The intracellular redox situation of

The intracellular redox situation of MC3T3-E1 Lenalidomide hydrochloride after exposure with fluoride has been investigated and results revealed that the generation of ROS increased at a concentration-dependent manner, which indicated that NaF induced oxidative stress damage in MC3T3-E1 cells. Fluoride-mediated inducement of oxidative stress brings out significant depletion of the activities of the antioxidant enzymes and augment of lipid peroxidation (Wang et al., 2014). Chronic fluorosis leads to the generation of free radicals and alterations in antioxidants or reactive oxygen species scavenging enzymes via oxidative stress abduction (Barbier et al., 2010; Suzuki et al., 2015). An increase of ROS production was also considered as a typical event in the process of mitochondrial-mediated early apoptosis (Huang et al., 2015). Previous studies have emphasized that fluoride induces apoptosis through elevating oxidative stress-induced lipid peroxidation, thereby causing mitochondrial dysfunction and the induction of downstream signaling pathways (Ameeramja et al., 2016; Cao et al., 2015; Inkielewicz-Stepniak et al., 2014; Ke et al., 2016). Therefore, the apoptosis parameters of MC3T3-E1 cells in response to fluoride treatment were evaluated in this study. Flow cytometer and Western blotting data indicated that NaF induces a significant increase in the apoptotic rate, and the significant upregulation of ratio of Bax and Bcl-2 expression, as well as the increase of protein expression of Cyt c and Caspase-3 suggested that fluoride induces mitochondria-dependent cell apoptosis. Furthermore, fluoride significantly increased protein expression of p53 and up-acetylated p53 at L379 site in MC3T3-E1 cells. p53 as a transcription factor regulates the expression of genes and miRNAs which are associated with a number of important cellular processes including proliferation, DNA repair, apoptosis, autophagy, metabolism, and cell migration (Liu et al., 2017; Tiwari et al., 2018). Acetylation of p53 increases the protein stability of p53, binding to low-affinity promoters, interaction with other proteins, antiviral potentials as well as checkpoint responses to DNA damage and activated oncogenes (Brooks and Gu, 2011; Munoz-Fontela et al., 2011; Reed and Quelle, 2014). It is widely reported that the inhibition of histone deacetylases (HDACs) may remove acetyl groups from p53 (i.e., HDAC1 and SIRT1) and promotes p53 acetylation and p53-dependent apoptosis and senescence (Schafer et al., 2017). In the present study, fluoride-induced acetylation of p53 might exert vital role in apoptosis promotion. p21, as a cyclin-dependent kinase inhibitor, exert significant negative regulator of proliferation, and it could be transcriptional upregulated by p53 in order to impel transient cell cycle arrest (Romanov et al., 2010). Using western blotting, we found that the protein expression of p21 upgrades firstly than descending lately, suggesting that p21 is involved in the regulation of cell cycle progression by fluoride. P21 has been shown not only to negatively modulate cell cycle progression and block DNA replication, but also have the novel roles in controlling ROS levels by positively regulating the transcriptional activity of Nrf2 (Deng et al., 2016; Chen et al., 2009). The previous study performed by the member in our research group indicated that p21 ameliorated cell death and ROS generation induced by Dexamethasone through activation activating of the Nrf2/HO-1 signaling pathway, playing a vital resistant role in the development and progress of osteoporosis (Han et al., 2018). Therefore, it is possible that high dose of NaF could increase the ROS generation and cell death through reducing the expression levels of p21 since the effect of NaF on osteoblast largely depends on fluoride concentration. The underlying mechanism would be explored in further investigation. Furthermore, cell cycle distribution was also analyzed after cells were exposed to NaF, which showed that fluoride caused an increase of cells in the S-phase, indicating that NaF arrest cell cycle of MC3T3-E1 cells in S-phase and interrupt the progress from S-phase to G2/M, blocking mitosis and inducing apoptosis. It has been suggested that fluoride has differential effects depending on the cell type. In agreement with these results, recently, analysis of the effects of fluoride on cell cycle phases in cultured rat osteoblasts indicated an increased number of cells at S phase and a decrease in cells at G2/M phase or G0/G1 (Liu et al., 2018). In our study, different concentrations of fluoride were used. As we known, fluoride has dual role which depends on its exposure doses and time. This might result in variation of G1 phase arrest which is not in accordance with general p53-dependent mechanism.