Neuroprotective Effects And Underlying Mechanism Of Niacinamide Phosphoribose Transferase In Traumatic Brain Injury

Background:Traumatic brain injury(TBI)is the main cause of death and disability in people around the world.Currently,there is no effective treatment method or strategy to rescue the damage caused by TBI.Therefore,in-depth research on the pathogenesis of TBI is of great significance for finding new treatment strategies and improving the neurological function of TBI patients.Niacinamide phosphoribose transferase(Nampt)is a key enzyme in the biosynthesis pathway of nicotinamide adenine dinucleotide(NAD+)cycle.The research shows that the Nampt specific agonist P7C3-A20 has anti apoptotic activity and has strong neuroprotective properties on the newborn neural precursor somatic cell cells and neurons in the hippocampus of adult mice.However,the role and possible mechanism of Nampt after TBI are still unclear.Neuron autophagy and apoptosis are important pathogenesis of secondary brain injury after TBI.Therefore,the purpose of this study is to explore whether Nampt activation can exert neuroprotective effects by reducing neuronal apoptosis through neuronal autophagy after TBI,and to elucidate its specific mechanisms.Methods:In order to explore the changes of Nampt after TBI,we detected the expression of Nampt at different time points after TBI through dual models in vivo and in vitro.Adult male ICR mice were randomly divided into 6 h,12 h,24 h,3 d,5 d and 7 d groups according to different time points after trauma.The TBI model was established by free fall method.The cell scratch model was used in the in vitro experiment,which was divided into the following groups according to different time points:Sham,3 h,6 h,12 h and 24 h.The changes of Nampt expression in brain tissue near the lesion and in vitro cell models were detected by Western blot.The experimental animals were randomly divided into Sham group,TBI group,TBI+placebo group(TBI+V)and TBI+A20 group.Neurological function score and brain edema were measured 24 hours after injury.In vitro experiments,permanent mouse hippocampal neuron cell lines(HT22)were randomly divided into control group(Sham),scratch group(scratch),scratch+placebo group(Scratch+V),scratch+administration group(Scratch+A20).Kit-8(CCK-8)and LDH activity kits were used to detect cell activity.To verify whether the neuroprotective effect of Nampt activation is related to autophagy,mice and HT22 cells were randomly divided into the same four groups as above.Immunofluorescence,Western blot,Nissl staining,electron microscopy and TUNEL staining were used to detect autophagy and apoptosis-related indexes.Autophagy related proteins(p-mTOR,mTOR,p-S6K1,S6K1,TSC2,p-TSC2,Sirt-1)were detected by Western blot analysis to explore the the molecular mechanism of autophagy enhancement induced by Nampt activation through regulation of TSC2/mTOR/S6K1 pathway.Results:The expression of Nampt in cerebral cortex after TBI and HT22 neurons after scratch treatment decreased first and then increased slowly.In vivo experiments,compared to the Sham group,the expression of Nampt protein reached its lowest level 24 hours after injury.In vitro experiments,compared with the Sham group,the expression of Nampt protein reached its lowest value at 6 hours after scratch injury.Immunohistochemistry and immunofluorescence staining showed that Nampt was expressed in the cytoplasm and nucleus of neurons,mainly concentrated in the nucleus.Activating Nampt can alleviate neurological damage,intracranial edema,and neuronal degeneration caused by TBI.Western blot analysis and TUNEL staining results suggest that activating Nampt can alleviate cell apoptosis in both in vivo and in vitro models of TBI.In the TBI in vitro and in vivo model,compared with the TBI group,the TBI+A20 group showed a significant increase in autophagy related proteins,while the ratios of p-mTOR/mTOR and p-S6K1/S6K1 were significantly increased compared to the Sham group.The TBI+A20 group showed a decrease,while the protein expression of p-TSC2 and Sirt-1 decreased.After treatment,their expression was significantly increased,indicating that the activation of Nampt can protect neurons by regulating the TSC2/mTOR/S6K1 pathway to activate autophagy.Conclusion:Activation of Nampt after TBI may reduce neuronal apoptosis and produce neuroprotective effects by enhancing autophagy,which may be related to the regulation of the TSC2/mTOR/S6K1 pathway.