Inactivation of Mst/Nrf2/Keap1 signaling flexibly mitigates MAPK/NQO-HO1 activation in the reproductive axis of experimental fluorosis.

Fluoride induced reprotoxicity through oxidative stress-mediated reproductive cell death. Hence, the current study evaluated the importance of the MST/Nrf2/MAPK/NQO-HO1 signaling pathway in fluorosis-induced reproductive toxicity. For this purpose, the reproductive toxicity of sodium fluoride (NaF) at physiological, biochemical, and intracellular levels was evaluated. In-vivo, NaF at 100 mg/L instigated physiological dysfunction, morphological, stereological, and structural injuries in the gut-gonadal axis of fluorosis mice through weakening the antioxidant signaling, Nrf2/HO-1/NQO1signaling pathway, causing the gut-gonadal barrier disintegrated via oxidative stress-induced inflammation, mitochondrial damage, apoptosis, and autophagy. Similar trends were also observed in-vitro in the isolated Leydig cells (LCs) challenging with 20 mg/L NaF. Henceforth, activating the cellular antioxidant signaling pathway, Nrf2/HO-1/NQO1, inactivating autophagy and apoptosis, or attenuating lipopolysaccharide (LPS) can be the theoretical basis and valuable therapeutic targets for coping with NaF-induced reproductive toxicity.

Efficient production of ε-poly-L-lysine from cassava bagasse hydrolysate used as carbon source by Streptomyces albulus US3-18.

The production of ε-poly-L-lysine (ε-PL) from cassava bagasse hydrolysate (CBH) by Streptomyces albulus US3-18 was investigated in this study. With 30 g/L glucose from CBH, 1.30 g/L ε-PL and 10.68 g/L biomass were obtained in shake flask fermentation. Interestingly, the two values were increased by 14.0% and 21.5%, respectively, compared to the control (1.14 g/L and 8.79 g/L). Simultaneously, the activities of four key enzymes of ε-PL synthesis during CBH fermentation were enhanced to varying degrees. In batch fermentation of 5-L bioreactor, 3.39 g/L ε-PL and 10.17 g/L DCW were harvested with 40 g/L glucose from CBH. The combination of fed-batch fermentation with two-stage pH strategy significantly increased ε-PL titer and biomass to 37.41 g/L and 41.0 g/L, respectively. Moreover, eleven volatile components were detected in CBH by GC-MS, and 6-pentyl-α-pyrone (6PP) was first identified as the most abundant volatile ingredient. The results in CBH fermentation demonstrated that S. albulus US3-18 exhibited high tolerance to these volatile byproducts. Using ICP-MS, the calcium concentration in CBH was determined as 195.0 mg/(kg hydrolyzate), and cobalt, copper, lead, chromium, mercury and arsenic were not detected. By adding 0.05 g/L CaCl2 to M3G medium, ε-PL yield was improved by 28.0%, indicating calcium was one of the factors for the enhanced ε-PL production. The study provides a reference for the efficient production of ε-PL from low-cost agricultural residues.

Arsenic induces autophagy in developmental mouse cerebral cortex and hippocampus by inhibiting PI3K/Akt/mTOR signaling pathway: involvement of blood-brain barrier's tight junction proteins.

