Effect And Mechanism Of Neuroglobin On Hypoxia/Reoxygenation Brain Injury Alleviated By Dexmedetomidine In Rats

Objective:Neonatal hypoxic-ischemic（H/I）brain injury is a frequently encountered clinical problem in survivors of perinatal asphyxia,which often caused different degrees of adverse neurologic damage,including cognitive disorder,epilepsy,cerebral palsy,and mental retardation.Transient ischemic hypoxia can cause severe acute brain injury,especially in hippocampus,which is highly susceptible to ischemic hypoxia.However,there is currently no effective therapy for neonatal H/I cerebral injury.Dexmedetomidine（DEX）is a selectiveα₂-recepter agonist that has been used clinically for analgesia,sedation and anxiolysis.Previously,DEX has been reported to produce its neuroprotective effect via theα₂-adrenoceptor.Although the neuroprotective effect of DEX has been demonstrated extensively,the molecular mechanism and mediated signaling pathways of DEX remains to be elucidated.Neuroglobin（Ngb）is a vertebrate,monomeric globin with abundant expression in neurons.It has high oxygen affinity,thus increasing the availability of oxygen to brain tissue.Recently,it is demonstrated that Ngb is overexpressed in H/I injury model and has protective effect on nerve cells from apoptosis,thereby protecting the neurons from H/I injury.Thus,we speculated that the neuroprotective effect of DEX may have potential association with Ngb,in which the DEX may induce the expression of Ngb to play an active role in the neuroprotective effect on H/I injury.Hypoxia induction factor 1 alpha（HIF-1α）,a master regulator of the response to hypoxia regulates the expression of a broad range of genes that facilitate the adaptation and survival of cells to low oxygen environments.It is a basic helix-loop-helix（bHLH）transcription factor that is degraded during normoxia.However,in hypoxic conditions,the hydroxylation modification declines and HIF-1αis stabilized for its transcriptional activities.Studies have shown that HIF-1αsignaling pathway has been found to be closely related to the pathogenesis of hypoxic encephalopathy in recent years,and its activation is believed to be related to the inhibition of neuronal apoptosis.Thus,in present study,the 7-day-old developing rat model of hypoxia/reoxygenation（H/R）injury was constructed to simulate the neonatal H/I brain injury.Then H/R model was exposed to different dose of DEX,and thereby to evaluate whether DEX contributed to reduce the neonatal H/I brain injury via regulating Ngb expression and further investigated whether this effect was related to the activation the HIF-1α/p53 signaling pathway via theα₂-adrenoceptor.Methods:1.One hundred and forty 7-day-old healthy SD rats weighing 12-16g were randomly divided into five groups（n=48 per group）:C group（control group）,H/R group（Hypoxia/Reoxygenation group）,D1 group（Hypoxia/Reoxygenation plus 25μg/kg DEX group）,D2 group（Hypoxia/Reoxygenation plus 50μg/kg DEX group）,and D3 group（Hypoxia/Reoxygenation plus 75μg/kg DEX group）.In addition to the C group,7-day-old Sprague Dawley（SD）rats were selected for hypoxia/reoxygenation treatment.The gas mixture of 8%oxygen and 92%nitrogen was inhaled for 2 hours.Then turn to supply 50%oxygen for 30min.After the treatment,except for the H/R group,the D1,D2and D3 groups were intraperitoneally injected with dexmedetomidine at 25μg/kg,50μg/kg and 75μg/kg,respectively.After 2h,24h,48h and 72h,the expression of Ngb was detected by Western blot and immunohistochemistry;the damage in the CA1 area of the hippocampus was observed by Nissl staining;the expression of apoptosis-related proteins Cyt-c,APAF-1 and Caspase-3 was monitored by Western blot;and the apoptosis rate of hippocampal cells was detected by TUNEL.2.Forty-eight 7-day-old healthy SD rats were randomly divided into four groups（n=12 per group）:C group（control group）,H/R group（Hypoxia/Reoxygenation group）,D group（Hypoxia/Reoxygenation plus50μg/kg DEX group）,and DY group（Hypoxia/Reoxygenation plus 50μg/kg DEX group and 0.5mg/kg yohimbine group）.After hypoxia/reoxygenation,group D was intraperitoneally injected with 50μg/kg dexmedetomidine,while group DY was intraperitoneally injected with 50μg/kg dexmedetomidine and 0.5mg/kg yohimbine,α₂receptor antagonist.After 24 hour,the expression of apoptosis-related proteins Cyt-c,APAF-1 and Caspase-3 was monitored by Western blot;the apoptosis rate of hippocampal cells was detected by TUNEL;the expression of Ngb and HIF-1αwas detected by Western blot,immunohistochemistry and immunofluorescence;and the expression of p53 was detected by Western blot.3.Forty-eight 7-day-old healthy SD rats were randomly divided into four groups（n=12 per group）:C group（control group）,H/R group（Hypoxia/Reoxygenation group）,D group（Hypoxia/Reoxygenation plus 50μg/kg DEX group）,and DM group（Hypoxia/Reoxygenation plus 50μg/kg DEX group and16mg/kg2-methoxyestradiol group）.After hypoxia/reoxygenation,in addition to intraperitoneal injection with 50μg/kg dexmedetomidine,the 7-day-old SD rats in the DM group were intraperitoneally injected with 16mg/kg 2-methoxyestradiol（2ME2）,an inhibitor of HIF-1α.After 24 hour,the expression of apoptosis-related proteins Cyt-c,APAF-1 and Caspase-3 