| ObjectiveThe purpose of this paper was to observe the effect of Kappa opioid receptors(KORs)agonist on postoperative cognitive dysfunction and the relationship with PKACREB pathway related molecules in rats after cardiopulmonary bypass,to explore the brain protective effect of KORs agonist and the possible mechanisms.MethodsForty adult male SPF grade SD rats weighting of 400~450g(provided by the General Hospital of Shenyang Military Department's animal experiment)were randomly divided into 5 groups(n=8 per group):Sham group(group S),CPB group(group C),CPB+KORs agonist U50488 H group(group K),CPB+KORs antagonist Nor-BNI+U50488H group(group N),CPB+PKA antagonist H89+U50488H group(group H).Rats were trained in water maze for five days,four times once a day before operation.Mechanical ventilation was performed after anesthesia in all groups.The model was not established and only arteriovenous punctured in group S.CPB was established and maintained for 60 min in the remaining four groups.Except for group S and C,rats were given U50488H(1.5mg/kg)by intravenous injection 30 min prior to CPB in other three groups.In group N,rats were received Nor-BNI(2.0mg/kg)by intravenous injection 60 min before CPB.In group H,rats were received H89(10m M,5?L)by intracerebroventricular injection 60 min before CPB.The Morris water maze experiment was used to test the cognitive function in each group 1 day after CPB.Then the rats were killed by bloodletting and the hippocampus samples were collected under anaesthesia.Hippocampal neuron apoptosis were quantitatively examined by TUNELstaining.The expressions of brain derived neurotrophic factor(BDNF),Amyloid ?-protein(A?)were measured by Western-blot and immunofluorescence method.The relative levels of p-PKA,p-CREB protein in hippocampus were measured by Westernblot.The m RNA levels of PKA,CREB,and BDNF were measured by Realtime-PCR.All quantitative data were expressed as the mean ± standard deviation(SD).All the statistical analyses were performed with SPSS 19 software.Multiple comparisons should be analyzed with one-way analysis of variance(ANOVA),followed by an appropriate multiple comparison test.A probability value of p<0.05 was considered as statistically significant.Results1.Comparison of the results of water maze test(1)the results of water maze test before CPBIn positioning cruise experiments,the average latency to find the platform and swim speed had no significant differences among all groups(p > 0.05).In space exploration experiments,there were no significantly differences among the average times of rats coming across the original platform,the distance and time of rats swiming in the target quardrant in five groups(p > 0.05).(2)the results of water maze test after CPBIn positioning cruise experiments,compared with group S,the average latency to find the platform were extended in all other groups(p<0.05).Compared with group C,the average latency were decreased in group K(p<0.05);there were no differences among group C,N and H(p>0.05);the swim speed had no significant differences among all groups(p>0.05).In space exploration experiment,compared with group S,the average times of rats coming across the original platform,the distance and time of rats swiming in the target quardrant were significantly reduced in all other groups(p<0.05).Compared with group C,the average times of rats coming across the original platform,the distance and time of rats swiming in the target quardrant were significantly increased in group K(p<0.05).There were no differences among group C,N and H(p>0.05).2.Comparison of the results of hippocampal neurons apoptosisCompared with group S,the rates of neuronal apoptosis were significantly increased in all other groups(p<0.05).Compared with group C,the rate of neuronal apoptosis was decreased in group K(p<0.05).There were no differences among group C,N and H(p>0.05).3.Comparison of the expressions of BDNF and A? protein in hippocampus(1)Western-blot method revealed that compared with group S,the expressions of BDNF protein were significantly decreased,A? protein were significantly increased in all other groups(p<0.05).Compared with group C,the BDNF protein was increased,A? protein was decreased in group K(p<0.05).There were no differences among group C,N and H(p>0.05).(2)Immunofluorescence method revealed that compared with group S,the levels and relative fluorescence intensity of BDNF protein positive cells were significantly decreased,A? protein positive cells were significantly increased in all other groups(p<0.05).Compared with group C,the levels and relative fluorescence intensity of BDNF protein positive cells were significantly increased,A? protein positive cells were significantly decreased in group K(p<0.05).There were no differences among group C,N and H(p>0.05).4.Comparison of the expressions of p-PKA,p-CREB protein in hippocampusCompared with group S,the relative expressions of p-PKA and p-CREB protein were significantly decreased in all other groups(p<0.05).Compared with group C,the relative expressions of p-PKA and p-CREB protein were increased in group K(p<0.05).There were no differences among group C,N and H(p>0.05).5.Comparison of the levels of PKA m RNA,CREB m RNA and BDNF m RNA in hippocampusCompared with group S,the levels of PKA m RNA,CREB m RNA,BDNF m RNA in rats' hippocampus were significantly decreased in all other groups(p<0.05).Compared with group C,the above indexes were increased in group K(p<0.05).There were no differences among group C,N and H(p>0.05).Conclusions1.CPB may changed cognitive function of rats,which is related to the decrease of BDNF protein and the increase of A? protein in hippocampus after operation.2.KORs agonists may increase the expression of BDNF protein,reduce the expression of A? protein and the rates of neuronal apoptosis in hippocampus through activated PKA-CREB signaling pathway,then plays a protective role in cognitive function after cardiopulmonary bypass. |
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