For the past decade, there has been an increased concern about the health risks from arsenic (As) exposure, because of its neurotoxic effects on the developing brain. The exact mechanism underlying As-induced neurotoxicity during sensitive periods of brain development remains unclear, especially the role of blood-brain barrier's (BBB) tight junction (TJ) proteins during As-induced neurotoxicity. Here, we highlight the involvement of TJ proteins in As-induced autophagy in cerebral cortex and hippocampus during developmental periods [postnatal day (PND) 21, 28, 35 and 42]. Here, the administration of arsenic trioxide (As2O3) at doses of 0.15 mg or 1.5 mg or 15 mg As2O3/L in drinking water from gestational to lactational and continued to the pups till PND42 resulted in a significant decrease in the mRNA expression levels of TJ proteins (Occludin, Claudin, ZO-1 and ZO-2) and Occludin protein expression level. In addition, As exposure significantly decreased PI3K, Akt, mTOR, and p62 with a concomitant increase in Beclin1, LC3I, LC3II, Atg5 and Atg12. Moreover, As exposure also significantly downregulated the protein expression levels of mTOR with a concomitant upregulation of Beclin 1, LC3 and Atg12 in all the developmental age points. However, no significant alterations were observed in low and medium dose-exposed groups of PND42. Histopathological analysis in As-exposed mice revealed decreased number of pyramidal neurons in hippocampus; and neurons with degenerating axons, shrinkage of cells, remarkable vacuolar degeneration in cytoplasm, karyolysis and pyknosis in cerebral cortex. Ultrastructural analysis by transmission electron microscopy revealed the occurrence of autophagosomes and vacuolated axons in the cerebral cortex and hippocampus of the mice exposed to high dose As at PND21 and 42. The severities of changes were found to more persist in the cerebral cortex than in the hippocampus of As-exposed mice. Finally, we conclude that the leaky BBB in cerebral cortex and hippocampus may facilitate the transfer of As and induces autophagy by inhibiting PI3K/Akt/mTOR signaling pathway in an age-dependent manner, i.e., among the four different developmental age points, PND21 animals were found to be more vulnerable to the As-induced neurotoxicity than the other three age points.

Alterations in epididymal proteomics and antioxidant activity of mice exposed to fluoride.

It is well known that high fluoride results in low fertility. Epididymis is the important place for spermatozoa maturation, which is essential for successful fertilization. In the previous studies, fluoride was reported to damage the epididymal structure of mouse and rabbit. However, the mechanism underlying sodium fluoride (NaF)-induced epididymal toxicity has not yet been well elucidated. The aim of this study is to explore the global protein alterations in epididymis of mice exposed to NaF using the iTRAQ technique. Results showed that 211 proteins were differentially expressed in both 25 and 100 mg/L NaF groups. Some of them have been proved to be important for reproduction, such as low-density lipoprotein receptor-related protein 2 (Lrp2), cytochrome c, testis-specific (Cyct), sorbitol dehydrogenase (Sord), glutathione S-transferases (GSTs), acrosin, beta-defensin 126, cysteine-rich secretory protein (Crisp) 1, and Crisp2. Gene ontology (GO) analysis suggested cellular process, organelle and catalytic activity account for high percent and number of differentially expressed proteins. 171 pathways were found after the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, among which the representative maps, such as ribosome, focal adhesion, and phagosome, were involved. Different functional categories post-translational modification, protein turnover, chaperones; translation, ribosomal structure and biogenesis; cytoskeleton; energy production and conversion are implicated in the Cluster of Orthologous Groups (COG) of proteins analysis. Subsequently, the effect of NaF on the antioxidant activity in epididymis, especially glutathione and glutathione-related enzymes, was evaluated. Results exhibited high fluoride caused low total antioxidant capacity (T-AOC), high methane dicarboxylic aldehyde (MDA), decreased reduced glutathione (GSH), and the glutathione-related enzymes [GSH peroxidase (GPx), GSH reductase (GR), and GSH S-transferase (GST)] changes in activity, protein, and mRNA expressions. In summary, NaF decreased the antioxidant activity of epididymis, especially glutathione and glutathione-related enzymes, as well as iTRAQ results, providing new explanations for the low sperm quality induced by fluoride.

Effect of sodium fluoride on the sperm mitochondrial DNA in mice.

