4-甲基丙烯酰氧基偏苯三酸酐對人類牙髓細胞的毒性反應研究

Abstract

目的：功能性單體4-甲基丙烯酰氧基偏苯三酸酐(4-methacryloxyethyl trimellitic anhydride, 4-META)被廣泛使用於牙科樹脂黏著劑(resin adhesives)中，這些樹脂材料填充在牙齒中、接觸牙齒表面，可能面臨聚合不完全、長期下來於口腔環境中水解的可能，過去的研究已證實許多牙科材料常見的樹脂單體都具有細胞毒性，會導致DNA破壞、細胞氧化壓力上升、干擾細胞週期運行、造成細胞死亡、誘導發炎反應等。然而，迄今為止對於4-META的研究相當匱乏，憑現有的研究我們仍無法了解4-META對於人類牙髓細胞的毒性及可能造成的影響。本研究目的在探討4-META對於人類牙髓細胞所造成的影響，包括：細胞活性、細胞週期的運行、細胞凋亡、氧化壓力及發炎反應的變化，並進一步研究調控細胞週期、氧化壓力、細胞凋亡及發炎反應相關之基因與蛋白質的表現，希望能了解4-META造成毒性的機制。

實驗方法：本實驗使用體外培養的初代人類牙髓細胞，加入0、0.5、1、 2.5、5及7.5 mM的4-META培養24小時，以MTT檢測細胞存活率，使用流式細胞術以PI分析細胞週期、以PI與Annexin V雙染分析細胞凋亡、以DCF螢光染色分析細胞活性氧的生成、以CMF螢光染色分析細胞穀胱甘肽的生成，並以即時定量聚合酶連鎖反應、西方點墨法及免疫化學螢光染色來檢測細胞週期調控因子(cyclin B1、cdc25C、cdc2、p21、p53、GADD45α)、細胞凋亡調控因子(BAX)、氧化壓力調控因子(Nrf2、HO-1)、發炎調控因子(COX-2、IL-6、IL-8)的基因與蛋白質表現之變化。

實驗結果：當人類牙髓細胞暴露在2.5 mM的4-META時會導致細胞存活率降低、細胞形態產生改變，本實驗測得的半抑制濃度應介於2.5 mM - 5 mM之間。在2.5 mM的4-META會使人類牙髓細胞的活性氧生成增加、並產生S phase停滯。當4-META濃度上升至5及7.5 mM時，GSH濃度下降、細胞凋亡的比例、sub G0/G1比例顯著增加。高濃度的4-META會使細胞週期調控因子的表現cdc2、cyclin B1、cdc25C表現下降，p21、p53、GADD45α表現上升。4-META會提高氧化壓力調控因子Nrf2、HO-1、細胞凋亡調控因子BAX、以及發炎相關因子COX-2、IL-6、IL-8的表現。

結論：由本實驗的結果，我們推測4-META造成人類牙髓細胞的毒性其成因是由於活性氧生成量增加，導致細胞的氧化壓力上升，進而造成DNA的受損而改變細胞週期調控因子的表現，擾亂細胞週期的運行。而隨著4-META濃度上升，細胞無法於細胞週期停滯時完成修復、穀胱甘肽的耗盡、細胞凋亡與壞死增加。而當氧化壓力上升，會活化Nrf2，因而提高HO-1的表現量。4-META造成的毒性也會使發炎相關因子的表現上升，誘導發炎反應。就目前我們的研究結果可以初步瞭解人類牙髓組織在接觸到4-META後會造成的影響。但對於4-META造成氧化壓力上升、干擾細胞週期、及如何誘導發炎等機制尚有許多未了解之處，還待未來更進一步的研究，希望未來能更了解這些機制，更能有助於臨床上的應用與牙科材料的改良。

Aim: As a functional monomer, 4-methacryloxyethyl trimellitic anhydride (4-META) is widely used in dental resin adhesives. These resin materials which are in long-term contact with dental tissues may face potential issues such as incomplete polymerization and hydrolysis, thus cause monomer releasing into the oral environment consequently. Previous studies have confirmed that many common resin monomers in dental materials have cytotoxicity, leading to DNA damage, increased cellular oxidative stress, disturbance of cell cycle progression, cell death, and induced inflammatory reactions. However, research on 4-META has been scarce so far. Based on existing few studies, the cytotoxicity and potential effects of 4-META on human dental pulp cells are poorly understood. The aim of this study is to investigate the effects of 4-META on human dental pulp cells, including cell viability, cell cycle progression, cell apoptosis, production of reactive oxygen species, and inflammatory responses. Furthermore, in order to understand the mechanisms of the toxicity caused by 4-META, the expression of some important factors regulating cell cycle, oxidative stress, apoptosis, and inflammation were also analyzed.

Material and methods: In this experiment, primary human dental pulp cells were cultured in vitro and treated with final concentrations of 0, 0.5, 1, 2.5, 5, and 7.5 mM of 4-META for 24 hours. Cell viability was assessed using the MTT assay. Flow cytometry with propidium iodide (PI) staining was used to analyze cell cycle distribution, while dual staining with PI and Annexin V was used to analyze cell apoptosis. The production of reactive oxygen species was analyzed using DCF fluorescence staining, and the production of cellular glutathione was assessed using CMF fluorescence staining. Real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunofluorescence staining were performed to examine the changes in gene and protein expression of cell cycle regulatory factors (cyclin B1, cdc25C, cdc2, p21, p53, GADD45α), apoptosis related factor (BAX), oxidative stress regulatory factors (Nrf2, HO-1), and inflammatory related factors (COX-2, IL-6, IL-8).

Results: Exposure of human dental pulp cells to 2.5 mM of 4-META resulted in decreased cell viability and cell morphological changes. At 2.5 mM of 4-META, the production of reactive oxygen species in human dental pulp cells elevated, and S phase cell cycle arrest occurred. When the concentration of 4-META increased to 5 mM and 7.5 mM, the glutathione concentration decreased, the proportion of cell apoptosis and sub G0/G1 phase raised significantly. High concentrations of 4-META lead to a decrease in the expressions of cdc2, cyclin B1, and cdc25C, while the expressions of p21, p53, and GADD45α were increased. 4-META also enhanced Nrf2, HO-1, and the apoptosis related factor BAX. It also enhances the expression of pro-inflammatory factors such as COX-2, IL-6, and IL-8.

Conclusions: The present in vitro experiment demonstrated that the toxicity of 4-META on human dental pulp cells may be related to the increase of reactive oxygen species, leading to elevated oxidative stress and subsequent DNA damage, which alters the expression of cell cycle regulatory factors and disturbs cell cycle progression. As the concentration of 4-META increases, cells may not be able to repair DNA completely during cell cycle arrest, leading to increased cell apoptosis and necrosis. 4-META may also trigger inflammatory responses by inducing the expression of inflammatory related factors. The results of this study provide a preliminary understanding of the toxic effect and mechanism of 4-META on human dental pulp tissue. Further studies are needed to verify the mechanisms of the toxicity of 4-META and develop safer materials in clinical use.