The Role And Mechanism Of Neuromodulation Receptor Degradation Protein(Nrdp1) In Post-operative Cognitive Dysfunction Caused By Cardiopulmonary Bypass In Aged Rats

Object:To establish the POCD model of aged rats through the CPB,preliminarily discussed the relationship between Nrdp1,the apoptosis of hippocampal neurons and POCD after CPB in elderly rats.With lentivirus to build Nrdp1 overexpression,Nrdp1 silencing and empty plasmid group of hippocampal neurons,to explore the regulation of Nrdp1 on hippocampal neuronapoptosis in hypoxia-reoxygenation model.Then through the gene interfering with the expression of Nrdp1 in aged rat hippocampus,explored the effect of Nrdp1 on POCD after CPB and it's regulatory mechanism at the carrier level.Methods:Part ?60 old Sprague-Dawley rats were concluded and randomly divided into the control group(Group C),the sham group(Group S),the model group(Group M),n=20,for five days of adaptation training.In Group M,CPB was established,Group S only made corresponding vessel ligature,and Group C were not done anything.In order to evaluate the effect of CPB on learning and memory function,we observed the behavioral changes on the 1st,3rd,and 7th day after CPB in three groups of elderly rats,including1.positioning cruise experiment(measuring escape latency and swimming speed),2.Space exploration experiment(measuring platform quadrant latency distance and platform quadrant latency time).After completion of the model,inflammatory cytokines TNF-?and IL-1?content in the hippocampus of rats in Groups C,S,and M were detected by ELISA(Enzyme Iinked immunosorbent assay,ELISA).The hippocampal tissues of rats in Groups C,S,and M were taken for TUNEL staining,and the CA1,CA3,and DG hippocampal neuronal apoptosis in each group were observed,and the apoptosis indexes were calculated.Western blot was used to detect the expression levels of Nrdp1,ErbB3,C.Caspase3 and p-AKT in the hippocampal tissues of Groups C,S and M after CPB.Part?The primary hippocampal neurons were cultured in vitro.In the 5~thh day,the recombinant virus were transfected into the primary hippocampal neurons to construct a specific Nrdp1 overexpression group,a specific Nrdp1 silencing group,and an empty plasmid group.After 3 days,the hypoxia-reoxygenation model of hippocampal neurons was constructed.The hippocampal neurons were divided into five groups according to the random number table method:blank control group(Group C),hypoxia/reoxygenation group(Group H/R),empty plasmid of hypoxia/reoxygenation group(Group H/R+NC),Nrdp1 Overexpression of hypoxia/reoxygenation group(Group H/R+Nrdp1),Nrdp1 silencing of hypoxia/reoxygenation group(Group H/R+shNrdp1).The viability of fives groups of neurons was detected by MTT assay,and the apoptosis of neurons was observed by flow cytometry.Western blot was used to detect the changes of Nrdp1,ErbB3,p-AKT,AKT and C.Caspase3 proteins.Part?The recombinant virus were injected into the hippocampal CA1 region of the rat through a craniocerebral locator to construct a Nrdp1overexpressing,silencing,and empty plasmid control rat.Then,establish the cardiopulmonary bypass modeland.The rats were divided into 5groups according to the random number table method:blank control group(Group C),cardiopulmonary bypass group(Group M),empty plasmid cardiopulmonary bypass group(Group M+NC),Nrdp1 overexpression cardiopulmonary bypass group(Group M+Nrdp1)and Nrdp1 silencing cardiopulmonary bypass group(Group M+shNrdp1).The Morris water maze test was used to observe postoperative behavioral changes,and to observe learning and memory function changes in five groups of aged rats,including 1.Positioning cruising experiment(measurement of escape latency and swimming speed),2.Space exploration experiment(measurement percentage of residence time and percentage of stay distance).TUNEL staining was used to observe the apoptosis of hippocampal neurons in hippocampal CA1 region in each group.Western blot assay was used to detect the changes of Nrdp1,ErbB3,p-AKT,AKT,and C.Caspase3 proteins in the hippocampus after CPB in the five groups of rats.Results:Part ?(1)There was no statistically significant difference in behavioral scores between the three groups of rats in the preoperative baseline test and Morris water maze training.Compared with the rats on the first day,the escape latency after training was shortened,and the percentage of staying distance and percentage of residence time increased(P<0.05).(2)After CPB surgery,in the test of cognitive function of the Morris water maze on days 1,3,and 7,compared with Group C,the escape latency,swimming speed,percentage of platform quadrant latency distance and platform quadrant latency time of the Group S,There were no statistical difference(P>0.05).But compared with Group C and Group S,the escape latency of Group M were increased,the percentage of platform quadrant latency distance and platform quadrant latency time of Group M were decreased,the difference was statistically significant(P<0.05).(3)TUNEL staining was used to observe the neurons apoptosis of three groups of rats after CPB surgery at hippocampal CA1,CA3,DG areas.the hippocampal neuronal apoptosis index of CA1 area in rats of Group C,S and M were:0.082±0.045,0.074±0.057 and 0.862±0.072.The hippocampal neuronal apoptosis index of rat DG area in Group C,S and M were:0.076±0.064,0.082±0.071,0.863±0.085.Compared with Group C,the neuronal apoptosis index of Group S in hippocampal CA1,DG areas had no statistical significance difference(P>0.05),compared with Group C,the apoptosis index of Group M in hippocampal CA1,DG areas were increased,the difference was statistically significant(P<0.05);compared with the Group S,the apoptosis index of Group M in hippocampal CA1,DG areas were increased,the difference was statistically significant(P<0.05).There was no statistical difference in the apoptosis of neurons in CA3 area of the three groups(P>0.05).