卒中后抑郁大鼠认知功能障碍与Cx36表达的相关性研究

Correlation between cognitive dysfunction and Cx36 expression in post-stroke depression rats

目的探讨卒中后抑郁(PSD)大鼠海马缝隙连接蛋白Cx36与认知功能障碍的相关性,探寻PSD认知功能障碍的潜在发病机制。方法将42只大鼠随机分为正常组、卒中组、抑郁组、PSD组、生理盐水组、甘珀酸(CBX)组和全反式维甲酸(ATRA)组,每组6只。抑郁组每天随机给予1种慢性不可预测的温和性刺激并孤养;向卒中组大鼠的运动皮层注射内皮素-1制备局灶性缺血型大鼠模型,PSD组、生理盐水组、CBX组和ATRA组在卒中组的基础上叠加抑郁刺激建立PSD模型。在PSD造模的同时,PSD组不进行干预;生理盐水组每天腹腔注射生理盐水1 ml;ATRA组每天腹腔注射浓度为1 mg/ml的1 ml乙醇和磷酸盐缓冲液(按1∶9配置);CBX组按照20 mg/kg的标准每天腹腔注射CBX。于卒中术后28 d,采用糖水实验、水迷宫实验、穿梭实验检测大鼠的兴趣缺失感、空间记忆能力、学习记忆能力,验证PSD认知障碍模型是否诱导成功;采用Real-time PCR检测大鼠海马Cx36 mRNA表达变化;采用Western blotting检测和免疫荧光检测大鼠海马Cx36蛋白的表达量和平均荧光强度。结果术后28 d,PSD组大鼠体重[(257.05±4.74)g]低于正常组[(352.00±7.99)g]、卒中组[(303.95±4.63)g],差异有统计学意义(P<0.05);PSD组大鼠24 h糖水饮用率[(61.92±3.12)%]低于正常组[(83.40±5.38)%]、卒中组[(88.03±3.65)%]、ATRA组[(71.15±4.55)%],高于CBX组[(49.62±5.85)%],差异有统计学意义(P<0.05);PSD组大鼠水迷宫潜伏时间[(51.18±4.14)s]长于正常组[(9.05±2.22)s]、卒中组[(9.06±2.25)s]、ATRA组[(32.92±2.29)s],短于CBX组[(91.13±3.27)s],差异有统计学意义(P<0.05);PSD组穿梭实验主动逃避次数[(10.67±1.51)次]少于正常组[(18.00±2.10)次]、卒中组[(16.5±1.87)次]和ATRA组[(13.83±1.17)次],多于CBX组[(7.00±1.26)次],差异有统计学意义(P<0.05)。术后28 d,PSD组海马区Cx36 mRNA表达量(0.50±0.03)低于正常组(1.04±0.07)、卒中组(1.07±0.10)和ATRA组(0.76±0.11),高于CBX组(0.20±0.06),差异有统计学意义(P<0.05);PSD组海马区Cx36蛋白相对表达量(0.60±0.07)低于正常组(0.86±0.05)、卒中组(0.88±0.03)和ATRA组(0.77±0.07),高于CBX组(0.56±0.02),差异有统计学意义(P<0.05);PSD组海马区Cx36蛋白平均荧光强度(0.36±0.03)低于正常组(1.00±0.03)、卒中组(0.97±0.03)和ATRA组(0.50±0.02),高于CBX组(0.21±0.03),差异有统计学意义(P<0.05)。结论PSD大鼠海马Cx36蛋白水平降低,CBX干预能降低Cx36蛋白表达并且大鼠认知功能障碍加重,ATRA干预能增加Cx36蛋白的表达并且大鼠认知功能障碍稍有好转。