Previous studies demonstrated that fluoride, as a widespread environmental pollutant, induced the reproductive toxicity at high dose. Besides the decrease of sperm characteristics like concentration, survival, and sperm motility, it was found that high fluoride induced the destructive mitochondrial ultrastructure and decreased ATP production from mitochondrial respiration. However, whether fluoride exposure can damage the mitochondrial DNA (mtDNA) of sperm is still unknown. In this study, 100 male mice were randomly divided into four groups 25 each, which were administrated with the distilled water containing 0, 25, 50 and 100 mg L-1 NaF, respectively for 60 days. After exposure, sperm mtDNA copy number was measured by real-time PCR, mtDNA integrity by acridine orange (AO) staining, and nuclear DNA (nDNA) integrity through long PCR amplification, as well as MTCYB and MTATP6 mutations by PCR and direct sequencing. The results showed that 100 mg L-1 NaF significantly increased sperm mtDNA copy number, and reduced nDNA integrity. There were no changes observed in mtDNA integrity, and mutations of MTCYB and MTATP6 between fluoride groups and the control group. These results indicated that along with low sperm quality, sperm mtDNA copy number is also a sensitive biomarker to reflect the sperm toxicity of fluoride.

Cell cycle arrest and gene expression profiling of testis in mice exposed to fluoride.

Exposure to fluoride results in low reproductive capacity; however, the mechanism underlying the impact of fluoride on male productive system still remains obscure. To assess the potential toxicity in testis of mice administrated with fluoride, global genome microarray and real-time PCR were performed to detect and identify the altered transcriptions. The results revealed that 763 differentially expressed genes were identified, including 330 up-regulated and 433 down-regulated genes, which were involved in spermatogenesis, apoptosis, DNA damage, DNA replication, and cell differentiation. Twelve differential expressed genes were selected to confirm the microarray results using real-time PCR, and the result kept the same tendency with that of microarray. Furthermore, compared with the control group, more apoptotic spermatogenic cells were observed in the fluoride group, and the spermatogonium were markedly increased in S phase and decreased in G2/M phase by fluoride. Our findings suggested global genome microarray provides an insight into the reproductive toxicity induced by fluoride, and several important biological clues for further investigations. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1558-1565, 2017.

In vivo influence of sodium fluoride on sperm chemotaxis in male mice.

Reproductive process covers lots of procedures, including capacitation, hyperactivation, chemotaxis and the acrosome reaction. Each plays an important role in the success of fertilization. Although multiple studies have reported the toxic effects of fluoride on the male reproduction, the effect of fluoride on sperm chemotaxis is little known. This study is to examine the effect of fluoride on the sperm chemotaxis and then to reveal the underling mechanisms of fluoride toxicity in sperm chemotaxis. 260 healthy Kunming male mice (8 weeks old) were randomly divided into four groups and exposed to 50, 100, 150 mg NaF/L in the drinking water for 8 weeks. At the end of the exposure, sperm chemotaxis was examined using a microchannel-based device. Ca(2+) concentration, adenylate cyclase (AC) content and mRNA expression of mACIII, mACVIII, Golf alpha, CatSper1, CatSper2 were measured to elucidate the possible molecular mechanisms. The results showed that the percentage of chemotactic sperm was decreased by NaF in a dose-dependent manner. In the 100 and 150 mg/L groups, Ca(2+) concentration and AC content were notably lower than the control group. Compared with the control group, mRNA expression of CatSper1 in the 100 and 150 mg/L treatment groups was decreased significantly, and other genes showed no statistical difference. These data suggested that excessive fluoride did adversely affect sperm chemotaxis. The alteration of Ca(2+) concentration, AC content and CatSper1 mRNA expression level may play a key role in the mechanism underlying the affection.

Effect of pubertal nano-TiO2 exposure on testosterone synthesis and spermatogenesis in mice.

