Melatonin protects TEGDMA-induced preodontoblast mitochondrial apoptosis via the JNK/MAPK signaling pathway

Abstract

Resin monomer-induced dental pulp injury presents a pathology related to mitochondrial dysfunction. Melatonin has been regarded as a strong mitochondrial protective bioactive compound from the pineal gland. However, it remains unknown whether melatonin can prevent dental pulp from resin monomer-induced injury. The aim of this study is to investigate the effects of melatonin on apoptosis of mouse preodontoblast cells (mDPC6T) induced by triethylene glycol dimethacrylate (TEGDMA), a major component in dental resin, and to determine whether the JNK/MAPK signaling pathway mediates the protective effect of melatonin. A well-established TEGDMA-induced mDPC6T apoptosis model is adopted to investigate the preventive function of melatonin by detecting cell viability, apoptosis rate, expressions of apoptosis-related proteins, mitochondrial ROS (mtROS) production, mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) level. Inhibitors of MAPKs are used to explore which pathway is involved in TEGDMA-induced apoptosis. Finally, the role of the JNK/MAPK pathway is verified using JNK agonists and antagonists. Our results show that melatonin attenuates TEGDMA-induced mDPC6T apoptosis by reducing mtROS production and rescuing MMP and ATP levels. Furthermore, mitochondrial dysfunction and apoptosis are alleviated only by the JNK/MAPK inhibitor SP600125 but not by other MAPK inhibitors. Additionally, melatonin downregulates the expression of phosphorylated JNK and counteractes the activating effects of anisomycin on the JNK/MAPK pathway, mimicking the effects of SP600125. Our findings demonstrate that melatonin protects mDPC6T cells against TEGDMA-induced apoptosis partly through JNK/MAPK and the maintenance of mitochondrial function, offering a novel therapeutic strategy for the prevention of resin monomer-induced dental pulp injury.

1. Introduction

Due to the superior performance, ease of operation and aesthetic properties, resin-containing compounds are being used for a wide variety of dental applications, such as restorations, sealants, bonding agents, and dental pulp capping [1‒3]. The most commonly used monomer compounds in dental resins are bisphenol-A-glycidyl methacrylate (Bis-GMA), 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) [4]. Depending on the application, different amounts of the resin compounds mentioned above depolymerize and release residual monomers into oral tissues [5]. Depolymerized resin monomers can diffuse across the dentin layer through the dentinal tubules, with concentrations ranging from 0.2 to 8 mM [6,7]. This diffusion can trigger a wide variety of cellular responses in the pulp tissue, including apoptosis, inflammation, and impairment of dental mineralization. Apoptosis is the primary manifestation of resin monomer-induced dental pulp injury, commonly observed as a result of cell toxicity [8‒11], which not only aggravates the existing damage but also obstructs the defense responses of the dentin-pulp complex. Therefore, exploring the mechanisms of TEGDMA-induced cell apoptosis and finding potential preventive/therapeutic strategies are essential for improving restorative materials for clinical application.

Mitochondria play vital roles in many cellular processes, including cell proliferation, metabolism, and apoptosis [12‒14]. Accumulating evidence has indicated that an excess of reactive oxygen species (ROS) in mitochondria promotes caspase-dependent apoptosis. As a result, mitochondria are identified as the center of apoptosis through the intrinsic pathway [15]. Furthermore, an excess of ROS causes respiratory dysfunction and reduces adenosine triphosphate (ATP) generation, which further promotes mitochondrial ROS (mtROS) production and oxidative damage, thereby creating a vicious cycle. Previous research has shown that the cytotoxicity of TEGDMA to oral tissues leads to an accumulation of mtROS and irreversible mitochondrial damage [16,17]. Our latest study also demonstrated that mitochondrial dysfunction is the major factor in preodontoblast apoptosis induced by TEGDMA [18]. However, the detailed mechanism, especially the signaling pathway that regulates mitochondrial dysfunction in TEGDMA-induced cell apoptosis, needs further exploration.