丁香油酚對人類牙髓細胞的細胞毒性和發炎反應的影響

Abstract

實驗目的：丁香油酚擁有多種生物學功能，包括抗菌、抗發炎、止痛、抗氧化、抗癌等特性。丁香油酚現今是牙科常用的間接覆髓材料成分之一，過去的研究顯示丁香油酚可經由牙本質緩慢的滲透進入發炎的牙髓腔中，達到抗發炎的效果；不過丁香油酚在高濃度下會對牙髓細胞產生細胞毒性，但各研究產生細胞毒性的濃度存在差異。另外，過去的研究很少著墨於丁香油酚對於健康人類牙髓細胞之發炎反應影響。本研究的目的是想探討丁香油酚對於人類牙髓細胞的細胞毒性和發炎反應以及相關細胞訊息傳導路徑的影響。 實驗方法：自拔除的牙齒(n=15)培養人類牙髓細胞，以不同濃度的丁香油酚(10µM~5mM)刺激細胞、並以光學顯微鏡觀察細胞形態的變化，再以MTT細胞存活率分析檢測細胞活性。之後將細胞分為兩大組做後續實驗:健康細胞組(n=12)和發炎細胞組(n=3)，其中發炎細胞組是以大腸桿菌之脂多醣(Lipopolysaccharide, LPS) (1 µg/ml)刺激細胞24小時誘發發炎反應。將兩大組別細胞加入不同濃度的丁香油酚(10µM~1.5mM)處理24小時或48小時後，以酵素結合免疫吸附分析法(ELISA)和西方墨點法(western blot)，觀察丁香油酚對於促發炎細胞激素的釋放、發炎路徑及細胞膜內訊息分子的影響。最後則以Mitogen-activated protein kinase kinase(MEK)/Extracellular Signal-regulated Kinase(ERK)的抑制劑(PD98059和U0126)預處理人類牙髓細胞1小時後，加入丁香油酚觀察抑制劑對於訊息傳導路徑的影響。實驗中分析的促發炎激素包含第六型介白質(interleukin-6, IL-6)、第八型介白質(interleukin-8, IL-8)、前列腺素E2(prostaglandin E2, PGE2)，訊息傳導路徑的蛋白包括Phosphoinositide 3-kinases(PI3K)/ Protein kinase B(PKB)、ERK 1/2、c-Jun N-terminal kinase(JNK)、p38 mitogen-activated protein kinases(p38)、nuclear factor-kappa B(NF-κB)、Cyclooxygenase-2(COX-2)和轉錄因子nuclear factor-kappa B(NF-κB)、Activator Protein-1 (AP-1)。 實驗結果: 丁香油酚在小於1mM的濃度下並不會影響人類牙髓細胞的細胞活性及細胞形態上的改變，但濃度大於2mM卻會開始導致細胞死亡。丁香油酚具有抑制LPS所誘發的發炎人類牙髓細胞之促發炎激素產生，包括: IL-6、IL-8、PGE2，10μM就具有很好的抗發炎的效果。另外，健康人類牙髓細胞加入不同濃度的丁香油酚處理後會造成COX-2的表現和ERK 1/2的磷酸化，且隨著丁香油酚的濃度上升有正向表現：COX-2在丁香油酚濃度500μM開始有統計學上顯著差異、並隨濃度持續上升趨勢。p-ERK則在濃度750uM有顯著的增加、而後慢慢地下降。抑制劑預處理一小時後的結果顯示，U0126和PD98059對於丁香油酚所引發之COX-2表現量的上升，具有抑制效果。然而，丁香油酚並不會造成健康人類牙髓細胞的促發炎激素IL-6、IL-8和PGE2的產生。 結論: 丁香油酚在小於1mM的濃度下並不會造成人類牙髓細胞的細胞毒性，但濃度大於2mM卻會開始導致細胞死亡。健康人類牙髓細胞加入丁香油酚後可能經由MEK/ERK訊息傳導路徑造成COX-2的表現；然而，丁香油酚並不會造成健康人類牙髓細胞的促發炎激素IL-6、IL-8和PGE2的產生。另外，丁香油酚濃度從10μM開始可以抑制LPS所誘發的人類牙髓細胞之發炎作用。現今丁香油酚廣泛用於牙科間接覆髓材料，因此丁香油酚對健康牙髓細胞所造成的影響對於牙科臨床治療相當重要，未來還需要更多的研究針對丁香油酚造成COX-2表現相關的訊息傳導路徑及下游所調控的蛋白做更進一步的探討。

