BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

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.

Objective:To determine the inhibitory effect of Rupixiao ( RPX) granule on mammary gland hyperplasia ( HMG) in rabbits and explore the possible mechanism to provide reference for clinical medication. Methods:Rabbit model of mammary hyperpla-sia was established by estradiol benzoate and progesterone. Xiaoyao pills and tamoxifen were used as the positive control, and the RPX granule group was respectively at low, medium and high dose (0. 525,1. 05,2. 1 g·kg-1). Each group was with intragastric adminis-tration for 30 days. The levels of E2 , PROG, FSH, LH and PRL in serum and the expression of VEGF in the homogenate tissue were determined by an Elisa method. The breast tissue of rabbits in each group was withdrawn to observe the structure changes after Hema-toxylin-eosin staining ( HE) . Results:Ovarian preservation-benzoic acid, estradiol combined with progesterone could be used to estab-lish mammary gland hyperplasia in rabbits. RPX granule could significantly decrease the serum levels of E2 and PRL (P<0. 01)and increase that of PROG (P<0. 01 or P<0. 001). The results of pathological section showed that the breast structure in RPX granule high dose group could be restored to the level of the blank control group, which showed its therapeutic effect on HMG was better than that of tamoxifen and Xiaoyao pills. The expression of VEGF in each treatment group significantly decreased compared with that in the model group by the Elisa reagent (P<0. 001). Conclusion:RPX granule has good efficacy in HMG rabbits. The inhibitory effects of RPX granule on HMG in rabbits maybe related to the inhibition of VEGF in rabbit breast tissues, and the inhibition of RPX granule was similar to that of tamoxifen and Xiaoyao pills.