| Objective: To study the protective effects of vitamin E(VE)on cadmium damaged cerebral cortical neurons and astrocytes on mice.Methods:?.In vitro study:(1)Primary culture the cerebral cortical neurons and the cerebral cortical astrocytes,the characterization and purity of the cells were determined by immunostaining of neuron marker(microtubule associated protein 2,MAP2),astrocyte marker(glial fibrillary acidic protein,GFAP)and microglia marker(type 3complement receptor,CR3/CD11b).(2)The neurons and astrocytes were treated with different concentrations of cadmium solution,respectively to observe the morphological changes under phase contrast microscope at different time points.After adding vitamin E,to observe morphological changes,to test the activity of superoxide dismutase(SOD)and the level of malonaldehyde(MDA)by spectrophotometry,to detect protein expression of Bcl-2/Bax and caspase-3 by Western blot in neurons and astrocytes.?.In vivo study:(1)Sixty healthy Kunming male mice were divided into three groups randomly,twenty mice in each group.In the chronic cadmium exposure group(Cd): the mice were injected with cadmium chloride solution(2 mg/kg)via subcutaneous of abdomen in twice a week.The chronic cadmium poisoning add vitamin E group(Cd+VE)were injected with the same content of chloride cadmium solution as the chronic cadmium exposure group,and adding VE(10 mg/kg)by intragastric administration in once a day.The control group were injected with saline solution.(2)After establish animal models for 13 weeks,learning-memory of all mice was measured by Y-maze,homogenate of the cerebral cortex was tested the activity of SOD and the level of MDA;to observe Nissl bodies changes of neurons on the frontal cerebral cortex by Nissl's staining;the ultrastructure of neurons were observed by the transmission electron microscope,and the positive cells of GFAP and caspase-3was examined by immunohistochemical staining;homogenate of the cerebral cortexwas tested the expressions of caspase-3 by Western blot.Results:(1)The purity of cultured primary cerebral cortical neurons and cortical astrocytes was more than 90 percent by immunofluorescent technology.(2)In vitro study,compared with control group,with the increase of concentration of cadmium and the extension of cadmium exposure time,the morphology damage was deteriorated gradually of primary neurons and astrocytes.The SOD activity were decreased and MDA level were increased of cells(vs control,P<0.05).The ratio of protein expression of Bcl-2/Bax was decreased,the expression of caspase-3 was increased(vs control,P<0.05).(3)Adding vitamin E to conduct interference treatment,compared with cadmium exposure group,the morphology damage was reduced of primary neurons and astrocytes.The SOD activity were increased and MDA level were decreased of cells(vs Cd,P<0.05).The ratio of protein expression of Bcl-2/Bax was increased,the expression of caspase-3 was decreased(vs Cd,P<0.05).Compared with control group,there are differences on VE intervention group(vs control,P<0.05).(4)The ability of learning and memory in the Cd group was lower than control group and the Cd+VE group(all P<0.05).The activities of SOD in cerebral cortex in the Cd group were significantly lower than those of controls and Cd+VE group(all P<0.05);the level of MDA in mice cerebral cortex in the Cd group were significantly higher than Control and Cd+VE group(all P<0.05).Comparing with the control group,Nissl bodies were decreased in neurons of Cd group,neurons' ultrastructure damage is serious,and expression of caspase-3 were significantly higher in Cd group(P<0.05).Comparing with the control group,Nissl bodies of Cd+VE group were slightly lower,and neurons' ultrastructure were mild injury;expression of GFAP and caspase-3 in Cd+VE group were significantly higher than control group.Conclusion: Vitamin E may protect mice cerebral cortical neurons and astrocyte on cadmium induced neurotoxicity damages,and the protective effect of vitamin E was correlated with role of anti-oxidant and reduce apoptosis. |
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