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Background Chronic excessive exposure to fluoride can cause damage to the central nervous system and a certain degree of learning and memory impairment. However, the associated mechanism is not yet clear and further exploration is needed. Objective Using 4D unlabelled quantitative proteomics techniques to explore differentially expressed proteins and their potential mechanisms of action in chronic excessive fluoride exposure induced brain injury. Methods Twenty-four SPF-grade adult SD rats, half male and half male, were selected and divided into a control group and a fluoride group by random number table method, with 12 rats in each group. Among them, the control group drank tap water (fluorine content<1 mg·L−1), the fluoride group drank sodium fluoride solution (fluorine content 10 mg·L−1), and both groups were fed with ordinary mouse feed (fluoride content<0.6 mg·kg−1). After 180 d of feeding, the SD rats were weighed, and then part of the brain tissue was sampled for pathological examination by hematoxylin-eosin (HE) staining and Nissl staining. The rest of the brain tissue was frozen and stored at −80 ℃. Three brain tissue samples from each group were randomly selected for proteomics detection. Differentially expressed proteins were screened and subcellular localization analysis was performed, followed by Gene Ontology (GO) function analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, cluster analysis, and protein-protein interaction analysis. Finally, Western blotting was used to detect the expression levels of key proteins extracted from the brain tissue samples. Results After 180 d of feeding, the average weight of the rats in the fluoride group was significantly lower than that in the control group (P<0.05). The brain tissue stained with HE showed no significant morphological changes in the cerebral cortex of the fluoride treated rats, and neuron loss, irregular arrangement of neurons, eosinophilic changes, and cell body pyknosis were observed in the hippocampus. The Nissl staining results showed that the staining of neurons in the cerebral cortex and hippocampus of rats exposed to fluoride decreased (Nissl bodies decreased). The proteomics results showed that a total of 6927 proteins were identified. After screening, 206 differentially expressed proteins were obtained between the control group and the fluoride group, including 96 up-regulated proteins and 110 down-regulated proteins. The differential proteins were mainly located in cytoplasm (30.6%), nucleus (27.2%), mitochondria (13.6%), plasma membrane (13.6%), and extracellular domain (11.7%). The GO analysis results showed that differentially expressed proteins mainly participated in biological processes such as iron ion transport, regulation of dopamine neuron differentiation, and negative regulation of respiratory burst in inflammatory response, exercised molecular functions such as ferrous binding, iron oxidase activity, and cytokine activity, and were located in the smooth endoplasmic reticulum membrane, fixed components of the membrane, chloride channel complexes, and other cellular components. The KEGG significantly enriched pathways included biosynthesis of secondary metabolites, carbon metabolism, and microbial metabolism in diverse environments. The results of differential protein-protein interaction analysis showed that the highest connectivity was found in glucose-6-phosphate isomerase (Gpi). The expression level of Gpi in the brain tissue of the rats in the fluoride group was lower than that in the control group by Western blotting (P<0.05). Conclusion Multiple differentially expressed proteins are present in the brain tissue of rats with chronic fluorosis, and their functions are related to biosynthesis of secondary metabolites, carbon metabolism, and microbial metabolism in diverse environments; Gpi may be involved in cerebral neurological damage caused by chronic overdose fluoride exposure.
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Objective To study the mechanism of central nervous system (CNS) injury in chronic fluorosis and the neuroprotective effect of chondroitin sulfate (CS).Methods Forty-eight female Sprague-Dawley rats weighting 90-120 g were divided into 8 groups according to body weight by random number table,6 rats in each group:control group,drinking tap water freely;low dose and high dose fluoride groups,freely drinking tap water with fluoride content of 10 and 50 mg/L,respectively;control + normal saline (NS),low dose fluoride + NS,and high dose fluoride + NS groups,each group was fed for 180 d,and treated with intraperitoneal injection of 0.66 mg/kg NS for 5 d (once a day);low dose fluoride + CS and high dose fluoride + CS groups,each group was fed for 180 d,0.66 mg/kg CS was injected intraperitoneally for 5 d (once a day).All groups were fed standard nutritive animal feed for 185 d and dissected for brain tissue.The pathologic change was observed after hematoxylin-eosin (HE)staining;the expression levels of phosphorylated extracellular signal-regulated protein kinase 1/2 (phospho-Erk1/2)and glutamate receptors 1,2 (GluR1,GluR2) in the brain cortex were detected by immunohistochemistry;the protein levels of Erk1/2,phospho-Erk1/2,GluR1,and GluR2 in the brain cortex were detected by Western blotting.Results Brain cortex of all rats in the fluoride groups showed eosinophilic degeneration,loss and disordered arrangement of neurons,and the brain morphological changes in each fluoride + CS groups were significantly improved compared with those in the fluoride groups.Immunohistochemistry results showed that compared with the control group [(0.44 ± 0.09)％,(1.49 ± 0.05)％,(2.51 ± 0.54)％],the expression levels of phospho-Erk1/2 [(1.47 ±0.09)％,(1.03 ± 0.05)％],and GluR2 [(2.37 ± 0.06)％,(3.38 ± 0.12)％] in the low dose and high dose fluoride groups were increased,and the expression levels of GluR1 [(1.49 ± 0.02)％,(0.99 ± 0.19)％] were decreased (P ＜ 0.05).Western blotting results showed that compared with the control group (1.00 ± 0.12,1.76 ± 0.33),the protein levels of Erk1/2 (3.10 ± 0.76,1.99 ± 0.01) and phospho-Erk1/2 (3.27 ± 0.25,2.67 ± 0.05) in low dose and high dose fluoride groups were significantly increased (P ＜ 0.05);compared with low dose fluoride group,the protein levels of Erk1/2,and phospho-Erk1/2 (1.30 ± 0.31,2.20 ± 0.34) in low dose fluoride + CS group decreased significantly (P ＜0.05).Compared with control group (1.86 ± 0.47,1.17 ± 0.27),the protein levels of GluR1 (1.09 ± 0.26,0.61 ± 0.14) in low dose and high dose fluoride groups decreased significantly,while the protein level of GluR2 (1.99 ± 0.42,3.38 ±0.27) increased significantly (P ＜ 0.05);compared with low dose and high dose fluoride groups,the protein levels of GluR2 in low dose fluoride + CS and high dose fluoride + CS groups (1.53 ± 0.41,2.65 ± 0.32) decreased significantly (P ＜ 0.05).The protein level of phospho-Erk1/2 was negatively correlated with GluR1 protein level (r =-0.975,-0.991,P ＜ 0.05) in low dose and high dose fluoride groups,and it was positively correlated with the protein level of GluR2 (r =0.986,0.993,P ＜ 0.05).Conclusion The CNS injury caused by chronic fluorosis may be related to GluR1 and GluR2 activated Erk1/2 signaling pathway,and CS has certain protection to the injury.