EGCG抑制溶血磷脂酸誘導人類牙齦纖維母細胞結締組織生長因子表現

Abstract

LPA（lysophosphatidic acid）是一種glycerophospholipids。當血小板受到活化時，會釋放LPA至血清中。血漿中LPA濃度大約為~100 nM，而血清中的濃度則高達5 μM。傷口癒合的過程中，LPA 調節很多纖維母細胞所行使的重要功能，包括增殖、移動和收縮。結締組織生長因子（CTGF）屬於CCN家族的一員，與許多組織的纖維化相關。在人類牙齦纖維母細胞中，LPA會刺激CCN2/CTGF表現。臨床上以牙齦切除術來治療牙齦過度生長有相當高的再發比例，推測初期傷口癒合過程中血小板中豐富的LPA刺激CTGF蛋白表現與牙齦過度生長有高度相關性。 實驗目的：本研究探討牙齦纖維母細胞中，LPA誘導CTGF表現的訊息傳遞路徑，希望可以藉由調控這些路徑中的成員，來尋找有效的化學預防物質抑制牙齦纖維母細胞中，LPA誘導的CTGF表現。 材料與方法：取在牙周手術過程中的健康牙齦組織進行牙齦纖維母細胞培養。使用15 μM LPA來刺激牙齦纖維母細胞並以西方墨點法分析EGCG對於LPA誘發CTGF蛋白、Smad3蛋白和JNK蛋白的影響。 結果：以TGF-β1 receptor ALK5 inhibitor（SB431542）、JNK inhibitor（SP600125）和Smad3 inhibitor（SIS3）作用後會有效地降低LPA所誘導的CTGF蛋白。另外以EGCG先處理3小時也可以有效地降低LPA所誘導的CTGF蛋白、磷酸化的JNK和Smad3蛋白。 結論：EGCG抑制LPA誘發之CTGF蛋白表現是透過抑制JNK和Smad3的路徑。 EGCG可以做為未來治療牙齦增生之潛力藥物。

Lysophosphatidic acid (LPA; 1-acyl-sn-glycerol-3-phosphate), the simplest of glycerophospholipids, is released into serum by activated platelets. Plasma LPA is ~100 nM with serum values as high as 5 μM. LPA regulates many important functions of fibroblasts in wound healing, including proliferation, migration, and contraction. Connective tissue growth factor（CTGF）belongs to CCN family and is a cysteine-rich polypeptide. It has been implicated in the onset and progression of fibrosis in most human tissue. LPA stimulate CCN2/CTGF expression in human fibroblastic cells. It has been reported that the re-growth of fibrotic gingival tissues in these patients is rapid following surgery. A potential mechanism for the rapid re-growth of gingival tissues is the early recruitment of platelets to the wound. Platelets are rich in LPA and also carry CCN2/CTGF protein. Objectives：We investigate the mechanism of LPA stimulation of CTGF in primary cultures of human gingival fibroblasts and identify unique signal pathways that mediate their effects. Therefore, we want to investigate the inhibition effect of chemopreventive agent in LPA induced CTGF expression in human gingival fibroblasts. Methods：Healthy gingival tissues were obtained from the patient under treatment of periodontal flap operation. Gingival fibroblasts were isolated by collagenase/dispase digestion method and used for the subsequent analysis. Gingival fibroblasts were stimulated with 15 μM LPA and the effect of EGCG（20 μM）on LPA induced expression of CTGF protein, phosphorylated Smad3 and JNK protein expression were analyzed by western blotting. Results：Pretreatment with TGF-β1 receptor ALK5 inhibitor（SB431542）, JNK inhibitor（SP600125）and Smad3 inhibitor（SIS3）significantly reduced LPA induced CTGF expression. Furthermore, pre-treatment with EGCG three hours before LPA stimulation demonstrated that EGCG reduced CTGF expression. EGCG also attenuated the phosphorylation of Smad3 and JNK induced by LPA. Conclusion：It is concluded that EGCG suppressed LPA induced CTGF expression probably through the interruption of JNK and Smad3 signaling. Further study for application of EGCG in clinical treatment is suggested.