| Part one: Establishment of Vascular Cognitive Impairment Rats Model and the Behavioral Effects of Yishen Prescription on Rats with Vascular Cognitive ImpairmentObjective: Vascular cognitive impairment model rats were established by two-vessel occlusion(2-vo)method,To observe the effects on cognitive and behavioral functions of rats.To explore whether the treatment of Yishen prescription can effectively improve the learning and memory ability of rats with vascular cognitive impairment.Methods: 1.Experimental group: before the experiment,the rats with blurred vision and swimming obstacle were eliminated.The rest were randomly divided into 5 groups : sham operation group,model 2 weeks group,model 4 weeks group,model 6 weeks group,and Yishen prescription treatment group by random number table method.2.Modeling method: 2-VO method was used as the modeling method in rats with vascular cognitive impairment.In the sham operation group,only separat rsts bilateral carotid arteries without occlusion.3.Method of treatment: the Yishen prescription treatment group was given Yishen prescription for 6 weeks.The other groups were given pure water for 6 weeks.4.Behavioral test: Morris water maze behavioral test was used for testing the sham operation group,model 6 weeks group,and Yishen prescription treatment group’s learning and memory behavior tests 1 week after modeling and 6 weeks after treatment.Results: 1.A week after modeling: during the orientation navigation experiment,the time for rats in the sham operation group to find the escape platform decreased from the first day to the fourth day.In the second and third days of training,compared with the sham operation group,the time to find the platform was longer in the model 6 weeks group and the Yishen prescription treatment group with statistically significant differences(P<0.05).There was significant difference on the fourth day of training(P<0.01).In the spatial search experiment,compared with the sham operation group,the retention time at the original platform,the times crossing the original platform and total movement distance at the original platform,the retention time at the effective area and total movement distance at the effective area in the model 6 weeks group and Yishen prescription treatment group were reduced,and there were significant differences(P<0.01).However,there was no significant difference between model 6 weeks group and Yishen prescription treatment group(P>0.05).2.After 6 weeks of treatment: during the orientation navigation experiment,the time to find the platform was continuously shortened with the increase of training days in the sham operation group and the Yishen prescription treatment group.Compared with the sham group,there was a significant difference in the escape latency from day 2 to day 4 in the model 6 weeks group(P<0.01).Compared with the model 6 weeks group,there were significant differences from day 2 to day 4 between the Yishen prescription treatment group(P<0.01).In the space exploration experiment,compared with the sham operation group,the retention time,crossing times and total movement distance at the original platform,the effective area retention time and total movement distance in the model 6 weeks group were significantly shorten(P<0.01).Compared with the model 6 weeks group,all the data in the space exploration experiment in Yishen prescription treatment group were improved to different degrees with statistical significance(P<0.05).Conclusion: 2-VO model can simulate the pathogenesis of vascular cognitive disorder well,and the behavioral changes after modeling are in line with clinical manifestations,so it is a relatively reliable animal model of VCI.After the treatment of VCI model rats with the Yishen prescription,their spatial learning ability and spatial memory ability were significantly improved.Part two:Protective effect of Yishen prescription on hippocampal neuron apoptosis in rats with vascular dementiaObjective: To investigate the damage of vascular cognitive impairment to hippocampal neurons and the protective effect of Yishen prescription on hippocampal neurons in rats with vascular cognitive impairment.Methods: TTC staining was used to observe the cerebral infarction area of rats.The morphology of neurons in hippocampus CA1 and CA3 was observed by HE staining.Immunohistochemistry was used to detect the percentage of Neu N and m TOR positive cells in the hippocampus of rats.Western Blot analysis of Bax,Bcl-2 and m TOR proteins in rats hippocampal.Results: TTC staining showed no obvious infarct area in the sham operation group.In the model 6 weeks group,rats showed Obvious ischemic area.While no obvious cerebral infarction was observed in the Yishen prescription treatment group.HE staining showed that the neurons in the hippocampus CA1 and CA3 of the sham operation group had complete structures,and the pyramidal cells were arranged in a neat and dense arrangement with complete nucleus.After modeling,the pyramidal cells layer was loosely arranged,with apoptotic bodies,nuclear shrinkage and vacuoles.The morphology of neurons was significantly improved