10-MDP對人類牙髓細胞之影響：細胞自噬與細胞焦亡之角色探討

Abstract

目的：10-(2-甲基丙烯酰氧基)磷酸單癸酯(10-Methacryloyloxydecyl Dihydrogen Phosphate, 10-MDP)是廣泛使用於牙科樹脂黏著劑(resin adhesive)中的功能性單體(functional monomers)。然而，由於鍵結不穩定、聚合不完全以及長期在口腔環境下的材料水解等因素，這些樹脂材料可能會釋放到口腔環境中，並在接觸牙齒後經由牙本質小管滲入牙髓，對牙髓細胞造成傷害。過去的研究已經證實10-MDP具有細胞毒性，會導致細胞氧化壓力上升、誘導發炎反應、DNA損傷，使細胞細胞週期受到干擾和細胞型態發生變化，最終導致細胞死亡。然而，對於10-MDP毒性造成的細胞死亡和其背後的作用機制等相關研究目前尚未完全明瞭。細胞自噬(Autophagy)被認為是細胞應對毒性壓力挑戰時，進行自我保護的適應性調節機制。細胞死亡的機制則有分很多種，其中細胞焦亡(Pyroptosis)是一種受調節的溶解性細胞死亡方式，對發炎反應和疾病進展有顯著影響。本研究旨在探討細胞自噬及細胞焦亡在10-MDP引發的人類牙髓細胞毒性中所扮演的角色，我們希望能進一步了解10-MDP造成毒性的機制，並且對未來在含有10-MDP的牙科黏著劑上的改良可以提供一些幫助。 實驗方法：本實驗使用體外培養的人類牙髓細胞，加入0、10、50、100、250和500 μM的10-MDP培養24小時，以即時定量聚合酶連鎖反應(real-time quantitative PCR)、西方點墨法(western blot) 和免疫化學螢光染色(immunofluorescence)來檢測細胞自噬調控因子(Beclin-1、LC3B、p62、ATG5、ATG12)和細胞焦亡調控因子(NLRP3、Caspase-1、GSDMD)的基因與蛋白質表現之變化。再分別加入Autophagy inhibitor Lys05 (5或10 μM、NH4Cl (10或20 mM)、Pyroptosis inhibitor MCC950 (25和50 μM) 做預處理，以MTT 檢測細胞的存活率。 實驗結果：隨著10-MDP濃度的上升，會使人類牙髓細胞自噬相關性因子Beclin-1、LC3B、ATG5和ATG12的表現上升。在500 μM時，會使人類牙髓細胞焦亡相關性因子NLRP3、Caspase-1和GSDMD有最大的表現。加入自噬抑制劑後，可以觀察到細胞存活率與對照組相比呈現下降，且細胞形態拉長變形、細胞核呈現縮小、空泡狀或碎裂；加入焦亡抑制劑後，細胞存活率與細胞型態則無顯著的差異。 結論：本實驗顯示，10-MDP對人類牙髓細胞可能誘導細胞自噬，導致自噬相關因子Beclin-1、LC3B、ATG5、ATG12的表現增加。加入NH4Cl或Lys05自噬抑制劑後，細胞存活率下降，且細胞型態發生破壞性改變，推測細胞自噬作為一種自我保護機制，可以減少10-MDP造成的毒性影響。隨著細胞焦亡相關因子NLRP3、Caspase-1、GSDMD在高濃度10-MDP暴露下的表達增加，我們推測10-MDP可能會誘導人類牙髓細胞發生細胞焦亡。然而，加入MCC950焦亡抑制劑後，細胞存活率和細胞型態沒有顯著變化，我們推測細胞焦亡可能不是10-MDP誘導人類牙髓細胞死亡的主要原因。其他10-MDP造成細胞死亡的機制形式的機制仍有許多未解之處，還待未來進一步的研究來深入探討。

Aims: Dental resin adhesives commonly use 10-MDP (10-Methacryloyloxydecyl Dihydrogen Phosphate) as a functional monomer due to its strong bonding capabilities. However, resin materials often suffer from unstable bonding, incomplete polymerization, and material hydrolysis, leading to monomer leaching into the oral cavity. This leached monomer can infiltrate the pulp through dentinal tubules, potentially causing damage to pulp cells. Previous studies have confirmed that 10-MDP increases oxidative stress, induces inflammatory responses, and causes DNA damage, cell cycle disruption, and cell morphology alterations, ultimately resulting in cytotoxicity and cell death. In this study, we investigate the effects of 10-MDP-induced cytotoxicity and human dental pulp cell death. Two mechanisms are proposed and confirmed: autophagy and pyroptosis. Autophagy is an adaptive regulatory mechanism that cells employ to protect themselves against toxic stress. Pyroptosis, on the other hand, is a regulated form of lytic cell death, significantly impacts inflammatory responses and the progression of various diseases. In the future, we aim to further elucidate the mechanisms underlying 10-MDP toxicity, thereby providing insights for the development of safer dental materials. Material and methods: Primary human dental pulp cells were cultured in vitro and treated with 0, 10, 50, 100, 250, and 500 μM of 10-MDP for 24 hours. Changes in the expression of autophagy regulatory factors (Beclin-1, LC3B, p62, ATG5, ATG12) and pyroptosis regulatory factors (NLRP3, Caspase-1, GSDMD) at both gene and protein levels were detected using real-time quantitative PCR, western blotting, and immunofluorescence staining. Additionally, cells were pre-treated with autophagy inhibitors Lys05 (5 or 10 μM) and NH4Cl (10 or 20 mM), as well as the pyroptosis inhibitor MCC950 (25 and 50 μM), to examine cell viability using the MTT assay. Results: As the concentration of 10-MDP increases, the expression of autophagy-related factors Beclin-1, LC3B, ATG5, and ATG12 in human dental pulp cells also increases. At 500 μM, the expression of pyroptosis-related factors NLRP3, Caspase-1, and GSDMD reaches its peak in human dental pulp cells. Upon the addition of autophagy or pyroptosis inhibitors, changes in the survival rate and morphology of human dental pulp cells exposed to 10-MDP can be observed. Conclusions: The present in vitro experiment demonstrated that the toxicity of 10-MDP may induce autophagy in human dental pulp cells, leading to the increase of autophagy-related factors Beclin-1, LC3B, ATG5, and ATG12. Following the addition of autophagy inhibitors NH4Cl or Lys05, a decrease in cell survival rate and destructive changes in cell morphology were observed. It is speculated that autophagy, as a self-protection mechanism, can reduce the toxic effects caused by 10-MDP. With the increased expression of pyroptosis-related factors NLRP3, Caspase-1, and GSDMD, we hypothesize that high concentrations of 10-MDP may induce pyroptosis in human dental pulp cells. However, when the pyroptosis inhibitor MCC950 was added, no significant changes in cell viability or morphology were observed. We speculate that pyroptosis may not be the primary cause of 10-MDP-induced cell death in human dental pulp cells. Other mechanisms of cell death induced by 10-MDP remain unclear and warrant further investigation.