(4)Western blot was used to detect the expression of Nrdp1,ErbB3,p-AKT and C.Caspase3 in the hippocampus of elderly rats.There was no statistical difference between Group C and Group S in the expression of Nrdp1,ErbB3,C.Caspase3 and p-Akt(p>0.05).Compared with Group C and Group S,the expression of Nrdp1 and C.Caspase3 in Group M were increased(P<0.05);the expression of p-AKT and Erb B3 in Group M were decreased,and the difference was statistically significant(p<0.05).Part?(1)The purity in the 5th day of cultured hippocampal neurons was more than 90%by NSE staining.According to the expression of GFP in the three groups of hippocampal neurons after transfection by lentivirs,the transfection efficiency was more than 80%when MOI=5.The results of real-time quantitative PCR and Western blot showed that there was a statistically significant difference in Nrdp1 between the empty plasmid control,Nrdp1 overexpression,and Nrdp1 silencing groups(P<0.05).(2)After hypoxic reoxygenation,The results of Western blot of the five hippocampal neurons were:compared with Group H/R,Group(H/R+NC),Group(H/R+Nrdp1),in the Group(H/R+sh Nrdp1)the expressions of Nrdp1 and C.Caspase3 were significantly decreased(P<0.05);The expressions of ErbB3 and p-AKT were significantly increased(P<0.05).Compared with Group C,Group H/R,Group(H/R+NC),Group(H/R+shNrdp1),in the Group(H/R+Nrdp1)the expressions of Nrdp1 and C.Caspase3 were significantly increased(P<0.05);The expressions of ErbB3 and p-AKT were significantly decreased(P<0.05).(3)MTT assay and flow cytometry showed:compared with Group C,Group H/R,Group(H/R+NC),Group(H/R+shNrdp1),the hippocampal neuronal activity of Group(H/R+Nrdp1)was decreased;the hippocampal neuronal apoptosis of Group(H/R+Nrdp1)was increased(P<0.05).compared with Group H/R,Group(H/R+NC),Group(H/R+Nrdp1),the hippocampal neuronal activity of Group(H/R+shNrdp1)was increased;the hippocampal neuronal apoptosis of Group(H/R+sh Nrdp1)was decreased(P<0.05).Compared with the Group H/R,Group(H/R+NC)had no significant difference in neuronal activity and apoptosis(P>0.05).Part?(1)There was no significant difference in the behavioral indexes of rats between the preoperative baseline behavior test and the Morris water maze training.No preoperative rats had learning and memory impairment.(2)Through injection of lentivirus into the hippocampus,three groups of rats were constructed:empty plasmid control,Nrdp1 overexpression,and Nrdp1 silencing group.The results of real-time quantitative PCR and western blot showed that:there was a statistically significant difference in Nrdp1 between the empty plasmid control,Nrdp1 overexpression,and Nrdp1 silencing groups(P<0.05).(3)The cognitive function of Morris water maze was test after cardiopulmonary.Compared with Group C,Group M,Group(M+NC),Group(M+shNrdp1),The escape latency of Group(M+Nrdp1)was prolonged;the percentage of the staying distance in the hidden quadrant and the percentage of the staying time in the hidden quadrant of Group(M+Nrdp1)were decreased(P<0.05).Compared with the Group M,Group(M+NC),Group(M+Nrdp1),The escape latency of Group(M+sh Nrdp1)was decreased;the percentage of the staying distance in the hidden quadrant and the percentage of the staying time in the hidden quadrant of Group(M+shNrdp1)were prolonged(P<0.05).(4)TUNEL staining was used to observe the neuronal apoptosis in CA1 region of the five groups.Compared with Group M,Group(M+NC),and Group(M+Nrdp1),the neuronal apoptosis in the hippocampal CA1region was decreased in the Group(M+shNrdp1)(P<0.05).Compared with Group C,Group M,Group(M+NC),Group(M+shNrdp1),the neuronal apoptosis in the hippocampal CA1 region was increased in the Group(M+Nrdp1)(P<0.05).(5)Western blot analysis showed that the expression of Nrdp1 and C.Caspase3 in the hippocampus of the rats in the Group(M+shNrdp1)were decreased and the expression of ErbB3 and pAKT were increased,when compared to the M,M+NC,and M+Nrdp1 groups.The difference were statistically significant(P<0.05).The expression of Nrdp1 and C.Caspase3in hippocampus of Group(M+Nrdp1)were increased and the expression of ErbB3 and p AKT were decreased,when Compared to the Group C,Group M,Group(M+NC),Group(M+sh Nrdp1).The difference were statistically significant(P<0.05).Conclusion:(1)CPB has obvious correlation with POCD in aged rats,the hippocampal neurons of POCD rats are obviously apoptotic,and the development of POCD may be closely related to the apoptosis of hippocampal neurons.(2)The expression of Nrdp1,C.caspase3 in the hippocampus of the aged rats after CPB was significantly increased.Nrdp1 may participate in the development of POCD by promoting the apoptosis process of hippocampal neurons.(3)The geneticengineering regulation of Nrdp1can affect the apoptosis process of hippocampal neurons at the cellular level.The relevant mechanism is that Nrdp promotes the ubiquitination and degradation of ErbB3,and reduces Erb B3 and the downstream signal pathway p-AKT,resulting in the occurrence of apoptosis.(4)Genetic engineering regulation of Nrdp1 can effectively prevent and improve cognitive impairment and hippocampal neuronal apoptosis after CPB in aged rats,and its mechanism may be related to the Nrdp1/ErbB3/p-AKT/C.Caspase3 signaling pathway.