4-META及10-MDP對人類牙髓細胞的影響：程序性壞死與內質網壓力之表現

Abstract

目的： 4-甲基丙烯酰氧基偏苯三酸酐 (4-Methacryloxyethyl trimellitate anhydride. 4-META)及 10-(2-甲基丙烯酰氧基)磷酸單癸酯 (10-methacryloyloxydecyl dihydrogen phosphate, 10-MDP)為牙科黏著系統中常用之功能性單體，未完全聚合之單體可能經由牙本質擴散至牙髓組織，進而影響牙髓細胞之生物活性。這兩種單體對人類牙髓細胞之細胞毒性及其相關細胞死亡機制尚未被完全了解。近年研究指出，內質網壓力 (endoplasmic reticulum stress, ER stress)與程序性壞死 (necroptosis)不僅參與細胞死亡調控，亦與發炎反應及組織損傷密切相關，因此在牙髓炎、牙髓修復及牙科材料生物相容性評估上具有重要意義。目前有關功能性單體是否透過內質網壓力及程序性壞死途徑影響人類牙髓細胞之研究仍相當有限。本研究旨在探討 4-META 與 10-MDP 對人類牙髓細胞之影響，並分析內質網壓力及程序性壞死相關訊號路徑之變化，希望能進一步了解這兩種單體造成毒性的機制，並且在未來可以對材料改良上有一些幫助。

實驗方法： 以不同濃度之 4-META 及 10-MDP 處理人類牙髓細胞 24 小時後，利用即時定量聚合酶連鎖反應 (real-time PCR)及免疫螢光染色 (immunofluorescence)檢測 ER stress 相關因子，包括 GRP78、PERK、eIF2⍺、ATF4、ATF6、XBP1u、XBP1s 及 CHOP，以及程序性壞死相關分子 RIPK1、RIPK3 及 MLKL 的表現變化。另外，分別加入 RIPK3 抑制劑 GSK872 後進行 MTT assay 評估細胞存活率，並以 Annexin V-FITC/PI flow cytometry 分析細胞死亡型態。

實驗結果： 在 ER stress 相關訊號方面，細胞暴露於 4-META 後，PERK、eIF2⍺ 及 ATF4 mRNA 表現在部分濃度有顯著增加，而 GRP78、ATF6、XBP1u、XBP1s 及 CHOP 則呈現上升趨勢；免疫螢光染色結果亦顯示相關蛋白質表現有不同程度的增加。另一方面，RIPK1、RIPK3 及 MLKL mRNA 表現皆有增加趨勢，且免疫螢光染色顯示 RIPK1、RIPK3 及 p-MLKL 蛋白表現增加。然而，加入 RIPK3 抑制劑 GSK872 後，細胞存活率並未獲得顯著改善，顯示程序性壞死可能並非造成細胞死亡之主要機制。在 10-MDP 處理組中，ATF4 及 CHOP mRNA 表現在部分濃度有顯著增加，而其餘 ER stress 相關基因則呈現上升趨勢；免疫螢光染色結果同樣顯示 GRP78、PERK、eIF2⍺、ATF4、ATF6、XBP1s 及 CHOP 蛋白表現有不同程度增加。RIPK1、RIPK3 及 MLKL mRNA 表現亦有增加趨勢，且免疫螢光染色顯示 RIPK1、RIPK3 及 p-MLKL 蛋白表現在部分濃度有增加趨勢。然而，加入 GSK872 後，細胞存活率仍未見明顯恢復。