Titanium dioxide nanoparticles (nano-TiO2) are frequently used in cosmetics, paints, sunscreens and the like. Recent studies have demonstrated that nano-TiO2 might be deleterious for the male reproductive function. However, the effects of pubertal nano-TiO2 exposure on testosterone (T) synthesis and spermatogenesis remained to be elucidated. Here, we investigated the effect of pubertal nano-TiO2 exposure on the synthesis of T and spermatogenesis. Nano-TiO2 was orally administered daily to Kunming male mice from 28th postnatal day (PND 28) to PND 70. The percentage of spermatozoa abnormality in epididymides was markedly increased in mice exposed to nano-TiO2; decreased layers of spermatogenic cells and vacuoles in seminiferous tubules were also observed in the nano-TiO2 treated group. In addition, pubertal nano-TiO2 exposure significantly decreased the serum T levels in male mice. Moreover, mice exposures to nano-TiO2 significantly reduced the expression of 17ß-hydroxysteroid dehydrogenase and P450 17α-hydroxysteroid dehydrogenase in the testis of mice, while the expression of cytochrome P450-19, a key enzyme for the translation of T to estradiol (E2), was increased. Taken together, these results indicated that nano-TiO2 could influence the levels of serum T through changes in both the synthesis and translation of T. Furthermore, the decreased serum T synthesis might contribute to the reduced spermatogenesis in mice exposed to nano-TiO2.

Altered sperm chromatin structure in mice exposed to sodium fluoride through drinking water.

This study investigated the effects of sodium fluoride (NaF) on sperm abnormality, sperm chromatin structure, protamine 1 and protamine 2 (P1 and P2) mRNA expression, and histones expression in sperm in male mice. NaF was orally administrated to male mice at 30, 70, and 150 mg/l for 49 days (more than one spermatogenic cycle). Sperm head and tail abnormalities were significantly enhanced at middle and high doses. Similarly, sperm chromatin structure was also adversely affected by NaF exposure, indicating DNA integrity damage. Furthermore, middle and high NaF significantly reduced the mRNA expressions of P1 and P2, and P1/P2 ratio, whereas the sperm histones level was increased, suggesting the abnormal histone-protamine replacement. Therefore, we concluded that the mechanism by which F induced mice sperm abnormality and DNA integrity damage may involved in the alterations in P1, P2, and histones expression in sperm of mice.

Zn(II) enhances the antimicrobial effect of chloroxine and structural analogues against drug-resistant ESKAPE pathogens in vitro.

The emerging antibiotic-resistant bacteria, especially the "ESKAPE" pathogens, pose a continuous threat to global health. In this study, we explored metalloantibiotics as promising therapeutics and innovative antimicrobial agents. The role of metal in the antimicrobial activity of chloroxine (5,7-dichloro-8-hydroxyquinoline), as a metalloantibiotic, was investigated by minimal inhibit concentration (MIC) assay and a series of assays, including growth curve, time-killing, and UV-visible spectroscopy and PAR (4-(2-pyridylazo)-resorcinol) competition assays. Both chloroxine and its structural analogues exhibited increased antibacterial potency against Gram-positive bacteria compared to Gram-negative bacteria. The introduction of exogenous manganese or zinc ions significantly boosted chloroxine's antibacterial efficacy against Gram-negative bacteria, including the notorious ESKAPE pathogens. However, the enhanced antibacterial activity induced by zinc ions could be negated in the presence of copper or ferrous iron ions, as well as changes in oxygen availability, highlighting the involvement of proton motive force, oxidative and antioxidative systems. Notably, chloroxine effectively inhibited the enzymatic activity of superoxide dismutase (SOD). In addition, chloroxine could reverse polymyxin and carbapenem resistance in E. coli in vitro. Therefore, these results suggested that chloroxine with zinc ions are promising therapeutics and antibiotics potentiator to combat multidrug-resistant ESKAPE pathogens.

Exercise Alleviates Fluoride-Induced Learning and Memory Impairment in Mice: Role of miR-206-3p and PREG.

