凝血酶誘導人類牙齦纖維母細胞結締組織生長因子表現之研究

Abstract

結締組織生長因子(CTGF)和許多人類器官組織的纖維化病變有很密切的相關性，在人類牙齦過度生長的組織中，也可以看到CTGF的過度表現；研究指出，即使在良好的回診及口腔衛生照顧下，牙齦過度生長之患者在接受牙齦切除術後十八個月的再發率仍高達34%。凝血酶曾被報導能誘導肺部纖維母細胞CTGF的表現，造成肺部的纖維化；過去研究也指出，在牙齦切除術後的早期愈合過程中，大量的血小板與凝血因子，會聚集在傷口處，可能和手術之後牙齦過度生長的快速再發有關。因此我們假設，凝血酶也扮演相同的角色。 本研究發現，凝血酶能以劑量與時間相關性來刺激人類牙齦纖維母細胞CTGF的表現。使用凝血酶受器PAR1之促效劑SFLLRN，和凝血酶有類似的刺激效果；而使用絲胺酸蛋白酶抑制劑PPACK，則可以完全抑制凝血酶誘導的CTGF表現，顯示凝血酶是透過其蛋白酶活性，酶切活化PAR1，來引發後續的訊息傳導。接著使用不同的訊息傳遞路徑抑制劑來前處理牙齦纖維母細胞，西方點墨法的結果顯示：mitogen-activated protein kinase (MAPK)訊息傳導路徑抑制劑中的ASK1抑制劑thioredoxin、JNK抑制劑SP600125 能顯著降低凝血酶誘導的CTGF表現，但使用PI3K抑制劑LY294002、ERK抑制劑PD98059、p38 MAPK抑制劑SB203580則無抑制效果。其次，使用抗氧化劑N-acetyl-L-cysteine (NAC)、Rac-GTPase抑制劑NSC-23766、NADPH oxidase (NOX)抑制劑apocynin和DPI亦能顯著降低凝血酶誘導的CTGF表現，而5-lipoxygenase activating protein (FLAP) 抑制劑MK886則無抑制效果。進一步使用AP-1抑制劑curcumin及茶多酚EGCG，對凝血酶誘導的CTGF表現也能夠得到劑量相關性的抑制效果。 總結之，在人類牙齦纖維母細胞中，凝血酶可能是透過 PAR1/ROS/ASK1/JNK/AP-1此一路徑來誘導CTGF的表現，且NOX可能是此路徑中ROS的來源。我們亦發現，EGCG可以抑制凝血酶誘導的CTGF表現，未來期望可以深入研究EGCG抑制纖維化的機制，提供預防或治療牙齦過度生長及其再發的新策略。

Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues and was found to overexpress in the tissue of gingival overgrowth. It has been reported that, in spite of a proper recall program after surgical treatment, the recurrent rate of gingival overgrowth in 18 months was still up to 34%. Thrombin has been implicated in lung fibrosis, with its capacity of inducing CTGF expression in lung fibroblasts. So we postulate that, the recruitment of platelets and normal clotting factors, like thrombin, early in the wound healing process could be responsible for the rapid re-growth of fibrotic gingival tissue. In this study, we showed that thrombin caused a concentration- and time-dependent increase in CTGF expression in human gingival fibroblasts (GFs). The effect of thrombin could be mimicked with the protease-activated receptor 1 (PAR1) agonist peptide, SFLLRN, and could be completely inhibited by a serine protease inhibitor, PPACK, indicating that thrombin mediated this effect via the proteolytic cleavage and activation of PAR1. We further used several inhibitors of mitogen-activated protein kinase (MAPK) pathway and reactive oxygen species (ROS) pathway, and a popular chemopreventive agent, EGCG, to investigate the key signaling molecules in thrombin-induced CTGF expression in GFs. The results of western blotting revealed that, the inhibitors of MAPK pathway, ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), but not PI3K inhibitor (LY294002), ERK inhibitor (PD98059), p38 MAPK inhibitor (SB203580), could significantly reduce the level of thrombin-induced CTGF. In addition, antioxidant N-acetyl-L-cysteine (NAC), Rac-GTPase inhibitor (NSC-23766), NADPH oxidase inhibitors (apocynin and DPI), but not 5-lipoxygenase activating protein (FLAP) inhibitor (MK886), had the inhibitory effects on the thrombin-induced CTGF. Furthermore, AP-1 inhibitor (curcumin) and EGCG could also attenuate the stimulatory effect of thrombin on CTGF expression with a dose-dependent manner. These results suggested that thrombin-induced CTGF expression in GFs could be mediated by PAR1, ROS, ASK1, JNK and AP-1 pathways, and the ROS-generating NADPH oxidase may play a part in the pathways as well. EGCG also displays a chemopreventive potential in treating or preventing the recurrence of gingival overgrowth.