxygen species, ROS）可以調節Cyclosporin A誘導牙齦上皮細胞Ca9-22及OECM-1中LOXL2的表現。薑黃素（Curcumin）可以降低Cyclosporin A與TGF-β1誘導牙齦上皮細胞Ca9-22及另一株牙齦上皮細胞OECM-1的LOXL2表現。當Curcumin濃度到達2.5μM時，即開始顯著抑制Ca9-22及OECM-1細胞中LOXL2表現，並具有劑量依賴效應。從這些結果中我們確認Cyclosporin A在口腔上皮細胞中可以經由TGF-β1誘導LOXL2促使EMT的產生。薑黃素可以經由抑制TGF-β1誘導LOXL2的表現，進而阻斷LOXL2所帶來對於口腔上皮細胞的EMT現象，而成為具有防治GO潛力的藥物。

Background: Cyclosporin A is an immunosuppressant commonly used in organ transplant patients. Approximately 70% of patients taking this medication experience side effects gingival overgrowth, which may impair their esthetic, function, and quality of life. Current research indicates that, Lysyl Oxidase-like protein 2 (LOXL2) is associated with epithelial-mesenchymal transition (EMT), leading to tissue overgrowth. Previous studies from our laboratory using chemical inhibitors have shown that Cyclosporin A led to EMT possibly through TGF-β and LOXL2 in gingival epithelium cell. The aim of this study is to find out the correlation between Cyclosporin A, TGF-β and LOXL2 during EMT in human gingival cells. Materials and methods: Two human gingival cell lines (Ca9-22 and OECM-1) were used in this study. The levels of LOXL2 and EMT associated markers, N-cadherin, vimentin and Snail were analyzed by Western blot. Result: TGF-β1 increased LOXL2 expression in Ca9-22 and OECM-1 cells in the dose of 2ng/ml and time-dependent manner. Pretreatment with EGFR inhibitor AG1478, NF-kB inhibitor BAY 11-7082(BAY) and PI3K inhibitor LY294002 suppressed TGF-β1-induced LOXL2 synthesis in Ca9-22. This indicate that LOXL2 expression is induced from TGF-β1 through EGFR, NF-kB and PI3K in noncanonical pathway. Reactive oxygen species (ROS) such as xanthine oxidase, NOX2, NOX4, NOS and mitochondrial ROS can regulate the expression of Cyclosporin A-induced LOXL2 expression in human gingival cells. Furthermore, curcumin decreased Cyclosporin A- and TGF-β1-induced LOXL2 expression in Ca9-22 and OECM-1 cells in a dose dependent manner. At concentration of 2.5μM, curcumin significantly inhibited LOXL2 expression in human gingival epithelial cells. Conclusions: TGF-β1 increased LOXL2 expression through EGFR, NF-kB and PI-3K signaling in human gingival epithelial cells. LOXL2 is required for TGF-β1-induced EMT in human gingival epithelial cells. Curcumin decreased Cyclosporin A- and TGF-β1-induced LOXL2 expression at a concentration of 2.5μM in human gingival epithelial cells.