| Objective: In this study,adult Kunming mice were used as the research object.First,D-galactose was used to make acute senescence model,and then exercise intervention was given to explore the protective effect of exercise on cerebral cortical neurons of aged animals from the perspective of cell apoptosis.Methods:1.Grouping of experimental animals: 40 healthy male Kunming mice were selected and kept clean.The animals were randomly divided into four groups(n=10),the control group(without any method),the control exercise group(with running wheels),the model group(with D-galactose injection),and the model exercise group(with running wheels after D-galactose injection).2.Model preparation: D-galactose(125mg/kg)was injected subcutaneously to the neck of Kunming mice once per day for 8 weeks in a row to prepare the acute senescence mouse model,and the control group was injected with normal saline of the same volume.In the exercise group,the intensity was 6m/min for 30 minutes per day.Exercise for 8 weeks with 2 days off per week..3.Specimen preparation: 5 mice were taken from each group for routine perfusion and fixation,brain extraction and paraffin embedding.A 5 m thick coronal section was performed.A coronal section containing the dorsal hippocampal plane was mounted on a polylysine-coated glass slide for HE,TUNEL and immunohistochemical staining.Five mice were taken from each group,and their heads were severed after anesthesia.50 mg of cerebral cortex corresponding to the dorsal hippocampus was taken to extract the protein.The protein concentration was determined and frozen in a refrigerator at-80℃ for western blot detection.4.Experimental methods1)The morphological changes of cerebral cortex in mice were observed by HE staining.2)TUNEL was used to detect the changes of apoptotic cells in the cerebral cortex of mice.3)The expressions of Bcl-2,Bax and Caspase3 in the cerebral cortex of mice were observed by immunohistochemical method.4)The expressions of Bcl-2,Bax and Caspase-3 in the cerebral cortex of mice were observed by Western Blot.Take-actin as internal reference.5.Statistical processing: SPSS 16.0 statistical software was used for statistical analysis of the experimental data,and the test data were expressed as mean ± SEM.One-way analysis of variance(ANOVA)and S-N-K test were used for pairwise comparison between groups.The test level was = 0.05,and P< 0.05 had statistical significance.Results:1.HE staining results: normal structure was dominant in the dorsal hippocampus plane cortical neurons of the normal control group and the control exercise group,and the number of denatuated cells per 100 cells was very small,which was 1.6±1.7 in the control group and 2.3±0.5 in the control exercise group,and there was no statistical significance between the two groups(P>0.05).A large number of denaturated neurons were observed in the model group,and the number of denaturated cells per 100 cells was up to34.5±6.5,with significant difference compared with the control group(P<0.01).Movement after exercise intervention model group degeneration dramatic decline in the number of neurons for every 100 cell degeneration of cell number was 23.2±4.0,significant difference compared with model group(P<0.01),significant differences compared with control group(P<0.01),suggesting exercise intervention can reduce degeneration in the aging model rat cerebral cortex neuron number,but could not return to normal levels.2.TUNEL staining results: The number of apoptotic cells per 100 cells in the dorsal hippocampus plane cerebral cortex was observed and counted: 4.8±1.8 in the control group,6.1±0.9 in the control group,31.4±1.2 in the model group,and 25.2±2.9 in the model exercise group.P > 0.05 showed no significant difference between the control group and the control exercise group.Compared with the control group and the model group,P < 0.01 was significantly different.There was significant difference between the control group and the model exercise group when P < 0.01.There was significant difference between model group and model exercise group when P < 0.01.The number of apoptotic cells increased in the model group and decreased after exercise,but the number was still higher than that in the control group.3.Immunohistochemical staining results of Bcl-2,Bax and Caspase-3 in the dorsal hippocampal plane cerebral cortex showed cytoplasmic staining for all three proteins.The positive expression of Bcl-2 was higher in the control group and the control exercise group,and lower in the model exercise group and the model exercise group.Count the number of bcl-2 positive cells per 100 cells:32.6±1.9 in the control group,31.3±3.7 in the control group,2.4±1.5 in the model group,and 16.5±1.4 in the model exercise group.Bax positive expression was lower in the control group and control exercise group,and higher in the model group and model exercise group.Count