The apoptosis is the main form of common resin composite materials induced dental pulp injury. Melatonin, a physiologically secreted anti-oxidant and mitochondrial protective product, able to efficaciously ameliorate mitochondrial dysfunction and inhibit cell apoptosis. However, there have no report on the role of melatonin in anti-resin monomer damage and its potential protective mechanisms. In our study, for the first time, we focus on the MAPK signaling, especially the JNK/MAPK pathway on the TEGDMA caused pre-odontoblasts mitochondrial apoptosis, and announce the pharmaceutical protection of the melatonin therefore representing a promising approach to the management of resin monomer induced dental pulp injury.
MtROS is an widely accepted initiator of apoptosis through numerous mechanisms, and it is not only related to damage of mitochondrial function, but also leading to an apoptotic signaling pathway cascade[9] [33] [34]. Notably, as the products of mitochondrial metabolism, mtROS comes from the respiratory chain when mitochondria are functionally disordered[12]. Mikulás et.al have found that TEGDMA inhibited Complex I in the respiratory chains of mitochondria isolated from guinea pig brain[35]. Nonetheless, our research demonstrated that TEGDMA damaged the Complex III activity of pre-odontoblasts, the disordered mitochondria manifested as ATP synthesis decrease and mtROS production increase[15]. Furthermore, mtROS generation is believed to be dependent on membrane potential of mitochondria. As the essential storage pool for electron chain, damaged mitochondria promote a reduction in electron flow when membrane potential across the inner membrane lost, hence ROS produce. As expected, our results found that the level of MMP and ATP significantly decreased, and the mtROS level was up-regulated, which confirmed the role of compromised mitochondrial function in TEGDMA-induced mDPC6T cells apoptosis.
Melatonin, a neurohormone mainly produced by the pineal gland, is a pleiotropic molecule with diverse physiological functions. In addition to its time-keeping function, melatonin acts as a potential mtROS scavenger and exhibits effective antioxidant properties. Studies have showed that this versatile hormonal compound has well-established anti-apoptotic activities[36] [37]. However, there is no report on the protective effect of melatonin on TEGDMA induced pro-odontoblasts apoptosis as well as its action mechanism. Here, we found that pretreatment with melatonin effectively inhibited TEGDMA-induced apoptosis of mDPC6T cells by mitigating the mitochondrial dysfunction. Notably, melatonin may act on cells through the following mechanisms. Firstly, because of its highly lipophilic nature, melatonin can penetrate cellular and membrane structure, and finally accumulates in mitochondria[38]. While it will enhance the activity of catalase, decreases Ca2+ influx, eliminates residual mtROS and maintains mitochondrial function[39]. On the other hand, the second way is that melatonin regulates the program cell death through a melatonin receptor. The G protein-coupled membrane receptors MT1 and MT2 are considered the primary molecules mediating the receptor-dependent pathways of melatonin[40] [41]. MT1 and MT2 trigger several signaling pathways, including the cAMP-response element binding protein (CREB), phosphatidylinositol 3-kinase (PI3K) and MAPKs signal pathways, and integrate different linear inputs to regulate cellular functions, such as circadian rhythm, cell differentiation, cumulus expansion and cell programed death[42] [43] [44]. However, which pathway is involved in the protective effect of melatonin on pro-odontoblastic cell apoptosis needs further verification.
Activated MAPKs exert various biological effects by promoting the phosphorylation of downstream substrates which then serve as signals in various cell responses including apoptosis. In our study, the phosphorylation of MAPKs was detected by Western blot, only p-JNK changed with time and concentration when mDPC6T was stimulated by TEGDMA. The results are different from those of previous studies[21] [22], as they found the phosphorylation of ERK1/2 and p38 MAPKs were more pronounced. Previous researches studied the effect of TEGDMA in mouse macrophages and treated cells for 24–48 hours, but we stimulated the mDPC6T with TEGDMA for about 6 hours. The distinct cell types and processing conditions may contribute to those inconsistences. Therefore, we specifically focused on JNK pathway regulated apoptosis. As expected, pretreatment with SP600125 notably abolished TEGDMA-induced apoptosis, increased mitochondrial function and suppressed mtROS production, and the opposite effect of Anisomycin on these results further proved that the JNK pathway is involved in the apoptotic effect of TEGDMA. JNK/MAPK not only is required for the release of Cytochrome C from the inner membrane space of mitochondria and the activation of the pro-apoptosis protein, like Bax and Caspase-3[45], but also leads to inhibition of mitochondrial respiration and electron transport, and the damaged mitochondria leads to the release of mtROS and the improvement of MMP[46]. Mechanistically, the directly disrupt of the interaction between JNK and mitochondria plays the important role in apoptosis. What’s more, Schweikl et.al[3] have reviewed the influence of monomer-induced oxidative stress on central signal transduction pathways including JNK/MAPK, which further confirmed that JNK singling pathway plays an important activator in TEGDMA-induced cell apoptosis. However, the pivotal role of JNK requires further validation through overexpression or silence by siRNA in our future study.
The release of melatonin in response to cellular stress by activating the JNK/MPAK pathway has been reported in various pathological processes[29] [42]. In our study, we found that melatonin antagonized mtROS and mDPC6T cells apoptosis caused by TEGDMA or Anisomycin alone. Meanwhile, melatonin significantly inhibited cell apoptosis induced by TEGDMA and Anisomycin together. Furthermore, melatonin mimicked the effects of the inhibitor SP600125 and abolished the suppressive effects of TEGDMA on p-JNK. Therefore, we proposed that melatonin played the role of mitigating mitochondrial dysfunction regulated apoptosis partly through the JNK/MAPK pathway. However, whether the receptors MT1 and MT2 act as upstream of JNK pathway in this process deserve further investigation.
Our current study, for the first time, demonstrated that melatonin exhibited excellent protective effect on pre-odontoblast cell apoptosis induced by TEGAMA, but there are still some limitations in the experiment. On one hand, a mouse pre-odontoblast cell line was only adopted, the primary dental pulp cell is needed to confirm the mechanism underlying the protective effect of melatonin on TEGDMA induced apoptosis. On the other hand, further in vivo studies should be performed to verify the preventive effect of TEGDMA on dental pulp injury and corroborate the role of JNK/MAPK signaling pathway.