Objective To investigate the correlation between hippocampal gap junction connexin Cx36 and cognitive dysfunction in post-stroke depression(PSD)rats,and to explore the potential pathogenesis of cognitive dysfunction of post-stroke depression.Methods A total of 42 rats were randomly divided into normal group,stroke group,depression group,PSD group,normal saline group,carbenoxolone(CBX)group and all trans retinoic acid(ATRA)group,with 6 rats in each group.The depression group was randomly given a chronic unpredictable mild stimulus every day and raised alone.The focal ischemic rat model was induced by injecting endothelin-1 into the motor cortex of stroke rats.PSD group,normal saline group,CBX group and ATRA group were superimposed with depression stimulation on the basis of stroke group to establish PSD model.At the same time of PSD modeling,PSD group did not intervene;In the normal saline group,1 ml of normal saline was injected intraperitoneally every day;ATRA group received intraperitoneal injection of 1 ml ethanol and phosphate buffer(1∶9)with a concentration of 1 mg/ml every day;CBX group was injected intraperitoneally every day according to the standard of 20 mg/kg.On the 28th day after stroke,sucrose preference test,Morris water maze test and shuttle test were used to detect the loss of interest,spatial memory,learning-memory ability of rats to verify whether the PSD cognitive impairment model was successfully induced.RT-PCR experiment was used to detect the expression changes of Cx36 mRNA in rat hippocampus.Western blot and immunofluorescence were used to detect the changes of Cx36 protein expression and average fluorescence intensity in rat hippocampus.Results On the 28th day after operation,the body weight of the rats in the PSD group[(257.05±4.74)g]was significantly lower than that of the normal group[(352.00±7.99)g]and the stroke group[(303.95±4.63)g],and the difference was statistically significant(P<0.05).The drinking rate of sugar water in PSD group[(61.92±3.12)%]was lower than that in normal group[(83.40±5.38)%],stroke group[(88.03±3.65)%]and ATRA group[(71.15±4.55)%],higher than the CBX group[(49.62±5.85)%],and the difference was statistically significant(P<0.05).The Morris water maze latency[(51.18±4.14)s]in the PSD group was significantly higher than that in the normal group[(9.05±2.22)s],the stroke group[(9.06±2.25)s]and the ATRA group[(32.92±2.29)s],lower than the CBX group[(91.13±3.27)s],and the difference was statistically significant(P<0.05).The times of active escapes in the shuttle experiment in the PSD group[(10.67±1.51)times]was lower than that in the normal group[(18.00±2.10)times],the stroke group[(16.5±1.87)times]and the ATRA group[(13.83±1.17)times],Higher than CBX group[(7.00±1.26)times],and the difference was statistically significant(P<0.05).The relative expression of Cx36 mRNA in hippocampus of PSD group(0.50±0.03)was lower than that of normal group(1.04±0.07),stroke group(1.07±0.10)and ATRA group(0.76±0.11),but higher than that of CBX group(0.20±0.06),and the difference was statistically significant(P<0.05).The relative expression of Cx36 protein in hippocampus of PSD group(0.60±0.07)was lower than that of normal group(0.86±0.05),stroke group(0.88±0.03)and ATRA group(0.77±0.07),but higher than that of CBX group(0.56±0.02),and the difference was statistically significant(P<0.05).The average fluorescence intensity of Cx36 protein in hippocampus of PSD group(0.60±0.07)was lower than that of normal group(1.00±0.03),stroke group(0.97±0.03)and ATRA group(0.50±0.02),but higher than that of CBX group(0.56±0.02),and the difference was statistically significant(P<0.05);The mean fluorescence intensity of Cx36 protein in the hippocampus of the PSD group(0.36±0.03)was lower than that of the normal group(1.00±0.03),the stroke group(0.97±0.03)and the ATRA group(0.50±0.02),but higher than that of the CBX group(0.21±0.03)(P<0.05).Conclusions The expression of Cx36 protein in the hippocampus of PSD rats was decreased.CBX intervention could reduce the expression of Cx36 protein and aggravate the cognitive dysfunction of the rats.ATRA intervention could increase the expression of Cx36 protein and the cognitive dysfunction of the rats was slightly improved.