SIRT3-dependent mitochondrial oxidative stress in sodium fluoride-induced hepatotoxicity and salvage by melatonin

Oxidative stress induced by fluoride (F) is associated with fluorosis formation, but the underlying molecular mechanism remains unclear. In this study, Melatonin pretreatment suppressed F-induced hepatocyte injury in HepG2 cells. Melatonin increases the activity of superoxide dismutase (SOD2) by enhancing sirtuin 3 (SIRT3)-mediated deacetylation and promotes SOD2 gene expression via SIRT3-regulated DNA-binding activity of forkhead box O3 (FoxO3a), indicating that melatonin markedly enhanced mROS scavenging in F-exposed HepG2 cells. Notably, melatonin activated the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α). PGC-1α interacted with the estrogen-related receptor alpha (ERRα) bound to the SIRT3 promoter, where it functions as a transcription factor to regulate SIRT3 expression. Furthermore, daily injection of melatonin for 30 days inhibited F-induced oxidative stress in mice liver, leading to improvement of liver function. Mechanistic study revealed that the protective effects of melatonin were associated with down-regulation of JNK1/2 phosphorylation in vitro and in vivo. Collectively, our data suggest a novel role of melatonin in preventing F-induced oxidative stress through activation of the SIRT3 pathway.

The EMSA assay was further performed to test the in vitro binding of ERRα and 1 8 3 SIRT3 fragments. A preliminary experiment was performed to test the binding of was regulated by F and melatonin. Moreover, a specific super-shift band was 1 8 7 detected with the ERRα antibody, thereby indicating that ERRα was bound to the 1 8 8 probe ( Fig. 7D-3). The results of EMSA also showed that melatonin increased the 1 8 9 binding of the exogenous consensus DNA oligonucleotide of SIRT3 with ERRα. To 1 9 0 further confirm the EMSA results and verify that ERRα physically occupies the 1 9 1 SIRT3 promoter, we performed the ChIP assay. As shown in Fig. 7E, a 3.6-fold 1 9 2 enrichment of ERRα was observed. Furthermore, we also investigate whether melatonin could prevent F-induced 1 9 5 oxidative stress in mice liver. Significant accumulation of F was observed in 1 9 6 F-toxicated mice liver (Fig. S3A). Liver functions were also measured based on 1 9 7 changes in the hepatic markers ALT and AST. The results showed that the serum 1 9 8 activities of ALT and AST were significantly increased in the F group when GSH level were decreased significantly in F-toxicated mice livers, which was 2 0 5 reversed by melatonin (Fig. S3F).

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Since apoptosis plays an important role in the pathogenetic mechanisms involved in anti-apoptotic factor in mice liver. Pretreatment with melatonin attenuated 2 1 0 caspase-3 activity and increased Bcl-2 protein expression in NaF-treated mice liver. Activation of mitogen-activated protein kinase (MAPK) has been implicated in 2 1 2 F-induced apoptosis and they are sensitive to oxidative stress, Western blot showed 2 1 3 that the melatonin significantly reduced the phosphorylation of JNK1/2 in 2 1 4 F-exposed mice liver.

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Oxidative stress is the cellular outcome of mitochondrial dysfunction. The MMP is  Oxidative stress have been demonstrated to activate a variety of signaling pathways, In summary, we obtained evidence for the first time to demonstrate mROS F-induced hepatotoxicity. This study was performed in strict accordance with the guidelines for the care and Medicine, Northwest A&F University.

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Chemicals and reagents 2 9 7 All chemicals and reagents were obtained from Sigma-Aldrich Chemical Co. (St.  and treated with or without NaF (2 mM) for an additional 12 h. Experimental 3 1 0 protocol are described in more details in Supporting information. Cell viability was analyzed using Cell Counting Kit-8 (Beyotime, Jiangsu, China).

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The absorbance was obtained with a microplate reader (Epoch, BioTek, Luzern, Switzerland) set at a wavelength of 450 nm. The concentrations of malondialdehyde (MDA) and glutathione (GSH), and the 3 1 7 activity of SOD2 were assayed by commercial assay kits purchased from Nanjing 3 1 8 Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).  Immunoblot analysis was performed as described in the protocols provided by the 3 2 9 primary antibody suppliers. NaCl; 1 mM DTT; 5 mM MgCl 2 ; 1 mM EDTA; 10 % glycerol; 0.1% NP-40). Tokyo, Japan). The fluorescence intensity was monitored at excitation and emission  The siRNA targeting SIRT3 and PGC-1α were purchased from Santa Cruz Supporting Information Table S3. Information Table S4. well as free access to food and water.

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Animals were randomly divided into four groups of 10 each. Group 1 (Control): Mice was provided distilled water and received daily injection vehicle for 30 days. The livers were homogenized in nine fold (w/v) cold normal saline using an indicated. Statistical analyses were performed with one-way ANOVA, followed by  The authors indicate no competing financial interest.  Confluent cells were pretreated for 2 h with various concentrations of melatonin.