Regulation And Its Mechanism Of Neuroglobin In Glutamate-induced Neurotoxicity Mediated By AMPK

Background and Objectives Glutamate(Glu)is a neurotransmitter extensively involved in organism's metabolism and nerve conduction,but high concentration of glutamate will cause excitotoxicity,oxidative damage and inflammatory reactions,which participate in the pathophysiological processes such as stroke,Parkinson's disease,amyotrophic lateral sclerosis and other nervous system diseases.It is of great significance to investigate effective therapeutic targets for preventing glutamate injury.The research of the mechanisms of glutamate toxicity showed that glutamate toxicity will lead to the activation of adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK),inhibiting AMPK maybe reduce the damage generates by glutamate.Few studies on the role and mechanism of AMPK in the central nervous system diseases have been conducted and the conclusions are not completely consistent.Clarifying mechanism of AMPK and appropriately regulating the activity of AMPK at the right time is expected to be a new therapeutic target for relevant nervous system diseases.Neuroglobin(Ngb)is a kind of oxygen-carrying globulin discovered in the present century and mainly located in the nervous system.The protein has a specific neuroprotective effect in nervous system diseases,however,the action mechanism is still unclear.Researches show that Ngb can inhibit the AMPK pathway and promote anabolism.Does Ngb play a protective role on glutamate injury? Does AMPK exert a regulating effect on glutamate injury? Does Ngb mediate the role of AMPK in glutamate injury? How does Ngb mediate the role of AMPK in glutamate injury? To counter these problems,this study was carried out to further reveal the neuroprotective mechanism of Ngb,hoping to provide a new target for neuroprotective therapy of central nervous system diseases.Methods 1.The HT22 stably transfected cell line with overexpression of Ngb was constructed.Based on the glutamate injury model,using methods such as Calcein AM and Western Blot,the cell morphology and cell viability after the intervention of glutamate were observed,so as to clarify the role of Ngb against glutamate injury in HT22 cells.2.AMPK activator(AICAR)and AMPK inhibitor(Compound C)were used to intervene the glutamate injury model of HT22 cells,and Calcein AM,ROS,Western Blot and other methods were applied to observe the cell viability,cell oxidative stress and related protein expressions after glutamate injury and activation or inhibition of AMPK,so as to clarify the role of AMPK on glutamate injury in HT22 cells.3.The HT22 stably transfected cell line with overexpression of Ngb was used to construct glutamate injury model,AMPK activator(AICAR)and AMPK inhibitor(Compound C)were respectively used to carry out pretreatment on the glutamate injury cells,Calcein AM,ROS,Western Blot and other test methods were applied to observe the cell viability,cell oxidative stress and related protein expressions after HT22-Ngb glutamate injury and activation or inhibition of AMPK,so as to clarify whether Ngb can regulate AMPK and play a protective role in glutamate injury,and to further investigate the mechanism.Results 1.The cell viability of HT22 cells decreased after the intervention of 1m Mol or above Glu for 16 hours.With the increase of glutamate concentration,the cell viability showed a gradually downward trend.The cell viability was 46.21±10.6% after the intervention of 4m M Glu,the cellular state was poor,the cellular morphology changed,the cell shape became small,the cytoarchitecture was disordered,the protuberances decreased,and the cell injury was medium.Therefore,in the follow-up experiments,we chose 4m M Glu for intervention of 16 hours to build the cell injury model.2.The HT22 stably transfected cell line with overexpression of Ngb was successfully constructed,on this basis,the glutamate injury cell model was built.The result showed that under the identical concentration of glutamate intervention,compared with HT22-GFP cells,HT22-Ngb cells had significantly increased in cell viability.The cell viability of HT22-Ngb cells was 62.44±5.47%(P<0.0001)higher than that of HT22-GFP cells after intervention of 4m M glutamate for 16 hours,suggesting that the over expressed Ngb has the function of anti glutamate injury in HT22 cells.3.AMPK activator(AICAR)and inhibitor(Compound C)were used to intervene the HT22 cells.It was found that 1m M AICAR and 5?M Compound C respectively exerted a favorable effect on the activation and inhibition of AMPK.The use of AMPK activator did not exert a significant effect on the cell viability with glutamate injury,but the cell viability increased by 84.26±5.16%(P<0.0001)after the use of AMPK inhibitor with glutamate injury,meanwhile,ROS was significantly reduced.These results suggested that the inhibition of AMPK has a protective effect on the HT22 cell injury induced by glutamate,which may be related to the inhibition of oxidative damage.4.Under the intervention of 4m M glutamate,compared with the HT22-GFP cells,the viability of HT22-Ngb cells increased by 62.44±5.47%(P<0.0001),but after the combined intervention of AMPK activator and Glu,the originally inhibited activity of AMPK in HT22-Ngb cells was reactivated,the cell viability decreased by 57.20±4.12%(P<0.0001),and ROS significantly increased.However,after the combined intervention of AMPK inhibitor and glutamate,compared with the glutamate interventional group,the level of AMPK in HT22-Ngb cells was not significantly inhibited and no significant difference was found in the cell viability and ROS level.5.Compared with HT22-GFP cells,the expression of HIF-1? in HT22-Ngb cells decreased by 32.69±12.12%(P<0.05).In HT22-Ngb cells,there was no significant difference in the expression of HIF-1? between glutamate intervention group and control group;compared with that in glutamate intervention group,the expression of HIF-1? in AICAR combined glutamate intervention group increased by 46.49±15.67%(P<0.05).6.For HT22-GFP cells,after 4m M glutamate intervention,the Bax level was up-regulated by 48.08±18.38%(P<0.05),and the phosphorylation-level of BCL-2 was down-regulated by 53.63±11.81%(P<0.05).For HT22-Ngb cells,the activation levels of Bax and BCL-2 had no significant change after 4m M glutamate intervention.Conclusion 1.Over expressed Ngb exerts a role of anti glutamate injury in HT22 cells.2.Inhibition of AMPK activity has a protective effect on glutamate induced HT22 cell injury.3.Ngb plays a neuroprotective role through the inhibition of AMPK.4.The Ngb role of anti glutamate injury by regulating AMPK activity may be related to the reduced level of oxidative stress,the regulation of HIF-1? and the apoptosis.