The Role And Mechanisms Of TRAF3 On Neuronal Apoptosis In Subarachnoid Hemorrhage

Background: Subarachnoid hemorrhage(SAH)is a serious stroke in neurosurgery practice,which is featured with high morbidity and mortality.SAH patients resulted from rupture of intracranial aneurysm account for about 85% of all SAH patients.In recent years,a large number of clinical and animal studies have shown that Early brain injury(EBI)is the main factor for poor prognosis after SAH.Neuronal apoptosis,inflammatory response,oxidative stress,ischemia,blood brain barrier disruption,energy metabolic disorders,brain edema,and calcium overload are all involved in the occurrence and development of the EBI,among which neuronal apoptosis plays a vital role in EBI.Although inhibition of a single pathway,such as Mitogen-activated protein kinases(MAPKs)and nuclear factor-kappa B(NF-κB)signaling pathways,could reduce neuronal apoptosis and improve the prognosis in experimental SAH,few of these research results have been translated into clinical practice.Accordingly,targeting multiple neuronal apoptosis pathways simultaneously is currently considered to be a novel treatment strategy for EBI.The tumour necrosis factor receptor-associated factor 3(TRAF3)can regulate multiple signaling pathways such as apoptosis,inflammation and oxidative stress via interacting with receptors and enzymes.In neurological diseases,TRAF3 has been found to regulate neuronal apoptosis via regulating TAK1-dependent MAPKs and NF-κB signaling pathways in ischemic stroke and spinal cord injury.Our preliminary experiment shows that TAK1 could regulate neuronal apoptosis in SAH-induced EBI.Thus,we supposed that TRAF3 could regulate MAPKs and NF-κB signaling pathways to regulate neuronal apoptosis by interacting with TAK1 during the SAH-induced EBI.We established the SAH mice and SAH primary neuronal cell culture to investigate the role and potential mechanism of TRAF3 in neuronal apoptosis after SAH.We investigated whether TRAF3 could be a novel therapeutic target for poor prognosis of the clinical SAH.Methods: Endovascular perforation was established in C57B/6J mice as a in vivo SAH mode.In vitro,primary cultured cortical neurons of mice were treated with oxygen hemoglobin to mimic SAH.Western Blot was used to detect the expression of TRAF3 protein at different time points after SAH both in vivo and in vitro.The cellular location of TRAF3 was evaluated by immunofluorescence staining.Then,we downregulated TRAF3 expression via injecting TRAF3 si RNA into the lateral ventricle in vivo and adding TRAF3 si RNA into neurobasal in vitro.Besides,we used many kinds of methods such as Western Blot,TUNEL staining,and NISSL staining to evaluate the role of TRAF3 in the neuronal apoptosis in SAH mice.we used Western Blot,LDH and other methods to evaluate the effect of TRAF3 on neuronal apoptosis in SAH primary neurons.In addition,we further explored the potential mechanism of TRAF3 regulating neuronal apoptosis after SAH via immunoprecipitation and TRAF3 adenovirus with mutation in the 267-376 amino acid domain.Results:1)The results revealed that the protein expression of TRAF3 upregulated after SAH,followed by a decrease after 24 h,and a return to normal levels at 72 h.Besides,TRAF3 was mainly located in neurons,with sparse expression in microglia and no expression in astrocytes in temporal cortex samples of SAH mice.2)After the downregulation of TRAF3 expression with TRAF3 si RNA,neuronal apoptosis induced by SAH was significantly improved both in vivo and in vitro.3)At the same time,we found that the downregulation of TRAF3 reduced the activation and phosphorylation of TAK1 after SAH both in vivo and in vitro.In addition,the MAPKs and NF-κB pathways,which paly a vital role in neuronal apoptosis were inhibited.4)The results of immunoprecipitation showed that there was a direct interaction between TRAF3 and TAK1 after SAH.5)The Ad TRAF3 significantly increased the activation of TAK1 and TAK1-dependent MAPKs and NF-κB pathways,whereas the Ad TRAF3-M couldn’t.Therefore,we confirmed that TRAF3 regulate neuronal apoptosis after SAH via directly interacting with TAK1 and promoting the activation of TAK1 and TAK1-dependent MAPKs and NF-κB signaling pathways.Conclusion: It has been revealed TRAF3 regulated the neuronal apoptosis via TAK1-dependent MAPKs and NF-κB pathways.Besides,downregulation of TRAF3 could inhibit the activation of MAPKs and NF-κB pathways via inhibiting the phosphorylation of TAK1,which effectively reduced neuronal apoptosis and neurological deficits in SAH.Thus,this research helps us to explore the specific mechanism of neuronal apoptosis in SAH,which provides powerful evidence that TRAF3 may serve as a potential therapeutic target for improving prognosis in SAH patients.