The Neuroprotective Effect And Mechanism Of Vagus Nerve Stimulation On Traumatic Brain Injury

Background and Aim:Traumatic brain injury(TBI)is a common clinical injury,which is mainly caused by traffic accidents,falls and violent events.According to statistics,more than50 million people worldwide suffer from TBI annually,which is a clinical emergency with a high incidence,disability rate and mortality rate,and has become a huge public problem threatening human health and life.The mechanism of TBI is complex,including primary injury and secondary injury.Primary injury referring to the direct forces leads to brain damage.Secondary injury includes a series of pathophysiological process,such as oxidative stress,edema,blood-brain barrier damage,inflammatory reaction and intracranial hematoma.However,the exact molecular mechanism of TBI is still not completely clear,and there is lack of effective rehabilitation treatment.Bioinformatics is an emerging frontier subject formed by the integration of life science and computer science.With the development of bioinformatics and the production of big data such as genomics and transcriptomics,we can mine useful information by biological and functional analysis,and use various graphical tools to visualize important information so as to understand the molecular mechanism of disease,and thus enrich our understanding of disease.Also,a new biological research model has been formed using the existing data information,we can make theoretical speculation,then carry out experimental verification,and finally apply it to clinical practice.With the deepening of TBI basic research and the improvement of clinical diagnosis and treatment technology,some basic research results are transformed into clinical application.Currently,there are many limitations for TBI clinical rehabilitation treatment.Therefore,it is of great significance to explore treatment options for patients with TBI,restore their functions,reduce the rate of disability and improve the quality of life of patients.VNS(vagus nerve stimulation)is a neuromodulation therapy that has shown significant efficacy in treatment-resistant epilepsy,persistent and recurrent depression,and cognitive dysfunction.Recently,several animal experiments and clinical studies have shown that VNS has the potential to ameliorate brain injury.However,the specific role and possible mechanism of VNS in TBI remains unclear.In view of this,our study intends to firstly study the potential molecular mechanism of TBI through bioinformatics,and then preliminarily explore the effect and possible mechanism of VNS on TBI.Methods:Download the TBI related datasets from the Gene Expression Omnibus(GEO).The differentially expressed genes were screened using the online analysis tool GEO2 R.For different genes at different time points,GO function enrichment analysis and KEGG pathway enrichment analysis were conducted by using David online database to explore the potential molecular mechanism of TBI.Cytoscape was used to construct a visual PPI network,and Cytoscape was used to screen the key genes in the PPI network.The key genes screened were verified by animal experiments.Then,a TBI rat model was constructed and stimulated by VNS.The changes of behavior and brain water volume of rats were detected.The morphological changes of brain tissues were observed by HE staining.The protein expressions of NLRP3,NF-κB,Caspase-3 and ASC were detected by Western Blot.The expression levels of TNF-α and IL-6 were detected by immunohistochemistry and quantitative real-time PCR.Results:We first explored the possible molecular mechanism of brain injury based on bioinformatics.The results showed that 109 genes were differentially expressed,including 67 up-regulated genes and 42 down-regulated genes when compared with the control group for 4 hours after TBI;66 genes were differentially expressed for 24 hours after TBI,including 39 up-regulated genes and 27 down-regulated genes.Then the differential genes of GO and KEGG were analyzed,and results showed that it was mainly enriched in MAPK signaling pathway,TNF signaling pathway,positive regulation NF-kappa B transcription factor activity.Further,identification of hub genes by constructing protein interaction networks of differentially expressed genes,and the top 5 differential genes were verified experimentally.The expression of key gene TNF-α、C-MYC、SPP1 and CXCL10 in TBI group were increased,and FN1 expression was decreased.Also,the expression of key gene PTPRC、EGF、MMP9and LCN2 in TBI group was also significantly increased,suggesting that these genes may be involved in secondary injury of early TBI.After that,the protective effect and mechanism of VNS on brain injury were studied.HE staining showed that VNS significantly reduced the brain tissue necrosis,the brain tissue edema,and the score of nerve function defect,suggesting that VNS had protective effect on early TBI rats.The possible mechanism of VNS was further explored.The results showed that VNS could promote the activity SOD、CAT、GSH antioxidant enzymes in brain tissue of TBI rats,and inhibit the product MDA activity of lipid peroxidation.Secondly,VNS can inhibit the expression Bax pro-apoptotic proteins and promote the synthesis of anti-apoptotic protein Bcl-2,thus reducing neuronal apoptosis in brain tissue of TBI rats.Additionally,VNS can promote the transfer of nuclear transcription factors NF-κB to the nucleus,inhibit the activation of inflammatory corpuscles(up-regulation of NLRP3、ASC、Caspase-1 expression),and then inhibit the synthesis and release of inflammatory factors such as IL-1β、IL-18、TNF-α、IL-6.Conclusion:Our results showed that inflammation-related signaling pathways play an important role in TBI injury,and TNF-α,c-MYC,SPP1,CXCL10,FN1,PTPRC,EGF,MMP9,and LCN2 genes may be involved in secondary injury of early traumatic brain injury.Moreover,VNS promotes brain tissue repair in TBI rats by inhibiting oxidative stress,inflammatory response,and apoptosis,and that the NF-κB/NLRP3 signaling pathway may be a molecular mechanism that mediated VNS in inhibiting the inflammatory response.Our results will provide theoretical support for the mechanism of VNS treatment TBI and provide new experimental data for promoting the transformation of clinical application of VNS.