Salubrinal Offers Neuroprotection Through Suppressing Endoplasmic Reticulum Stress,Autophagy And Apoptosis In A Mouse Traumatic Brain Injury Model

Objective: Traumatic brain injury(TBI)is a complex disease process,which can lead to very serious disability or even death.TBI can induce secondary injury cascades,including but not limited to endoplasmic reticulum(ER)stress,apoptosis and autophagy.Although the investigators has previously shown that Salubrinal,the selective phosphatase inhibitor of p-e IF2?,ameliorated neurologic defificits in murine TBI model,the neuroprotective mechanisms of Salubrinal need further research to warrant the preclinical value.The purpose of this study was to explore the protective effect of salubrinal therapy after TBI on the consequences of neurological injury in mice and its possible mechanism.Methods: TBI Model of ICR mice established by improved free fall device.In order to study the expression of ERS-related protein after brain injury,the mice were killed 6 hours,1 day,2 days,3 days,7 days,10 days and 14 days after operation,and the control mice were killed on the second day.The co-expression of CHOP in NVU cells(including neurons,astrocytes,microglia,vascular endothelial cells and peripheral cells)was observed by immunofluorescence(2 days after TBI).In order to control the non-specific effect of the operation,the animals in the sham operation group received the same craniotomy without damaging the cortex.In order to explore the influence of salubrinar on TBI,the experimental groups are as follows: vehicle-treated sham group,Salubrinaltreated sham group,vehicletreated TBI group and Salubrinal-treated TBI group.As mentioned before,salubrinar(1mg/kg,first dissolved to 100 mg/kg in DMSO,then dissolved to the final concentration in brine)was intraperitoneally injected 2 hours after TBI.The solvent control mice received intraperitoneal injection(1%DMSO saline)at the same time point after TBI.The sham operated group also received Salubrinal or solvent injection.PI staining(2 days after TBI)was used to observe the damage of neurons(including cortex and CA1,CA3 and DG areas in hippocampus)after salubrinal treatment.The long-term protective effect of salubrinal on TBI was evaluated by lesion volume(LV).Behavioral experiments(including climbing pole experiment and Morris water maze experiment)were carried out to observe the changes of motor and learning memory function of mice after salubrinal treatment.In order to study the protective mechanism of salubrinal on TBI,the expression of ERS related protein(p-e IF2?,e IF2?,GRP78,CHOP),autophagy related protein(P62,LC3,Beclin-1)and apoptosis related protein(Bcl-2,caspase-12,caspase-3)were detected by Western blot,and the co-expression of CHOP and LC3,TUNEL in nerve cells was detected by immunofluorescence staining(2 days after TBI).Result:(1)In this study,the TBI Model of mice was established firstly,and the expression of ERS related proteins p-e IF2?,GRP78 and CHOP was found to be temporal expression by Western blotting,and the peak expression was found at 2d and 3d after TBI.(2)Immunofluorescence(2d)showed that CHOP protein could be co-located and expressed with neurons,astrocytes,microglia,vascular endothelial cells and peripheral cells.(3)Continuous administration of ERS inhibitor Salubrnal can repair damaged plasma membrane(2d),improve motor function(1-5d),learning and memory function(10-14d)and reduce brain lesion volume(21d).(4)Western blotting showed that Salubrinal(2d)could inhibit TBI induced expression of ERS related proteins p-e IF2?,ATF-4,GRP78 and CHOP,inhibit autophagic death(LC3II,Beclin-1 and P62),and inhibit autophagic death(LC3II,Beclin-1 and P62).(5)Immunofluorescence showed that Salubrinal(2d)could reduce CHOP and TUNEL,CHOP and LC3 co-positive cells after TBI.Conclusion: ERS related proteins are expressed in a time-sequential manner,CHOP can be co-located with NVU cells,Salubrinal can respair cytoplasmic membrane damage and neurological defects after TBI.Its protective mechanism involves the inhibition of ERSautophagy-apoptosis pathway.Therefore,Salubrinal may be an effffective therapeutic agent for the treatments of TBI and perhaps other acute central nervous system injuries and disorders.