薑黃素對Sphingosine 1-Phosphate誘發牙齦纖維母細胞表現結締組織生長因子之抑制效果及機制探討

Abstract

本研究進行S1P對影響人類牙齦纖維母細胞表現CCN2的探討， 進而對S1P在牙齦纖維母細胞中訊息傳遞路徑的調控有更深入的瞭解。 實驗目的: S1P為一種存在血清中且具生物活性的鞘脂 (sphingolipid)， 主要是由活化的血小板所分泌， 可調控包括增殖、 遷移、 分化、 血管新生和細胞外間質之重塑等多種細胞反應。 結締組織生長因子 (CTGF/CCN2) 和許多人體組織纖維化的發生和發展有關， 包括口腔頰側黏膜以及牙齦組織。 薑黃素(Curcumin) 在活化之肝臟星狀細胞可抑制CTGF基因的表現， 抑制膠原蛋白的合成以及細胞外間質的堆積， 而達到抑制肝臟纖維化的效果。 近期研究發現S1P藉由與 TGF-β1訊息傳遞途徑的相互影響， 可能在纖維化的過程扮演了一個重要的角色。 然而， S1P在牙齦纖維母細胞中的訊息傳遞機制仍不清楚。 材料與方法: 在牙周手術中收集健康的牙齦組織， 進行牙齦纖維母細胞的培養， 利用西方墨點分析法， 測定CCN2的表現量， 以及不同藥物對抑制CCN2表現的效果。 實驗結果: S1P在人類牙齦纖維母細胞中可誘發CCN2基因表現。 使用SIS3 (Smad3抑制劑) 以及 SP600125 (JNK抑制劑) 對牙齦纖維母細胞做前處理， 可明顯抑制由S1P所誘發之CCN2蛋白合成。 然而使用SB431542 (ALK5抑制劑)、 PD98059 (MEK抑制劑)、 SB203580 (p38 MAPK抑制劑) 進行前處理則並無明顯抑制S1P誘發CCN2蛋白合成之效果。 另外， 薑黃素對於由S1P所誘發之CCN2蛋白合成以及Smad3的活化有顯著抑制效果， 但是對JNK活化則無抑制。 因此， 我們推測S1P可經由Smad3及 JNK 路徑調控並誘導CCN2蛋白合成， 薑黃素則是透過抑制Smad3路徑而非JNK路徑， 來抑制S1P誘發之CTGF表現。 期望將來可研發薑黃素成為治療牙齦纖維化之潛力藥物。

This study was undertaken to examine the effect of S1P on the expression of CCN2 in human gingival fibroblasts (GF) to gain further insight into the regulation of the S1P pathway in GFs. Objectives: Sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid secreted predominantly by activated platelets, regulates a variety of cellular responses including proliferation、 migration、 differentiation、 angiogenesis, and extracellular martrix remodeling. Connective tissue growth factor (CTGF/CCN2) is associated with the onset and progression of fibrosis in many human tissues including buccal mucosa and gingiva. Curcumin inhibits CTGF gene expression in hepatic stellate cells, it further inhibits synthesis of collagen and accumulation of extracellular matrix to control liver fibrosis. Recent studies have shown that S1P may play an important role in fibrosis through crosstalk with the TGF-beta pathway. However, the effects of S1P on gingival fibroblasts remain unknown. Material and methods: Healthy gingival tissues were obtained from patients during periodontal surgery. Gingival fibroblasts were isolated and were used for subsequent analysis. Western blot analyses were used to assess CCN2 expressions and its inhibitory effects by different reagents. Results: We found that S1P stimulated CCN2 synthesis in human gingival fibroblasts. Pretreatment with SIS3 (Smad3 inhibitor) and SP600125 (JNK inhibitor), but not SB431542 (ALK5 inhibitor)、 PD98059 (MEK inhibitor)、 SB203580 (p38 MAPK inhibitor), significantly reduced S1P-induced CCN2 synthesis. Furthermore, curcumin completely inhibited S1P-induced CCN2 synthesis and Smad3 activation, but not JNK activation. Conclusions: These results indicated that S1P-induced CCN2 synthesis was mediated by Smad3 and JNK pathways and curcumin could be a useful agent in controlling gingival fibrosis.