The Role Of Src-PSD95-NR2A Signal Module In Hepatic Ischemia-reperfusion Induced Hippocampus Injury

Objective: Hepatic ischemia-reperfusion(HIR)is a common pathophysiological process during liver surgery.It can not only cause liver damage,but also induce brain damage,and even lead to cognitive impairment.The developing nervous system is very fragile,and vulnerable to external stimulations which may even leave long-term sequelae in central nerves system.N-methyl-D-aspartate(NMDA)receptors are the primary excitatory glutamatergic receptors expressed in brain,the functions of which are closely associated with hippocampal learning and memory functions.And NMDA receptors mediated excitotoxicity is one of the key mechanisms of brain injury.Src,a member of non-receptor protein tyrosine kinases has been reported to up-regulate the tyrosine phosphorylation of NMDA receptor subunit 2A(NR2A),mediate excessive activation of NMDA receptors which can induce a large influx of Ca2+ leading to calcium overload and excitotoxicity of neurons.Postsynaptic density protein 95(PSD95)has been found to assemble NR2 A subunits and Src which acts as a bridge linking Src kinases with its substrate—NR2A subunits,and enhances the interaction between NR2 A and Src.This study aims to evaluate the effects of HIR on hippocampal neurons and long-term cognitive function in young mice,and explore the mechanisms related with Src-PSD95-NR2 A signal module.Methods: 2-week C57 mice were chosen to make 70% HIR model.The serum brain injury biomarkers S100β and NSE were observed after reperfusion of 6 h,1 d,3 d and 7 d respectively in young mice through enzyme-linked immunosorbent assay(ELISA).The expression level of Src,p-Src,NR2 A,p-NR2 A were observed in hippocampal tissues of mice through western blot.Then we chose HIR 3d mice to further randomly divide into six groups: vehicle pretreatment+sham-operated(sham)group,Src inhibitor PP2 pretreatment +sham-operated(sham(PP2))group,NR2 A inhibitor NVP-AAM077 pretreatment +sham-operated(sham(NVP))group,vehicle pretreatment+HIR(HIR)group,PP2 pretreatment +HIR(PP2)group and NVP-AAM077 pretreatment + HIR group(NVP group).Hippocampal histology and neurons apoptosis were determined by HE and TUNEL staining respectively.Western blot was used to detect the expression level of c-Src、p-Src、NR2A、p-NR2A、PSD95、cleaved caspase-3 in hippocampus.Interactions among Src,PSD95 and NR2 A were detected by immunoprecipitation.Biochemical analyzer was used to detect the serum ALT and AST,ELISA was conducted to detect the serum tumor necrosis factor(TNF)-α,interferon(IFN)-γ and interleukin(IL)-6.Another part of mice were randomly divided into four groups: sham group,HIR group,PP2 group and NVP group.Morris water maze was used to detect the long-term cognitive function after reperfusion of 30 d.Then the mice wre killed,western blot was used to detect the expression level of c-Src、p-Src、NR2A、p-NR2A、PSD95 in hippocampus,ELISA was used to detect serum TNF-α,IFN-γ and IL-6 in mice.Results: Serum S100β and NSE concentrations were significantly higher compared with sham group and the HIR3 d group had the highest level(P<0.05).The expression of c-Src and NR2 A in hippocampus didn’t have significant difference over time(P>0.05),while p-Src/c-Src and p-NR2A/NR2 A were significantly higher than sham group(P<0.05).And p-Src/c-Src reached highest after reperfusion of 1 d and 3 d,p-NR2A/NR2 A reached highest after reperfusion of 3 d.Therefore,we chose HIR 3 d group to further detect the mechanisms.The hippocampal tissues were edema,cells arrangement were disorder,neurons apoptosis index(AI)was increased in HIR group compared with sham group(P<0.05).PP2 and NVP-AAM077 pretreatment attenuated hippocampal pathological injury and decreased the AI(P<0.05).HIR not only up-regulated the p-Src/c-Src and p-NR2A/NR2 A,but also increased interactions of Src with NR2 A or PSD95,while both PP2 and NVP-AAM077 pretreatment decreased the interactions of Src with NR2 A or PSD-95(P<0.05).The data proved a interaction between Src and NR2 A.Serum ALT,AST,IFN-γ and IL-6 concentrations were significantly higher compared with sham group(P<0.05),PP2 and NVP-AAM077 pretreatment didn’t affect their concentrations(P>0.05),which indicates that PP2 and NVPAAM077 have a direct protective effect on hippocampus without affecting hepatocellular function and the release of inflammatory cytokines.The results of morris water maze showed that both escape latency and swimming speed didn’t have significant difference among the four groups(P>0.05).The mice in HIR group had less time spent in the target quadrant compared with sham group,while PP2 and NVP-AAM077 pretreatment increased the time spent in the target quadrant compared with the HIR group(P<0.05).The serum TNF-α、IFN-γ、IL-6 concentrations and the expression level of c-Src、p-Src、NR2A、p-NR2A、PSD95 in hippocampus didn’t have significant difference among the four groups(P>0.05).Conclusion: In summary,the present data suggest that HIR can lead to hippocampal injury and long-term cognitive dysfunction which is associated with the excitotoxicity caused by excessive phosphorylation of NR2 A,and the autophosphorylation of Src functions by up-regulating the activity of NR2 A via binding to PSD95.By exploring this pathway,we hope to put forward new ideas for the prevention and treatment of brain injury and long-term cognitive impairment after pediatric liver transplantation.