Effect And Mechanism Study Of Recombinant Osteopontin In Early Brain Injury After Subarachnoid Hemorrhage

Objective:To determine the effect of osteopontin(OPN)on autophagy and apoptosis after subarachnoid hemorrhage(SAH)and potential mechanisms involved.Methods:A total of 133 adult male Sprague-Dawley rats weighed 290～330 were subjected to sham or SAH operation,and were randomly divided into 5 groups:Sham,SAH+Vehicle(PBS,phosphate buffered saline),SAH+rOPN(5μg/rat recombinant OPN),SAH+rOPN+Fib-14(30mg/kg of FAK inhibitor-14),and SAH+rOPN+DMSO(dimethyl sulfoxide).Short and long-term neurobehavior tests were performed and followed by collection of brain samples for assessment of neuronal deaths,autophagy-apoptosis interaction,and hippocampal injury.The methods used included Western Blot,double immunofluorescence staining for cell location and autophagy-apoptosis interaction,Fluoro-jade C staining,terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)assay,and Nissl staining.Results:Endogenous OPN and autophagy-related proteins(Beclin 1,ATG 5 and the ratio of LC-3Ⅱ to LC-3 Ⅰ)all increased significantly after SAH.rOPN administration improved neurobehavior results,attenuated neuronal deaths 24 hours after SAH via elevating autophagy,suppressing apoptosis,and influencing autophagy-apoptosis interaction.Moreover,early administration of rOPN improved long term neurobehavior results,possibly by alleviating hippocampal injury.The effects of rOPN were reversed by Fib-14 injection in SAH+rOPN+Fib-14 group compared with SAH+rOPN+DMSO group.Conclusions:The results suggest that rOPN inhibits neuronal apoptosis through activating autophagy and regulating autophagy-apoptosis interaction.In addition,the FAK-ERK signaling pathway may be involved in upstream regulation.Early administration of rOPN may be a preventive and therapeutic strategy against delayed brain injury.