Previous studies have reported that excessive fluoride exposure induced liver damage. However, the underlying mechanism of fluoride-induced hepatic toxicity is still unclear. Hence, this study was aimed to evaluate the fluoride-induced apoptosis, autophagy, and IL-17 signaling pathway-related genes to explore the possible mechanisms of NaF-induced liver injury in mice. For this, 48 male mice were allotted randomly to four groups, treated with deionized water, 25, 50, 100 mg/L NaF for 150 days continuously. Our results suggested that treatment with NaF decreased the PAS staining-positive area, with a concomitant increase in liver score, and serum ALT and AST levels which indicated that NaF induced the liver injury. In addition, the qRT-PCR, immunohistochemistry, and western blotting results indicated that NaF exposure activated IL-17 signaling, apoptosis, and autophagy pathways. In summary, these results suggested that NaF induced apoptosis and autophagy in liver by activating the IL-17 signaling pathway, eventually leading to impaired liver function.
Apoptosis, characterized by nuclear fragmentation, chromatic condensation, and caspase activation, is a major molecular mechanism to eliminate cells (Oropesa-.vila et al. 2017 ). Apoptosis is observed widely in liver diseases (Cui et al. 2018 ; Potz et al. 2016 ). Fluoride, as an exogenous toxicant, has been reported to cause apoptosis in the liver (Khan et al. 2018 ). Song et al. (2015 ) reported that chronic fluoride exposure induced the liver apoptosis through activating the apoptotic pathways. Zhan et al. (2006 ) treated the pig with 400 mg/kg fluoride for 50 days, and found that fluoride induced apoptosis by elevating the oxidative stress and further activating caspase-9 and caspase-3. Bcl-2, Cyt-c, Apaf-1, caspase3, caspase12, and p53 are key genes in the apoptosis. Among them, Bcl-2 is an anti-apoptotic protein and the others are proapoptotic protein (Ryu et al. 2018 ; Zhu et al. 2017 ). In this study, we evaluated the mRNA and protein expression of these genes to determine the effects of fluoride on the apoptosis. The mRNA expressions of caspase3, caspase12, and p53 were significantly increased; the mRNA levels of Cyt-c and Apaf-1 were significantly in the 50 and 100 mg/L NaF. However, the mRNA expression of Bcl-2 was significantly decreased in the NaF treated group. The results of protein levels of caspase3 and Cyt-c were similar with the mRNA. The above results indicated that fluoride exposure induced apoptosis by activating the apoptotic pathway in the liver.
… Autophagy, as an important modulator for cellular homeostasis, is known to eliminate the intracellular cell contents (Sungwoo et al. 2016 ). Recent studies have demonstrated that autophagy is closely related to liver injury (Lv and Qiao 2018 ). LC3 is a marker indicating the formation of autophagic vesicles, which plays the role through LC3I converting to LC3II. The increase in the ratio of LC3BII/ LC3BI reflects the levels of autophagic activity (Sungwoo et al. 2016 ). Benlin1 promotes the formation of autophagosomes by combining with PI3K (Cheng et al. 2017 ). As an autophagic substrate, the enhancement of autophagosome formation or the blockage of autophagosome degradation leads to the accumulation of p62, eventually p62 enter into mature autophagosomes, and degrade in autophagosomes, so the levels of p62 are negatively correlated with autophagy (Zhao et al. 2018 ). Because of the important role of these factors in autophagy, many studies have used them as indicators of autophagy (Zhang et al. 2018a ). In the present study, TEM results showed that autophagosomes were significantly increased in the cells after 50 and 100 mg/L NaF treated. Moreover, it was observed that fluoride induced an increase in the mRNA and protein expressions of LC3 and Beclin1, while led to a decrease in p62 mRNA and protein levels. MDC is an acidic dye, which stains autophagosomes and lysosomes. Hence, MDC is used to reflect the changes of autophagy in many studies (Cristina et al. 2015 ; Li et al. 2017 ). In our study, MDC staining showed that autophagic vacuoles increased significantly in the liver after fluoride exposure. These results suggested that fluoride induced the autophagy in the liver.