4. Discussion

Cellular senescence is a pathophysiologica l phase in which the cell cycle is arrested and cells stop growing. In recent years, cellular senescence has been widely highlighted as one of the pathogenesis mechanisms that regulate various diseases such as tumors, degenerative diseases, and chronic diseases [20]. The reason and mechanism of cell senescence are not fully understood. Oxidative stress is the main external cause of cell senescence that is more generally accepted. Cells undergoing senescence have characteristic features, displaying a large and flat morphology: an increase in SA-?-gal, DNA damage, and a stable cell cycle arrest executed by interplaying between the RB and p53 tumor suppressor pathways (often accumulate the p16INK4a, p15INK4b, and p21Cip1/Waf1 cyclin-dependent kinase inhibitors that engage the RB pathway or mediate p53 effects accordingly) [21, 22]. Previous studies have shown that there are senescent cells in the liver of patients with non-alcoholic fatty liver (NAFLD) and cirrhosis. The increase in the number of senescent cells can promote the occurrence of NAFLD, but the underlying mechanism is not fully elucidated [23, 24].

It has been verified that the increased oxidative stress induced by fluoride was associated with DNA fragmentation in rat hepatocytes, which was one of the main mechanisms leading to liver damage. As oxidative stress is one of the main causes of cell senescence, these studies suggested that fluorosis may cause liver damage by inducing cell senescence. However, there are very few studies on the toxic effects of fluoride by inducing cell senescence. The present study investigated the effects of fluoride on hepatocyte senescence through in vitro and in vivo experiments.

The results showed that fluoride could induce cell senescence by ?-galactosidase staining on frozen sections of liver tissues of chronic fluorosis rats and L02 cells treated with different concentrations of NaF. With the increase of fluoride concentration, the SA-?-gal-positive cells were increased obviously, suggesting that the degree of senescence was positively correlated with the concentration of fluoride. Concerning the levels of p16 and p21, the hallmarks of cell senescence, it was found that the expression levels of p16 and p21 were increased in a concentration-dependent manner in fluorosis rats’ liver and NaF treated L02 cells, which further confirmed that fluoride could induce hepatocyte senescence. As p16 was the main tumor suppressor, which could induce cell senescence and cycle arrest. After exposure NaF to L02 cell, the cell cycle was arrested in the G2 phase by flow cytometry. At the same time, the level of IL-8 in the liver tissue of fluorosis rats and culture medium of fluoride-treated L02 cells were significantly increased. Taken together, our results confirmed that fluoride could induce hepatocyte senescence, which might play an important role in the fluoride induced liver damage.

It is clear now that SASP components actively participate in the senescence process. For example, key SASP factors (IL-6 and IL-8) act in an autocrine feedback loop to reinforce the senescence growth arrest. Nuclear factor-?B (NF-?B) is a master regulator of SASP and is used to further probe the impact of SASP biology on the senescence program [25]. A large number of studies suggested that NF-?B signaling was highly activated in cellular senescence [15]. NF-?B gene expression was also enhanced in primary rat hippocampal neurons by fluoride treatment in a dose-dependent manner, which meant fluoride might regulate cell senescence by activating NF-?B signaling [26]. The NF-?B system is an evolutionarily conserved signaling pathway, which can be triggered by not only immune activation but also diverse external/internal danger signals associated with senescence and even the aging process (oxidative and genotoxic stresses). IKBKG is generally called NEMO, important regulatory component of IKK complex linked upstream to genotoxic signals and IL-1 and TNF receptor mediated signaling [16]. Recent studies have shown that IKBKG protein plays a major role in the activation of NF-?B signals caused by DNA damage, which suggests that IKBKG may be involved in the process of cell senescence [17, 27]. Studies have found that IKBKG can also regulate liver diseases at different functional levels [28] [29]. This study further explored the role of IKBKG in fluoride-induced hepatocyte senescence, hoping to provide a theoretical basis for the pathogenesis of fluorosis.

The results showed that the expression of IKBKG increased in liver tissues of chronic fluorosis rats and liver L02 cells exposured with NaF in a concentration-dependent manner. The results suggested that fluoride might induce hepatocyte senescence via up-regulating the expression of IKBKG. To further verify the role of IKBKG in fluoride-induced hepatocyte senescence, tail vein injection of AAV-IKBKG and transfected sh-IKBKG to L02 cells were adopted to knock down the level of IKBKG. The results showed that compared with the fluorosis rat or L02 cells treated with fluoride, the SA-?-gal-positive cells were decreased, the expression levels of P16 and P21 decreased, the IL-8 level decreased in rat liver or L02 cells treated with NaF and down-regulation of IKBKG, and the number of L02 cells arrested in the G2 phase was also reduced.

In conclusion, the chronic fluorosis rats and human L02 cells exposed with NaF as to evaluate whether fluoride could induce hepatocyte senescence and its potential regulated mechanism. The results indicated that fluoride might cause liver damage by inducing cell senescence, and its mechanism might be related to the up-regulation of IKBKG expression in hepatocytes. This research might shed more light on the underlying pathogenesis of fluorosis, the senescence is complex pathophysiological process, IKBKG just as a pathological factors and further researches should be designed to fully elucidate the regulation signal pathway of IKBKG in the role of cell senescence in the toxicity of fluoride.

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