the number of Bax positive cells per 100 cells: 1.5±0.5 in the control group,2.4±1.2 in the control group,33.1±2,2 in the model group,and19.2±1.8 in the model group.Positive expression of Caspase-3 was lower in the control and control exercise groups,and higher in the model and model exercise groups.Count the number of caspase-3 positive cells per 100 cells: 3.8±2.3 in the control group,3.3±1.3 in the control group,33.3±2.1 in the model group,and21.2±2.9 in the model group.The data of the three groups were analyzed by one-way an OVA,all P <0.01,indicating that the difference between the groups was significant.Further pairwise comparison,P > 0.05 showed no significant difference between the control group and the contraindication group.Compared with the control group and the model group,P < 0.01 was significantly different.There was significant difference between the control group and the model exercise group when P < 0.01.There was significant difference between model group and model exercise group when P < 0.01.It was suggested that aging would be accompanied by decreased expression of bcl-2,the most important antiapoptotic protein in the Bcl-2 family,and increased expression of Bax,the pro-apoptotic protein.And increased expression of caspase-3,a hydrolyzed protein that performs apoptosis.Exercise intervention can increase the expression of Bcl-2 and decrease the expression of Bax and Caspase-3 in the cerebral cortex of aging model,but it is still different from the control group.4.Western blot results of dorsal hippocampus plane cerebral cortex:1)Bcl-2 protein: the gray ratios of the control group,control exercise group,model group and model exercise group were 1.063±0.201,0.891±0,160,0.531±0.079 and 0.688±0.065,respectively.The model group and model exercise group were significantly lower than that of the control group(P<0.01),and the model exercise group was significantly higher than that of the model group(P<0.01),suggesting that aging may cause decreased expression of Bcl-2 protein,while exercise can improve the expression of Bcl-2 protein in cortical neurons of the aging model rats,but it was still lower than that of the control group.2)Bax protein: The gray ratios of the control group,control exercise group,model group and model exercise group were 0.506±0.747,0.561±0.573,0.898±0.767 and 0.776±0.092,respectively.The model group and model exercise group were significantly higher than the control group(P<0.01),while the model exercise group was significantly lower than the model group(P<0.01),indicating that aging would cause increased expression of Bax protein,while exercise could reduce the expression of Bax protein in cortical neurons of aging mice,but it was still higher than the control group.3)Caspase-3 protein: the gray ratios of the control group,control exercise group,model group and model exercise group were 0.601±0.211,0.630±0.203,1.044±0.148 and 0.874±0.113,respectively.The model group and model exercise group were significantly higher than that of the control group(P<0.01),while the model exercise group was significantly lower than that of the model group(P<0.05),suggesting that aging can cause increased expression of caspase-3 protein,while exercise can reduce the expression of caspase-3 protein in cortical neurons of aging mice,but still higher than that of the control group.4)Changes in the ratio of Bcl-2/Bax: The ratio between bcl-2 /Bax proteins is a key factor in the regulation of apoptosis,and the increased ratio of Bcl-2/Bax can inhibit the occurrence of apoptosis.The ratios of the control group and the transport group were 2.12±0.42,1.62±0.41,all greater than 1,model group 0.59±0.08 and model exercise group 0.90±0.41,both less than 1.The model group and model exercise group were significantly lower than that of the control group(P<0.01),and the model exercise group was significantly higher than that of the model group(P<0.01),suggesting that aging would cause a decrease in the Bcl-2/Bax ratio,while exercise could improve the Bcl-2/Bax ratio in the cerebral cortex neurons of the aging model rats,but the ratio was still lower than that of the control group.Conclusions:1.The number of degenerated neurons and apoptotic cells in the cerebral cortex of the acute senescence model mice increased,and the number of degenerated neurons and apoptotic cells in the cerebral cortex of the acute senescence model mice decreased by exercise intervention.2.Decreased bcl-2 expression,increased Bax and Caspase-3 expression,and decreased Bcl-2/Bax ratio in cerebral cortex of acute senescence mice;After exercise intervention,the expression of Bcl-2 increased,the expression of Bax and Caspase3 decreased,and the ratio of Bcl-2/Bax increased,suggesting that exercise intervention could play a neuroprotective role by inhibiting the implementation of cell apoptosis. |
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