Fluorosis decreases the learning and memory ability in humans and animals, while exercise can reduce the risk of cognitive decline. However, the effect of exercise on learning and memory in fluoride-exposed mice is unclear. For this purpose, in this study, mice were randomly allotted into four groups (16 mice per group, half male and half female): control group (group C), fluoride group (group F, 100 mg/L sodium fluoride (NaF)), exercise group (group E, treadmill exercise), and E plus F group (group EF, treadmill exercise, and 100 mg/L NaF). During 6 months of exposure, exercise alleviated the NaF-induced decline in memory and learning. In addition, NaF induced injuries in mitochondria and myelin sheath ultrastructure and reduced the neurons number, while exercise restored them. Metabolomics results showed that phosphatidylethanolamine, pregnenolone (PREG), and lysophosphatidic acid (LysoPA) were altered among groups C, F, and EF. Combined with previous studies, it can be suggested that PREG might be a biomarker in response to exercise-relieving fluorine neurotoxicity. The miRNA sequencing results indicated that in the differently expressed miRNAs (DEmiRNAs), miR-206-3p, miR-96-5p, and miR-144-3p were shared in groups C, F, and EF. After the QRT-PCR validation and in vitro experiments, it was proved that miR-206-3p could reduce cell death and regulate AP-1 transcription factor subunit (JunD) and histone deacetylase 4 (HDAC4) to alleviate fluoride neurotoxicity. To sum up, the current study reveals that exercise could alleviate NaF-induced neurotoxicity by targeting miR-206-3p or PREG, which will contribute to revealing the pathogenesis and therapeutic method of fluoride neurotoxicity.

MiRNA-seq and mRNA-seq revealed the mechanism of fluoride-induced cauda epididymal injury.

The widespread presence of fluoride in water, food, and the environment continues to exacerbate the impact of fluoride on the male reproductive health. However, as a critical component of the male reproductive system, the intrinsic mechanism of fluoride-induced cauda epididymis damage and the role of miRNAs in this process are still unclear. This study established a mouse fluorosis model and employed miRNA and mRNA sequencing; Evans blue staining, Oil Red O staining, TEM, immunofluorescence, western blotting, and other technologies to investigate the mechanism of miRNA in fluoride-induced cauda epididymal damage. The results showed that fluoride exposure increased the fluoride concentration in the hard tissue and cauda epididymis, altered the morphology and ultrastructure of the cauda epididymis, and reduced the motility rate, normal morphology rate, and hypo-osmotic swelling index of the sperm in the cauda epididymis. Furthermore, sequencing results revealed that fluoride exposure resulted in differential expression of 17 miRNAs and 4725 mRNAs, which were primarily enriched in the biological processes of tight junctions, inflammatory response, and lipid metabolism, with miR-742-3p, miR-141-5p, miR-878-3p, and miR-143-5p serving as key regulators. Further verification found that fluoride damaged tight junctions, raised oxidative stress, induced an inflammatory response, increased lipid synthesis, and reduced lipid decomposition and transport in the cauda epididymis. This study provided a theoretical basis for developing miRNA as potential diagnostic markers and therapeutic target drugs for this injury.

Integrating network pharmacology with pharmacological research to elucidate the mechanism of modified Gegen Qinlian Decoction in treating porcine epidemic diarrhea.

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to neonatal piglets, particularly due to the limited efficacy of existing vaccines and the scarcity of efficacious therapeutic drugs. Gegen Qinlian Decoction (GQD) has been employed for over two millennia in treating infectious diarrhea. Nonetheless, further scrutiny is required to improve the drug's efficacy and elucidate its underlying mechanisms of action. In this study, a modified GQD (MGQD) was developed and demonstrated its capacity to inhibit the replication of PEDV. Animal trials indicated that MGQD effectively alleviated pathological damage in immune tissues and modulated T-lymphocyte subsets. The integration of network analysis with UHPLC-MS/MS facilitated the identification of active ingredients within MGQD and elucidated the molecular mechanisms underlying its therapeutic effects against PEDV infections. In vitro studies revealed that MGQD significantly impeded PEDV proliferation in IPEC-J2 cells, promoting cellular growth via virucidal activity, inhibition of viral attachment, and disruption of viral biosynthesis. Furthermore, MGQD treatment led to increased expression levels of IFN-α, IFN-ß, and IFN-λ3, while concurrently decreasing the expression of TNF-α, thereby enhancing resistance to PEDV infection in IPEC-J2 cells. In conclusion, our findings suggest that MGQD holds promise as a novel antiviral agent for the treatment of PEDV infections.

