| Objective:To investigate the alteration of glial fibrillary acidic protein（GFAP）and neuronalα-Synuclein in astrocytes during neurological injury in chronic fluorosis and the related mechanisms.Methods:24 adult SD（Sprague-Dawley,SD）rats of SPF（Specific pathogen Free）class were randomly coded after 1 week of acclimatization feeding and divided into two groups,control（given tap water with fluoride content less than 0.5 mg/L）and fluoride-stained（given fluoridated tap water with fluoride content of 10 mg/L）,for 6 months.The rats that died during feeding were removed from the experiment and supplemented After feeding,brain tissues of SD rats in each group were removed intact and stained with Hematoxylin-eosin staining（HE）to observe the morphological changes of brain tissue,and the expression and distribution of GFAP andα-Synuclein were observed by immunofluorescence staining and immunohistochemical staining.Astrocytes were extracted from the brain cortex of adult SD rats at SPF level for primary culture,and the well-grown astrocytes were identified by cell immunofluorescence staining,and then the well-grown primary cells were grouped into control and fluoride-stained groups.The control group was placed in the same volume of astrocyte medium for 24h.GFAP protein andα-Synuclein protein were detected by fluorescence immuno-and immunocytochemical staining;GFAP m RNA and its protein level were detected by real-time fluorescence quantitative PCR and Western Blot;the calcium content of astrocytes was detected by flow cytometry;the calcium content of astrocytes was detected by flow cytometry.The astrocyte apoptosis was detected by flow cytometry.Results:The brain structure of SD rats in the control group was clear and intact by HE staining,while the fluoride-stained group showed eosinophilic changes in neurons,disorganized neuronal arrangement,partial neuronal loss,swelling and nuclear consolidation of neuronal cells compared with the control group;the results of immunohistochemistry showed that the expression of GFAP andα-Synuclein was observed microscopically,and the fluoride-stained group showed brown positive expression in astrocytes.GFAP expression was higher in the fluoride-stained group（0.240±0.050,0.195±0.020）compared with the control group（P<0.05）,and the brown positive results in theα-Synuclein-dyed group were lighter in color,smaller in area and more sparsely distributed compared with the control group,showing that the expression ofα-Synuclein was significantly lower in the fluoride-stained group compared with the control group（P<0.05）.The results showed that the expression ofα-Synuclein in the fluoride-stained group was significantly lower than that in the control group（P<0.05）;after protein immunoblotting analysis,it could be found that the expression of GFAP protein increased in the fluoride-stained group（P<0.05）;the expression of GFAP andα-Synuclein was observed under fluorescence microscopy by immunohistochemical staining,and the fluorescence area was counted,and GFAP in the fluoride-stained group（0.25±0.12,0.44±0.20）fluorescence area was increased（P<0.05）andα-Synuclein（0.24±0.05,0.15±0.03）fluorescence area was decreased（P<0.05）.After fluorescent immunostaining of primary cultured astrocytes,GFAP showed green fluorescence;real-time quantitative PCR was performed,and the results showed that the m RNA expression of GFAP in the fluoride-dyed group（0.134±0.005,2.78±0.12）was elevated（P<0.05）;after protein immunoblotting assay analysis,it could be found that GFAP in the fluoride-dyed group（1.38±0.05,2.76±0.11）protein expression increased in the fluoride-stained group（P<0.05）,α-Synuclein protein location did not see significant bands（0.97±0.03,0.005±0.004）,and the difference was statistically significant（P<0.05）;flow cytometric detection of changes in calcium ion content showed that the number of calcium ions in the fluoride-stained group（54±9）was lower than that in the control group（72±13）（P<0.05）;flow cytometric detection of astrocyte apoptosis showed that the number of apoptotic cells in the fluoride-stained group（3.5±0.6）was higher than that in the control group（55±1）（P<0.05）.Conclusions:After the nervous system was damaged by chronic fluorosis,the expression of GFAP in astrocytes increased;astrocytes proliferated and hypertrophied,affecting synaptic transmission,decreasing intracellular Ca2+content and increasing apoptosis;α-Synuclein expression decreased. |
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