was monitored by Western blot;the apoptosis rate of hippocampal cells was detected by TUNEL;the expression of pathway-related proteins Ngb and HIF-1αwas detected by Western blot,immunohistochemistry and immunofluorescence;and the expression of p53 was detected by Western blot.4.Five groups of SD rats（n=10 per group）:C group,H/R group,D group,DY group and DM group were tested by Morris water maze only on the 28th day of neonatal rats in order to evaluate long-term spatial learning and memory ability of rats.Results:1.Western blot analysis showed that the Ngb expression was up-regulated in H/R injury rat model at each time point compared to the control（p<0.05）.Medium and high doses of DEX significantly up-regulated the Ngb expression compared to the H/R group（p<0.05）,while low dose of DEX had no effect on the expression of Ngb（p>0.05）.Consistently,similar results were observed in the IHC analysis.Nissl staining showed that at 2h,24h after hypoxia/reoxygenation in the control group,neurons had abundant Nissl bodies.In the H/R model group,Nissl bodies reduced,disintegrated and even disappeared,and the number of neurons decreased compared to the control group,which suggested that hippocampal neurons were injured after H/R model establishment.When H/R model treated with low dose of DEX,Nissl bodies still reduced and disintegrated with small amount of neurons.When H/R model treated with medium and high doses of DEX,Nissl bodies reappeared and increased,meanwhile the number of neurons increased compared to the H/R group,and neurons showed a granular distribution of Nissl bodies.At 2h,24h after hypoxia/reoxygenation,the expressions of Cyt-c,APAF-1,Caspase-3 increased significantly in H/R group compared to the control（p<0.05）.After low dose of DEX exposure,the expressions of Cyt-c,APAF-1,Caspase-3 were not changed obviously（p>0.05）.When H/R model treated with medium and high doses of DEX,the expressions of Cyt-c,APAF-1,Caspase-3 significantly inhibited compared to the H/R group（p<0.05）.There was no significant difference in the expressions of Cyt-c,APAF-1,Caspase-3 among five groups at 48h,72h after hypoxia/reoxygenation（p>0.05）.TUNEL assay showed that after H/R model establishment,cell apoptosis rate was enhanced compared with the control（p<0.05）.Although D1 group has no effect on cell apoptosis（p>0.05）,D2 and D3 groups significantly inhibited the cell apoptosis at 2h,24h after hypoxia/reoxygenation（p<0.05）.There was no significant difference in the cell apoptosis among five groups at 48h,72h after hypoxia/reoxygenation（p>0.05）.2.Compared with D group,the expression of apoptosis-related proteins Cyt-c,APAF-1 and Caspase-3 were all increased after yohimbine treatment and TUNEL results showed that the staining intensity of D group was lower than that of DY group,and the corresponding apoptosis was also less than that of DY group（p<0.05）.In D group,up-regulated protein expressions of Ngb,HIF-1αand p53 were observed when compared with H/R group（p<0.05）.After treatment with yohimbine,however,the protein expressions of Ngb,HIF-1αand p53 in DY group were markedly decreased in comparison to D group（p<0.05）.Consistently,similar results were observed in the IHC and immunofluorescence analysis of Ngb and HIF-1α.3.The Western-blotting results showed that when 2ME2 was added,the protein expression of Cyt-c,APAF-1 and Caspase-3 in DM group were significantly increased in comparison with D group（p<0.05）.TUNEL results showed that the staining intensity of D group was lower than that of DM group,and the corresponding apoptosis was also less than that of DM group（p<0.05）.The Western-blotting results showed that the protein expressions of Ngb,HIF-1αand p53 were decreased significantly（p<0.05）.A similar pattern change was observed in the immunohistochemical and immunofluorescence of Ngb and HIF-1α.4.To investigate the effect of DEX on long-term learning and memory on H/R injure rats,the MWM test was performed on the 28th day of neonatal rats.It was found that in D group,the fifth day of the rats escape latency was significantly shortened,and the number of crossing platforms on the sixth day increased significantly in comparison with H/R group（p<0.05）.While compared with D group,the escape latency on the fifth day was significantly prolonged and the number of crossing the platform was significantly reduced in DY group and DM group（p<0.05）.Conclusion:DEX could induce the Ngb expression pharmaceutically in a dose-dependent manner,the DEX-mediated up-regulation of Ngb inhibited apoptosis by inhibiting Cyt-c,APAF-1,and Caspase-3.With theα₂ receptor antagonist-yohimbine intervention,the expression of Cyt-c,APAF-1 and Caspase-3 was significantly increased and the expression of Ngb was decreased.When the HIF-1αsignaling pathway was inhibited,the promotion of DEX on HIF-1α,p53 and Ngb disappeared.Furthermore,application of yohembine,or 2-methoxyestradiol（2ME2）,reversed the improvement of dexmedetomidine on spatial learning and memory function in H/R injure rats.Thus it can be seen that DEX can play a role in the treatment of neonatal hypoxic encephalopathy by activating the HIF-1α/p53 signaling pathway and up regulating the expression of Ngb via theα₂-adrenoceptor.