Screening and characterization of a novel linear B-cell epitope on orf virus F1L protein.

Background: Orf, also known as contagious ecthyma (CE), is an acute, contagious zoonotic disease caused by the orf virus (ORFV). The F1L protein is a major immunodominant protein on the surface of ORFV and can induce the production of neutralizing antibodies. Methods: The prokaryotic expression system was used to produce the recombinant F1L protein of ORFV, which was subsequently purified and used to immunize mice. Positive hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity and specificity of the monoclonal antibody (mAb) were verified through Western blot and indirect immunofluorescence (IFA). The linear antigenic epitope specific to the mAb was identified through Western blot, using truncated F1L proteins expressed in eukaryotic cells. A multiple sequence alignment of the ORFV reference strains was performed to evaluate the degree of conservation of the identified epitope. Results: After three rounds of subcloning, a mAb named Ba-F1L was produced. Ba-F1L was found to react with both the exogenously expressed F1L protein and the native F1L protein from ORFV-infected cells, as confirmed by Western blot and IFA. The mAb recognized the core epitope 103CKSTCPKEM111, which is highly conserved among various ORFV strains, as shown by homologous sequence alignment. Conclusion: The mAb produced in the present study can be used as a diagnostic reagent for detecting ORFV and as a basic tool for exploring the mechanisms of orf pathogenesis. In addition, the identified linear epitope may be valuable for the development of epitope-based vaccines.

Identifying Serum Metabolites and Gut Bacterial Species Associated with Nephrotoxicity Caused by Arsenic and Fluoride Exposure.

Co-contamination of arsenic (As) and fluoride (F) is widely distributed in groundwater, which are known risk factors for the nephrotoxicity. Emerging evidence has linked environmentally associated nephrotoxicity with the disturbance of gut microbiota and blood metabolites. In this study, we generated gut microbiota and blood metabolomic profile and identified multiple serum metabolites and gut bacteria species, which were associated with kidney injury on rat model exposed to As and F alone or combined. Combined As and F exposure significantly increased creatinine level. Abnormal autophagosomes and lysosome were observed, and the autophagic genes were enhanced in kidney tissue after single and combined As and F exposure. The metabolome data showed that single and combined As and F exposure remarkably altered the serum metabolites associated with the proximal tubule reabsorption function pathway, with glutamine and alpha-ketoglutarate level decreased in all exposed group. Furthermore, phosphatidylethanolamine (PE), the key contributor of autophagosomes, was decreased significantly in As and F + As exposed groups during the screen of autophagy-animal pathway. Multiple altered gut bacterial microbiota at phylum and species levels post As and F exposure were associated with targeted kidney injury, including p_Bacteroidetes, s_Chromohalobacter_unclassified, s_Halomonas_unclassified, s_Ignatzschineria_unclassified, s_Bacillus_subtilis, and s_Brevundimonas_sp._NA6. Meanwhile, our analysis indicated that As and F co-exposure possessed an interactive influence on gut microbiota. In conclusion, single or combined As and F exposure leads to the disruption of serum metabolic and gut microbiota profiles. Multiple metabolites and bacterial species are identified and associated with nephrotoxicity, which have potential to be developed as biomarkers of As and/or F-induced kidney damage.

Exposure to Fluoride From in Utero to Puberty Alters Gonadal Structure and Steroid Hormone Expression in Offspring Rats.

