Curcumin Inhibits TGF-β-induced CTGF Expression in Human Gingival Fibroblasts

許多纖維化的病變都可以偵測到轉化生長因子β (TGF-β)的上升。而轉化生長因子β不僅會促進間質蛋白的合成，同時也會增加蛋白酶抑制劑的分泌，使得膠原蛋白堆積。結締組織生長因子(CTGF)是一種間質蛋白，屬於CCN家族的一員，為富含cysteine的多胜肽，由轉化生長因子β所調控，與許多組織之纖維化有關，也會促進轉化生長因子β所產生纖維化之效果，因此認為抑制結締組織生長因子的表現，可以有效抑制纖維化病變。薑黃素(curcumin)為咖哩中主要的黃色成分，已被證實可以抑制肝臟星狀細胞的活化及膠原蛋白的合成。實驗目的：第一，探討牙齦增生組織，CTGF免疫染色的表現；第二，探討薑黃素對TGF-β1誘發正常牙齦纖維母細胞產生CTGF之抑制效果及其機制。材料與方法：經過同意書的簽署後，收集臨床上七例牙齦增生的組織，進行CTGF免疫組織染色。接著取自年齡介於25-40歲，拔牙時之健康牙齦組織，進行牙齦纖維母細胞培養，利用細胞活性(MTT assay)、反轉錄聚合酶連鎖反應(RT-PCR)及西方點墨法(western blot)，完成下列實驗。結果發現：在免疫組織染色方面，藥物引起之牙齦增生組織中，上皮細胞、纖維母細胞、及微血管之內皮細胞與平滑肌細胞，有明顯的CTGF陽性染色。以5 ng/mL TGF-β1刺激正常牙齦纖維母細胞6小時，CTGF的表現達到高峰。MTT assay顯示，Curcumin的濃度在10 μM以下，對正常牙齦纖維母細胞的活性不會產生影響。若先以10 μM curcumin處理1小時，再以5 ng/mL TGF-β1刺激6小時，可發現會明顯抑制CTGF的表現。此外以10 μM curcumin先處理1小時會抑制TGF-β1所引發之Smad2/3、ERK、JNK及p38磷酸化，且抑制Smad2進入細胞核，但加入20 μM ERK inhibitor (U0126)、JNK inhibitor (SP600125)、p38 inhibitor (SB203580)先處理1小時，再以5 ng/mL TGF-β1刺激6小時，卻沒有抑制CTGF之表現。同時以10 μM curcumin 先處理1小時也不會抑制TGF-β receptor type I及type II的表現。因此，推測薑黃素抑制TGF-β1誘發之CTGF表現主要是透過抑制Smad2/3的路徑。Many fibrotic pathologies are associated with increasing levels of TGF-β. TGF-β not only increases synthesis of matrix proteins but also enhances secretion of protease inhibitors while reducing secretion of proteases and resulted in matrix accumulation. Connective tissue growth factor (CTGF) belongs to CCN family and is a cysteine-rich polypeptide. It acts as a down stream profibrotic effector of TGF-β and has been proved to be associated with the onset and progression of fibrosis in many human tissues. Curcumin, a yellow pigment from turmeric, has been shown to reduce matrix accumulation in pulmonary and hepatic fibrosis. Objectives: One of the purposes of this study is to compare CTGF expression between normal gingival tissue and overgrowth gingival tissue. The other one is to investigate the inhibition effect of curcumin in TGF-β induced CTGF expression in normal primary human gingival fibroblasts. Methods: Tissue specimens from 7 cases of gingival overgrowth were examined by immunohistochemistry with the expression of CTGF protein. Healthy gingival tissues were obtained from patients under treatment of odontectomy. Gingival fibroblasts were isolated by collagenase/dispase digestion method and used for the subsequent analysis in the third passage. The effect of curcumin on cell viability was tested by MTT assay. Gingival fibroblasts were stimulated with 5 ng/mL TGF-β1 and the effects of curcumin (10 μM) on TGF-β1 induced CTGF gene and CTGF protein, TGF-β1 receptors protein, phosphorylated Smad2/3 proteins and MAPK proteins expression were analyzed by RT-PCR and western blotting. Results: Drug-induced gingival overgrowth was found with positive CTGF staining in epithelial cells, fibroblasts, endothelial cells and smooth muscle cells. Curcumin was nontoxic at doses of 0 to 10 μM. Pre-treatment with curcumin one hour before TGF-β1 stimulation demonstrated that curcumin reduced CTGF expression. Curcumin also attenuated the phorsphorylation of Smad2/3, MAPK and translocation of Smad2 induced by TGF-β1. Interruption of MAPK signaling by inhibitors did not reduce the CTGF expression. Conclusion: It is concluded that curcumin suppressed TGF-β1-induced CTGF expression probably through the interruption of Smad2/3 signaling. Further study for application of curcumin in clinical treatment is suggested