| Objectives:To study the relationship between the severity of cognitive impairment and the content of srxn1 in cerebrospinal fluid and the protein expression of srxn1 in hippocampus of rats with vascular cognitive impairment by Western blot,ELISA and RT-PCR.Method:1.Model making: 66 normal male SD rats,6 weeks old,230-270 g in body weight,were fed in separate cages for 5 days,under normal light,free water and food,temperature 22 ?,relative humidity 50%-70%.The rats were divided into five groups randomly,which were indicated by the letters a,B,C,D and e respectively.Group A was the blank control group,six SD rats were selected,and the rats in this group were not given any treatment;Group B was a sham operation group.Six SD rats were selected.The treatment of this group was to separate bilateral common carotid arteries;Group C was a routine operation group,18 SD rats were selected.The rats were treated with modified 2-V0 method to ligate bilateral common carotid arteries,so as to establish VCI rat model;Group D was srxn1 si RNA operation group,and 18 SD rats were selected.In this group,the modified 2-v0 method was also used to ligate bilateral common carotid arteries.However,24 hours before ligation of bilateral common carotid arteries,the interference fragment of srxn1 gene was injected into the lateral ventricle of rats;Group E was Nrf2 si RNA operation group,and 18 SD rats were selected.The treatment method was the same as that of group D,and the difference between group E and gene interference fragment was Nrf2 gene interference fragment.2.Model selection: The rats in each group were fed and observed for 30 days after modeling,and then the behavioral performance of rats in Morris water maze navigation experiment and space exploration experiment was observed to evaluate the spatial learning and memory ability of rats in each group,so as to select the target VD model of modeling success.3.Specimen preparation: Rats in each group were anesthetized with excessive chloral hydrate and decapitated.The skull was quickly peeled off on the ice,the cerebrospinal fluid was collected and the hippocampal tissue was separated.The hippocampal tissue was rinsed with pre refrigerated normal saline,put into a clean EP tube,and then put into an ultra-low temperature refrigerator at-80 ? after marking.4.Experimental method: according to the instructions of SOD,MDA and srxn1 kits,the concentrations of SOD,MDA and srxn1 in CSF of each group were measured with enzyme labeling instrument;the protein tissue solution was extracted after the protein lysate was added to the hippocampus of each group,and then the srxn1 and Nrf2 protein in hippocampus of each group were detected with Western blot method,and the m RNA expression level and si RNA interference of srxn1 in hippocampus of each group were detected with RT-PCR method Nrf2 mRNA level in hippocampus after sequence;Results:1.Establishment of animal model: After excluding the dead rats from 66 rats,we used Morris water maze to observe the behavioral changes of rats.We screened 46 successful VCI rats,and the success rate of VCI rats was more than 85%.2.In the navigation experiment,the blank control group rats were taken as the reference objects,the escape latency of rats in the sham operation group was not significantly prolonged,there was no significant difference in escape latency between the two groups,no statistical significance(P>0.05),compared with the control group,the rats in the routine operation group,the escape latency of the rats was longer than that of the blank control group,and the difference was statistically significant(P<0.05);Taking the rats in the conventional operation group as the reference object,the escape latency of srxn1 si RNA operation group was significantly longer than that of conventional operation group(P<0.05),Compared with the conventional operation group,the escape latency of Nrf2 si RNA operation group was also prolonged,with significant difference(P<0.05);In addition,srxn1 si RNA operation group rats and Nrf2 si RNA operation group rats were compared.There was no difference in the escape latency between the two groups(P>0.05).3.In the space exploration experiment,the blank control group rats were taken as the reference objects,compared with the blank control group,there was no significant difference in the number of times of crossing the platform and the time of staying in the platform quadrant between the sham operated group and the blank control group(P>0.05),compared with the blank control group,the number of times of crossing the platform in a unit time in the conventional operation group was significantly reduced,with statistical significance(P<0.01),compared with the blank control group,the retention time in the platform quadrant of the rats in the routine operation group decreased significantly,with statistical significance(P<0.01);The rats in the conventional operation group were taken as the reference objects,compare the crossing times of platform and the retention time of platform quadrant in unit time,In the srxn1 si RNA operation group,these two indexes were reduced,compared with the rats in the conventional operation group,the differences of the two indexes are significant,and they are all statistically significant(P<0.05),Compared with the conventional operation group,the Nrf2 si RNA operation group has the same number of platform crossing times and the retention time of platform quadrant as the srxn1 si RNA operation group.The difference between the two indexes is also significant(P<0.05);There was no significant difference between the two groups in the times of platform crossing and the time of platform quadrant retention,and the difference was not statistically signi ficant(P>0.05).4.In the space exploration experiment,the blank control group was taken as the reference object,and there was no significant difference in the retention time of the other four groups except the platform quadrant(P>0.05).5.There was significant difference of srxn 1 content in CSF of each group(P < 0.05),There was no significant difference in the expression of srxn 1 protein in the hippocampus of each group(P > 0.05),There was no significant difference in Nrf2 protein expression in hippocampus of each group(P > 0.05),There was no significant difference in the expression of srxn1 m RNA in the hippocampus of each group(P > 0.05),There was no significant difference in Nrf2 m RNA expression in hippocampus of each group(P > 0.05),There was no significant difference in Nrf2 m RNA level in hippocampus after si RNA interference.6.The difference of SOD activity in CSF of each group was statistically significant(P< 0.05)There was no significant difference in the content of MDA in CSF(P > 0.05).Conclusion:1.The expression of srxn 1 may not be related to the pathogenesis of vascular cognitive impairment. |
PDF Full Text Request