The reproductive toxicity of fluoride has been proven by a large number of studies. While the underlying mechanism of reproductive toxicity during pregnancy is still unclear. Hence, in this study, we investigated the effects of fluoride exposure on ovarian and testicular steroid hormone synthesis in young and adult rat offspring. We established a model of fluoride-exposed rat pups from in utero to puberty to explore the mechanisms of fluoride impacts on reproductive toxicity in the offspring. The results showed that NaF exposure did not affect the 3 weeks of age offspring. Whereas the body weight in both sexes significantly decreased, and the ovarian and testicular tissue structures were damaged at 11 weeks of age. In females, the total number of secondary follicles and mature follicles were significantly reduced after NaF exposure. Moreover, estradiol (E2) and follicle-stimulating hormone (FSH) levels in the females were significantly reduced in the 100 mg/L NaF exposure group. In males, the sperm viability and testosterone (T) were significantly decreased in the NaF exposure groups. Additionally, during steroidogenesis in ovaries and testes, fluoride remarkably decreased the expression levels of genes and proteins, including acute regulatory protein (StAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 17a-hydroxylase (CYP17A1), and cholesterol side-chain cleavage enzyme (CYP11A1), while the mRNA levels of 17ß-hydroxysteroid dehydrogenase (17ß-HSD) decreased only in the testes. These results indicated that fluoride exposure disrupted the steroid hormone balance by changing several important steroidogenic-related genes associated with the development of the gonads, and damage the normal structure of the gonads in rat offspring.

Effects of Treadmill Exercise on Liver Apoptosis in Fluoride-Exposed Mice.

Hepatotoxicity induced by excessive fluoride (F) exposure has been extensively studied in both humans and animals. Chronic fluorosis can result in liver apoptosis. Meanwhile, moderate exercise alleviates apoptosis caused by pathological factors. However, the effect of moderate exercise on F-induced liver apoptosis remains unclear. In this research, sixty-four three-week-old Institute of Cancer Research (ICR) mice, half male and half female, were randomly divided into four groups: control group (distilled water); exercise group (distilled water and treadmill exercise); F group [100 mg/L sodium fluoride (NaF)]; and exercise plus F group (100 mg/L NaF and treadmill exercise). The liver tissues of mice were taken at 3 months and 6 months, respectively. Hematoxylin-eosin (HE) staining and situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) results showed that nuclear condensation and apoptotic hepatocytes occurred in the F group. However, this phenomenon could be reversed with the intervention of treadmill exercise. The results of QRT-PCR and western blot displayed NaF- induced apoptosis via tumor necrosis factor recpter 1 (TNFR1) signaling pathway, while treadmill exercise could restore the molecular changes caused by excessive NaF exposure.

Intestinal microbiota regulates colonic inflammation in fluorosis mice by TLR/NF-κB pathway through short-chain fatty acids.

Intestinal inflammation and microbial dysbiosis are found simultaneously in patients with fluorosis. However, whether the inflammation derived from fluoride exposure only or intestinal microbial disorders has not been clarified. In this study, 100 mg/L NaF exposure for 90 days significantly elevated the expressions of inflammatory factors (TNF-α, IL-1ß, IL-6, IFN-γ, TGF-ß, and IL-10), and the levels of TLR4, TRAF6, Myd88, IKKß, and NF-κB P65 in mouse colon, while the above factors were reduced in pseudo germ-free mice with fluorosis, hinting that disordered microbiota might play a more direct role in the development of colonic inflammation than fluoride. Fecal microbiota transplantation (FMT) lowered the levels of inflammatory factors and inactivated the TLR/NF-κB pathway in fluoride-exposed mice. In addition, supplementing short-chain fatty acids (SCFAs) exhibited the identical effects to the model of FMT. In summary, intestinal microbiota may alleviate the colonic inflammatory of mice with fluorosis by regulating TLR/NF-κB pathway through SCFAs.