in the Yishen prescription group,and the phenomenon of nuclear pyknosis was significantly reduced.The arrangement of pyramidal cells was closer and more complete than that in the model group,and the cell morphology was become normal.Immunohistochemical results showed that,in the hippocampal CA1 and CA3 regions of rats,compared with the sham operation,the percentage of Neu N positive staining cells in each model group was significantly reduced,with a significant difference(P<0.01),and compared with the model 6 weeks group,the percentage of Neu N positive staining cells was significantly increased after the treatment with Yishen prescription for 6 weeks(P<0.01).In the hippocampus CA1 region,the percentage of m TOR positive staining cells in each model group was significantly higher than that of the sham group(P<0.01)and increased with time.Compared with the model 6 weeks group,the percentage of m TOR positive staining cells in the Yishen prescription treatment group was significantly lower(P<0.01).The percentage of m TOR positive staining cells in the model 2 weeks group was lower than the model 6 weeks group,and the difference was statistically significant(P<0.05).In the hippocampal CA3 region,the percentage of m TOR positive staining cells in rats in each model group increased significantly,and showed a trend of increasing with the number of days.Compared with the sham operation,the percentage of m TOR positive staining cells was decreased in the model 2 weeks group,and the difference was statistically significant(P<0.05),while it was significantly reduced in the model 4 weeks group and the model 6 weeks group(P<0.01).The percentage of positive staining cells was significantly reduced in the Yishen prescription treatment group,which was significantly different from that in the model 6 weeks group(P<0.01).Compared with the model 6 weeks group,the percentage of m TOR positive cells was significantly reduced at the model 2 weeks group(P<0.05).Western Blot results showed that,Compared with the sham operation group,the expression of bcl-2 protein in the hippocampus of the model 2 weeks group,model 4 weeks group and model 6 weeks group was significantly down-regulated(P<0.01),which decreased with the increase of days.Compared with the model 6 weeks group,the expression of bcl-2 protein in the model group was significantly increased(P<0.01).After 6 weeks of treatment from Yishen prescription,bcl-2 protein expression was significantly increased.Compared with the model 6 weeks group,the expression of bcl-2 protein in the hippocampus of rats in the Yishen prescription treatment group was significantly increased(P<0.01).Compared with the sham operation group,the expression of Bax protein in the hippocampus of rats in each model group was significantly increased(P<0.01),and the model groups showed an increase trend with the increase of weeks.Compared with the model 6 weeks group,the model 2 weeks group showed a decrease,and the difference was statistically significant(P<0.05).After 6 weeks of treatment with Yishen prescription,Bax protein expression showed a significant downward trend.Compared with the model 6 weeks group,the expression of Bax protein in hippocampus of rats in the Yishen prescription treatment group was significantly reduced(P<0.01).Compared with the sham operation group,bcl-2 /Bax in the hippocampal area of rats in each model group decreased significantly(P<0.01),while that in the model groups decreased with the increase of days,but the comparison between groups was not statistically significant(P>0.05).After 6 weeks of Yishen prescription treatment,bcl-2 /Bax protein expression showed an obvious increase trend.Compared with the model 6 weeks group,bcl-2 /Bax expression was significantly increased in the hippocampus of rats in the Yishen prescription treatment group.(P<0.01).In the hippocampus of rats,compared with the sham group,the model groups of m TOR protein expression significantly increased(P<0.01),with the increase of number of days,in the rat hippocampus m TOR expression showed a trend of increase,compared with the model 6 weeks group,the model 2 weeks group had lower expression model,the difference was statistically significant(P<0.05);After the treatment with Yishen prescription treatment for 6 weeks,the expression of m TOR in the hippocampal area of rats in the Yishen prescription treatment group was decreased,which was significantly different from that in the model group(P<0.01).Conclusion: Cerebral ischemia and hypoxia lead to apoptosis and loss of neurons in VCI model rats,inducing apoptosis-related factor generation.Meanwhile,m TOR pathway,which is closely related to neuron formation,apoptosis and autophagy,is overexpressed in hippocampus CA1 and CA3,resulting in decreased learning and memory ability.However,Yishen prescription can inhibit the apoptosis of neurons in the hippocampal area of rats,and its mechanism may be through the regulation of m TOR signaling pathway,ultimately improving the learning and memory ability of rats with vascular cognitive impairment. |
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