p as a cyclin dependent kinase
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 mma f 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. SIRT1 has deacetylated regulation on numerous nonhistone protein substrates [Atgs, Foxo1, Foxo3, PGC-1α, NF-kB, E2F1 and p53] (Conrad et al., 2016) to play a key role in protecting against cell stress. Therefore, the role of SIRT1 in fluorine-induced oxidative stress was explored. The results revealed that group pretreated with SRT1720 significantly reduced the intracellular ROS level compared with NaF group; whereas group pretreated with Ex-527 presented a more significant increase of ROS level. It suggested that SIRT1 activation attenuated the increase of intracellular ROS level. Here, the role of SIRT1 in fluorine-induced apoptosis and cell cycle was also investigated. The Annexin V-FITC/PI dual staining results showed that apoptosis rate was attenuated after pretreatment with SRT1720 and exacerbated after pretreatment with Ex-527 when compared with group treated with NaF alone. In addition, pretreatment of SRT1720 caused an inhibition at the S-phase arrest. Furthermore, the protein expression of Ac-p53, p21 and Caspase-3 presented a significant reduction with pretreatment of SRT1720, and p53 protein expression remained unchanged， when compared with treatment with NaF alone. Acetylation increases p53 protein stability, binding to promoters, and association with signaling proteins. Ac-p53 is indispensable for cell cycle checkpoint responses to DNA damage. Additionally, Ac-p53 develops its regulation effect on cell cycle arrest via cyclin-dependent kinase inhibitor 1A/p21 (CDKN1A/p21) and modulates apoptosis (Bao et al., 2016). SIRT1 deacetylates p53 at Lys379 to inhibit p53-dependent apoptosis (Suzuki et al., 2018). The results of the study demonstrate that SIRT1-mediated p53 deacetylation is critical to alleviating mitochondrial-mediated intrinsic cell apoptosis and growth inhibition during fluoride toxicity. The regulation of p53 acetylation by fluoride and SIRT1 in the current study was shown in Fig. 10.