Molecular Mechanisms And Potential Functions Of Sirtuin 1 In The Brain Injury After Experimental Subarachnoid Hemorrhage

Background:Subarachnoid hemorrhage(SAH)is a fatal neurological injury with high morbidity and mortality rates.Accumulating studies indicated that early brain injury(EBI)instead of cerebral vasospasm(CVS)is acknowledged as the primary cause of a poor outcome for patients with SAH.Therefore,treatment of EBI has been considered to be the main goal in the management of patients with SAH.However,despite intense researches have been carried out to elucidate its mechanism,the exact molecular mechanism of EBI is still not well understood.It has hindered the development of effective and specific treatment paradigms for EBI.SIRT1 is a type of histone deacetylase whose activity is dependent on NAD.Multiple lines of studies have revealed that SIRT1 could modulate a variety of biological functions,such as oxidative stress,immune response,mitochondrial biogenesis.and apoptosis/autophagy.In the brain,SIRT1 is expressed with high levels in the cortex and predominantly expressed in neurons nucleus.Mounting evidence has demonsrated that SIRT1 could protect against cerebral ischemia,traumatic brain injury,spinal cord injury,Alzheimer's disease,Parkinson's disease,and Huntington disease.However,few studies are performed to elucidate the potential role of SIRT1 in SAH and the underlying molecular mechanisms.Our group previously reported that the expression of SIRT1 in the brain cortex was increases at day 1 after SAH.In conjunction with other sutides,we proposed that SIRT1 activation may confer brain protection against SAH.Thus,the aim of this study is to investigate the potential role of SIRT1 in the brain injury after SAH and the underlying molecu ar mechanisms.Methods:We performed two SAH models in our study,including a prechiasmatic cistern injection model in vivo and an oxyhemoglobin-stimulated SAH model in vitro.In in vivo experiments,we used western blot to detect the protein expression of SIRT1 in the cerebral cortex in different time points after SAH.In addition,immunohistochemistry and double immunofluorescence staining were performed to evaluate the cellular distribution of SIRT1 in the cerebral cortex after SAH.We further used a SIRT1-specific inhibitor sirtinol and activators(activator 3 and salvianolic acid B(SalB))to decrease or increase SIRT1 expression.Western blot,double immunofluorescence staining,ELISA,and other biochemical methods were performed to evaluate the change of oxdative stress,inflammatory response,neuronal apoptosis,neurological functions,as well as the nuclear factor-erythroid 2-related factor 2(Nrf2)siganling pathway activation after SAH.In in vitro experiments,we further used western blot,double immunofluorescence staining,ELISA,lactate dehydrogenase(LDH)activity,and TUNEL to investigate neuronal damage after SAH.Results:Our data suggested that the protein level of SIRT1 was markedly elevated at the early stage of SAH,peaked at 24 h,and remain elevated at 72 h after SAH.The expression of SIRT1 was mainly located in neuronal nucleus and both cytosolic and nucleic immunoreactivities of SIRT1 in neurons were enhanced after SAH.In addition,SIRT1 can also be located in microglia,but not in astrocytes after SAH.Administration of sirtinol inhibited the expression of SIRT1 and increased acetylation of FoxOl,nuclear factor-kappa B(NF-?B),and P53 after SAH.Concomitant with these,inhibition of SIRT1 could exacerbate oxidative damage,neuroinflammation.neuronal apoptosis,brain edema,and neurological impairment leading to the aggravated brain injury after SAH.In contrast,activator 3 could increase SIRT1 expression and inhibit FoxOl-induced.NF-KB-induced,and P53-induced oxidative,inflammatory,and apoptotic pathways to ameliorate barin injury after SAH.We also evaluated the neuroprotective role of Sa1B in SAH and the underlying molecular mechanisms.Our data showed that SalB could upregulate SIRT1 expression,modulate the downstream Nrf2 signaling pathway activation,and mitigate oxidative stress and the secondary brain injury after SAH.However,SIRT-specific inhibitor sirtinol pretreatment significantly suppressed Sa1B-induced SIRT1 activation and Nrf2 expression and reversed the antioxidant and neuroprotective effects of SalB after SAH.Conclusion:Our data suggested that SIRT1 activation might play an endogenous brain protection role after SAH.Pharmacologically elevating SIRT1 expression could ameliorate brain injury after SAH.These indicated that enhancing SIRT1 signaling might be a promising treatment strategy for brain injury after SAH.