Aim: 4-allyl-2-methoxyphenol (eugenol) is a phenolic compound and the main component of clove. Eugenol has several pharmacological activities, including antibacterial, anti-inflammatory, analgesic, antioxidative and antitumor activities. Dental materials containing zinc oxide–eugenol (ZOE) have been widely used in indirect pulp capping therapy. Eugenol released from ZOE material can penetrate through dentinal tubules into pulp chamber and have anti-inflammatory effect upon inflamed pulp tissue. However, eugenol present some toxicity on human dental pulp cells (hDPCs) in the high concentrations. The effect of eugenol on inflammatory process of healthy hDPCs has not been well investigated in the previous studies. This study is aimed to obtain the cytotoxicity concentration of eugenol and to evaluate inflammatory effect of eugenol on hDPCs. Materials and methods: Primary-cultured hDPCs from extracted teeth (n=15) were exposed to different concentrations of eugenol (10µM to 5mM). Cell morphology was observed under optical microscopy and cytotoxicity was measured by a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After that, hDPCs were divided into two groups: the healthy hDPCs group (n=12) and the inflamed hDPCs group (n=3). hDPCs that were stimulated with the LPS of E. coli (1 µg/ml) for 24 hours was considered as the inflamed hDPCs group. Two experimental groups were treated with different concentrations of eugenol(10µM to 1.5mM) for 24 or 48 hours. Culture medium was collected for determination of prostaglandin E2 (PGE2), interleukin-6 (IL-6) and interleukin-8 (IL-8) production by enzyme-linked immunosorbent assay (ELISA). The cellular proteins were extracted for western blot analysis. For understanding the possible signal pathway of the eugenol regulation, hDPCs were pretreated with either PD98059 or U0126 (inhibitors of MEK/ERK) 1 hour before the treatment of eugenol and LPS stimilation. Then, the cellular proteins were extracted for western blot analysis. Results: Eugenol in the concentration less than 1 mM showed little cytotoxicity and morphologic changes in hDPCs. Cell depth could be observed when the concentration of eugenol was elevated beyond 2 mM. In LPS-stimulated hDPCs, treatment with eugenol (10~900 μM) significantly suppressed the production of IL-6, IL-8 and PGE2. In healthy hDPCs, treatment with eugenol (10~1500 μM) enhanced the protein expression of COX-2 in a concentration-dependent manner. After eugenol treatment (10~1500 μM), the highest protein expression of p-ERK 1/2 was at 750 mM then began to drop down. The enhancement of COX-2 which induced by eugenol was repressed by U0126 and PD98059. However, eugenol (10~1500 μM) did not induced the proinflammatory cytokines such as IL-6, IL-8 and PGE2 in healthy hDPCs. Conclusion: Eugenol in the concentration less than 1 mM showed little cytotoxic effect. Cell depth could be observed when the concentration of eugenol was elevated beyond 2 mM. In the LPS-stimulated hDPCs, eugenol in the concentration over 10 μM exhibited a significant inhibition of IL-6, IL-8 and PGE2 formation that indicates eugenol have anti-inflammatory effects. Eugenol did not stimulate proinflammatory cytokines such as IL-6, IL-8 and PGE2 production in healthy hDPCs. However, eugenol significantly induced the expression of COX-2 in healthy hDPCs with dose-dependently through ERK pathway. A further investigation on the effect of eugenol on inflammatory process in healthy hDPCs is necessary in the future.