結論： 本實驗顯示，4-META 及 10-MDP 可誘導人類牙髓細胞產生內質網壓力反應，導致 GRP78、PERK、eIF2⍺、ATF4、ATF6、XBP1s 及 CHOP 等相關分子表現有不同程度增加。顯示兩種功能性單體在人類牙髓細胞中可能活化部分內質網壓力相關訊號路徑。另外，4-META及10-MDP也造成RIPK1、RIPK3及MLKL等程序性壞死相關分子表現增加，推測兩種功能性單體可能有活化一些程序性壞死相關訊號。然而，加入RIPK3抑制劑GSK872後，細胞存活率並未獲得顯著改善，因此推測程序性壞死可能並非4-META及10-MDP誘導人類牙髓細胞死亡之主要機制，比較可能是細胞受到壓力刺激後所產生的綜合反應。綜合上述結果，本研究推測內質網壓力及程序性壞死在4-META及10-MDP所造成之細胞毒性中有一定的影響，但可能不是細胞死亡的主要原因。關於4-META及10-MDP細胞毒性之分子機制，未來仍需要進一步研究加以釐清。

Aims: 4-Methacryloxyethyl trimellitate anhydride (4-META) and 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) are commonly used functional monomers in dental adhesive systems. Recent studies have suggested that endoplasmic reticulum (ER) stress and necroptosis are involved in the regulation of cell death and inflammatory responses and may play important roles in pulp tissue homeostasis and biocompatibility evaluation of dental materials. However, the effects of 4-META and 10-MDP on ER stress- and necroptosis-related signaling pathways in human dental pulp cells remain unclear. Therefore, the aim of this study was to investigate the effects of 4-META and 10-MDP on cell viability, cell death, ER stress, and necroptosis-associated signaling pathways in human dental pulp cells.

Materials and methods: Primary human dental pulp cells were cultured in vitro and treated with various concentrations of 4-META and 10-MDP for 24 hours. The expression levels of endoplasmic reticulum (ER) stress-related markers, including GRP78, PERK, eIF2⍺, ATF4, ATF6, XBP1u, XBP1s, and CHOP, as well as necroptosis-related molecules, including RIPK1, RIPK3, and MLKL, were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. Furthermore, the involvement of necroptosis was investigated using the RIPK3 inhibitor GSK872, and cell viability was evaluated by MTT assay. Apoptotic cell death was analyzed by Annexin V-FITC/propidium iodide (PI) flow cytometry.

Results: Regarding ER stress-related signaling, exposure to 4-META significantly increased the mRNA expression levels of PERK, eIF2⍺, and ATF4, whereas GRP78, ATF6, XBP1u, XBP1s, and CHOP showed increasing trends. Immunofluorescence staining further demonstrated elevated protein expression levels of these ER stress-related markers. In addition, the expression levels of RIPK1, RIPK3, and MLKL were upregulated, and increased protein expression of RIPK1, RIPK3, and phosphorylated MLKL (p-MLKL) was observed. However, treatment with GSK872 failed to significantly restore cell viability, suggesting that necroptosis may not be the predominant mechanism responsible for cell death. In the 10-MDP-treated groups, ATF4 and CHOP mRNA expression levels were significantly increased, while the other ER stress-related genes exhibited upward trends. Immunofluorescence staining also revealed increased protein expression of GRP78, PERK, eIF2⍺, ATF4, ATF6, XBP1s, and CHOP. Furthermore, the expression levels of RIPK1, RIPK3, and MLKL, as well as their corresponding proteins, were elevated. Nevertheless, inhibition of RIPK3 by GSK872 did not significantly improve cell viability. Flow cytometric analysis demonstrated increased apoptotic cell populations in both 4-META- and 10-MDP-treated groups, accompanied by concentration-dependent reductions in cell viability.

Conclusion: The present study demonstrated that 4-META and 10-MDP induced ER stress in human dental pulp cells and increased the expression of necroptosis-related molecules. However, inhibition of RIPK3 failed to rescue cell viability, suggesting that necroptosis is unlikely to be the major mechanism underlying monomer-induced cytotoxicity. Instead, ER stress may play a central role in the cytotoxic effects of 4-META and 10-MDP and may promote apoptotic cell death through the PERK/eIF2⍺/ATF4/CHOP signaling pathway. Further studies are required to elucidate the precise role of necroptosis and other potential mechanisms involved in monomer-induced cytotoxicity.