The Study On Adenosine Kinase In Neuronal Apoptosis After Traumatic Brain Injury

Objective The study was designed to determine the expression and distribution alteration of adenosine kinase (ADK) in traumatic brain injury (TBI), and to explore whether ADK participates in neuronal apoptosis after TBI.Methods1. Sharp instrument induced rat TBI model was performed in vivo. Western Blot (WB) and immunohistochemistry (IHC) were employed to test the expression and distribution exchanges of ADK.2. Double-immunofluorescent staining (IF) was employed to investigate the co-localization of ADK with different cellular markers.3. WB was performed to investigate the expression files of GFAP, Ki-67and iNOS. IF was performed to test the co-localization of ADK with GFAP and Ki-67, in order to explore the association of ADK with astrocyte activation.4. TUNEL was employed to test neuronal apoptosis in rat brain cortex after TBI.5. Astrocyte activation was induced in vitro via lipopolysaccharide (LPS) treatment. WB was employed to investigate the expression of ADK, GFAP and iNOS in astrocytes after LPS treatment. Besides, ELISA was performed to investigate the release of TNF-α, IL-1β and IL-6. Then, conditioned medium (CM) was collected for the further research.6. CM collected from activated astrocytes was used to stimulate neurons. WB, IF, DAPI staining and cytotoxicity assay were performed to investigate neuronal apoptosis.7. ADK siRNA was built and tested, then it was employed to explore whether ADK interfernce affects astrocyte activation.8. WB, IF, DAPI staining and cytotoxicity assay were perfromed to investigate whether ADK interfernce affects neuronal apoptosis.Results1. ADK was up-regulated and peaked at day5after TBI, then it gradually decreased to the normal level.2. ADK was co-localized both with GFAP and CD11b in the rat brain cortex, only the co-localization of ADK with GFAP was increased after TBI.3. The expression of iNOS, Ki-67and GFAP were up-regulated, and then gradually decreased after TBI at day5. Co-localization of Ki-67with GFAP and ADK were found after TBI.4. TUNEL positive neurons were found after brain trauma.5. The expression of ADK, GFAP, iNOS, TNF-α, IL-1β and IL-6altered in astrocytes under the time-and dose-dependent way of LPS treatment.6. The expression of active-caspase-3and LDH altered in a time-and dose-dependent way in neurons under the CM treatment.7. Down-regulation of ADK decreased inflammatory factors released by astrocytes, and relieved neuronal apoptsis.Conclusions1. ADK participated in the pathophysiological processes of TBI2. Both astrocyte activation and neuronal apoptosis were found in TBI.3. Astrocyte activation induced by inflamatory factor can promote neuraonal apoptosis.4. ADK took part in the regulation of neuronal apoptosis via astrocyte activation.