Interleukin-17A knockout or self-recovery alleviated autoimmune reaction induced by fluoride in mouse testis.

Fluoride (F) is usually treated as a hazardous material, and F-caused public health problem has attracted global attention. Previous studies demonstrate that interleukin-17A (IL-17A) plays a crucial role in F-elicited autoimmune orchitis and self-recovery reverses F-induced testicular toxicity to some extent, but these basic mechanisms remain unclear. Thus, we established a 180 d F exposure model of wild type (WT) mice and IL-17A knockout mice (C57BL/6 J background), and 60 d & 120 d self-recovery model based on F exposure model of WT mice, and used various techniques like qRT-PCR, western blot, immunohistochemistry and ELISA to further explore the mechanism of F-induced autoimmune reaction, the role of IL-17A in it and the reversibility of F-caused toxicity in testis. The results indicated that F exposure for 180 d caused the decreased sperm quality, the damaged testis histopathology, the enhanced mRNA and protein expression levels of inflammatory cytokines, the changes of autoantibody such as the appearance and increased content of anti-testicular autoantibodies in sera and the autoantibody deposition in testis, the alterations of autoimmune related genes containing the decreased mRNA and protein expressions of AIRE and FOXP3 with an increase of MHCII, and the reduced protein expressions of CTLA4, and the activation of IL-17A signaling cascade like the elevated mRNA and protein expressions of IL-17A, Act1, NF-κB, AP-1 and CEBPß, and the increased protein expressions of IL-17RC, with a decrease of IκBα. After IL-17A knockout, 29 of 35 F-induced changes were alleviated. In two self-recovery models, all F-caused differences except fluorine concentration in femur were gradually restored in a time-dependent manner. This study concluded that IL-17A knockout or self-recovery attenuated F-induced testicular injury and decrease of sperm quality through alleviating autoimmune reaction which was involved with the activation of IL-17A pathway, the damage of self-tolerance and the enhancement of antigen presentation.

α-Lipoic acid improves mitochondrial biogenesis and dynamics by enhancing antioxidant and inhibiting Wnt/Ca2+ pathway to relieve fluoride-induced hepatotoxic injury.

Fluoride (F), widely present in water and food, poses a serious threat to liver health, and oxidative damage and mitochondrial damage are its main causes. As a natural mitochondrial protector and antioxidant, α-lipoic acid (ALA)'s alleviating effect on fluorosis liver injury and its underlying mechanism are still unclear. Therefore, this study established a fluorosis ALA intervention mice model to explore the mechanism of mitochondrial biogenesis, mitochondrial dynamics, and Wnt/Ca2+ pathway in ALA attenuating fluorosis liver injury. The results showed that ALA mitigated F-induced weight loss, hepatic structural and functional damage, hepatocytes mitochondrial damage, and decreased antioxidant levels. However, ALA did not reduce F accumulation in the femur. Further mRNA and protein detection results showed that F increased the expression levels of key genes in the mitochondrial fission (Drp1, Mff, and Fis1), mitophagy (Parkin, Pink1, and Prdx3), Wnt/Ca2+ pathway (Wnt5a and CaMK2), and rised the number and intensity of fluorescent spots of Drp1, but decreased the expression levels of key genes in the mitochondrial biogenesis (Sirt1, Sirt3, and PGC-1α) and fusion (OPA1, Mfn2, and Mfn1), and reduced the number and intensity of fluorescent spots of PGC-1α in the liver. However, the intervention of ALA relieved the F-induced changes in the expressions of the above genes. In conclusion, ALA mitigated F-induced hepatic injury through enhancing antioxidant capacity and inhibiting Wnt/Ca2+ pathway to improve mitochondrial biogenesis and dynamics disturbance. This study further reveals the hepatotoxic mechanism of F and the protective mechanism of ALA, and provides a theoretical basis for ALA as a potential preventive and palliative agent for F-induced hepatotoxic injury.