E64d Attenuating Nerve Cell Plasmolemma Permeability And Neurological Dysfunction Through Apoptotic And Autophagic Pathways After Traumatic Brain Injury In Mice

Objective: To investigate the effects of E64d (a cathepsin B andμ-calpain inhibitor) on TBI induced nerve cell plasmolemma permeability and neurological dysfunctionMethods: TBI model was established by weight drop device in adult mice based on procedures previously reported. Two hundreds mature male mice were randomly divided into DMSO (1μl) control group and E64d-(0.5μg/1μl) pretreated group for time course works of TBI. Fifty were used to assess the number of cell death by nuclear staining with Propidium Iodine (PI) for 1 h, 6 h, 12 h, 24 h, and 48 h. Mice post-TBI were pretreated with intraperitoneal injection of PI (0.4mg/ml) 1 h before sacrificed. Other animals were sacrificed at 1 h, 6 h, 12 h, 24 h, and 48 h time point after suffering TBI. The brain tissues were undergone immunofluorescence or Western blot analyses for caspase-3 activity, tbid, bcl-2, bax, lysosomal enzyme cathepsin B, and autophagy related protein LC3Ⅱ, P62. Brain tissue defect was assessed by Lesion Volume Test. Neurological function was detected by Motor Test and Morris Water Maze. E64d - and DMSO -pretreated mice subjected to TBI were also compared with each other respect to neurological function.Results: Lateral cerebral ventricle administration of E64d before TBI in mice resulted in improvement of neurological function when compared with DMSO control groups, as assessed by PI cell count, lesion volume and behavior test after TBI. caspase-3 activity, tbid, bcl-2, bax, lysosomal enzyme cathepsin B, and autophagy related protein LC3Ⅱ, P62 were quantified by immunoblotting. E64d pretreatment drastically reduced TBI induced caspase-3 activation, bax, tBid, P62 protein levels in the injured cortex and hippocampus (P < 0.05), and quantity analysis of LC3Ⅱ/LC3Ⅰshowed that TBI-induced activation of autophagy remarkably enhanced by E64d treatment(P < 0.05).Conclusion: 1. Lateral cerebral ventricle administration of cathepsinB andμ-calpain inhibitor E64d (0.5μg/1μl) could significantly attenuate nerve cell plasmolemma permeability and TBI-induced nerve cell death and dysfunction. 2. The effect of E64d on TBI induced nerve cell plasmolemma permeability and neuronal dysfunction may due to the crosstalk regulatory mechanism between autophagy and apoptosis.