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    • PBIT br Materials and methods br

    2023-10-25


    Materials and methods
    Results
    Discussion Fluoride can readily penetrate cellular membranes and seriously damage the structure and physiological function of cells (Zhou et al., 2015). Several studies have shown that excessive fluoride can decrease the viability of cells and that the growth of cells is inhibited by the excess presence of fluoride ion (Chae et al., 2016, Yan et al., 2015, Yang et al., 2015). Our previous work confirmed this inhibition in HeLa cells (Wang et al., 2017a). In the present study, the viability of C2C12 cells was significantly decreased with increasing fluoride concentrations, and the IC50 was shown to be 3.209 mmol/L. The IC50 of fluoride exposure was 1.9 mmol/L in human bronchial epithelial cells (BEAS-2B) and was 5.8 mmol/L in human liver cells (HL-7702) (Ying et al., 2017). These results suggested that high concentrations of fluoride affect cell viability of all types and inhibit cellular growth, though the tolerance of different cells exposed to fluoride is different. This finding provides evidence for fluoride-induced toxicology in C2C12 cells. To explore the theory that fluoride would decrease the viability of C2C12 cells, the histopathological and ultrastructural changes were observed. The C2C12 cellular structures were obviously changed from the control group, with nuclear pyknosis and deformities seen in the fluoride group. The number of C2C12 cells was also reduced by the presences of excess fluoride. This result was consistent with the fluoride-induced decrease in viability of the C2C12 cells. Under TEM observation, the mitochondria were observed to be swollen and vacuolised, with indistinct ridges. Furthermore, the endoplasmic reticulum was dilated, and nuclear pyknosis was visible in the C2C12 cells in the fluoride group. The above histopathological characteristics were due to the beginnings of apoptosis. Several studies indicated that the fluoride-induced mitochondrial damage and endoplasmic reticulum swelling were highly associated with the apoptosis process (Wang et al., 2017b, Zhang et al., 2015, Deng et al., 2016). Moreover, the increasing number of positive cells observed in the Hoechst staining provided direct evidence in this case for fluoride-induced apoptosis in C2C12 cells. PI3K, PDK1 and AKT, proteins in the PI3K/AKT pathway, play an important role in the regulation of PBIT progression (Chang et al., 2003). The phosphorylation of AKT is an important link in the cell apoptosis process (Gu et al., 2016). Phosphorylated AKT could facilitate cell survival and proliferation and regulate various signaling pathways (Sussman, 2007). The present study found that with the significant decrease of the expression level of PDK1, the P-AKT1 expression level significantly decreased in C2C12 cells exposed to fluoride. Due to the week suppression to BAD by P-AKT1 made it possible to induce apoptosis in C2C12 cells with the fluoride treatment. Bcl-2 is a widely studied modulator of programmed cell death. Moreover, Bax, as a member of the Bcl-2 family, can form heterologous dimeric complexes with Bcl-2 and accelerate programmed cell death (Lou et al., 2014). Recent studies reported that the Bcl-2 levels decreased and Bax levels increased in presence of excessive fluoride (Liu et al., 2013, Cao et al., 2013). However, the present study showed that the expression levels of Bcl-2 and Bax both increased in the C2C12 cells exposed to excessive fluoride. The increases in both Bcl-2 and Bax expression might be a feedback effect following the C2C12 cells apoptosis caused by fluoride (Lou et al., 2014). Furthermore, Bax, an opposition partner of Bcl-2, its over expression promotes the level of Bcl-2 increased. Our results indicated that Bax and Bcl-2 play a pivotal role in the PI3K/AKT pathway involving the fluoride-induced apoptosis of the C2C12 cells. Caspase-9 is the key initiating caspase for the intrinsic pathway to cell death (Yan et al., 2009). In the present study, the level of caspase-9 expression in C2C12 cells significantly increased in the fluoride group. It is reported that caspase-9 can be activated through Apaf1 binding to cytochrome c in the cytoplasm, thereby forming a multi-protein complex known as an apoptosome that leads to apoptosis (Wu et al., 2014). BAD, a pro-apoptotic protein, heterodimerises with anti-apoptotic proteins such as Bcl-2 and Bcl-xL to promote cell death (Martelli et al., 2006, Franke et al., 2003, Li et al., 2013). It was reported that the increased expression of BAD promoted the apoptosis induced by fluoride in human oral squamous cell carcinoma HSC-2 cells (Otsuki et al., 2011). In the present study, the expression levels of BAD significantly increased promotes the fluoride-induced apoptosis in C2C12 cells. Several studies suggested that a deficit of BAD can result in diminished mitochondria-based glucokinase activity and blunted mitochondria respiration in response to glucose, which may be associated with apoptosis (Danial et al., 2003, Ranger et al., 2003, Seo et al., 2004), thus this mechanism requires further study. These results, taken together, indicated that the PI3K/AKT pathway is involved in the fluoride-induced apoptosis of C2C12 cells.