The Mechanism Of TLR4 In Early Brain Injury And Anti-inflammatory And Neuroprotective Effects Of Naloxone After Subarachnoid Hemorrhage

Background: Subarachnoid hemorrhage(SAH)is a cerebrovascular disease with high rates of mortality and disability thereby having devastating consequences.Although SAH only accounts for 5%-10% of all strokes,it has a heavy burden on the society due to its onset at a young age,high mortality,disability rates and poor clinical outcomes.In spite of researchers have studied SAH for decades,it is still a serious life-threatening problem worldwide.To date,the mechanisms contributing to brain injury after SAH remain unclear despite decades of study focusing on early brain injury(EBI)and delayed brain injury(DBI).But with the failure of clinical research on DBI,researchers have begun to shift the focus of research to EBI.EBI encompasses any kind of acute pathophysiological event that occurs in the brain within the first 72 hours of SAH,including elevation of intracranial pressure,reduced cerebral blood flow and perfusion pressure,blood-brain barrier(BBB)disruption,brain edema,oxidative stress,neurological deficient,neuronal apoptosis,acute vasospasm,and impaired cerebral auto-regulation.EBI is a very complicated pathophysiological process,and the molecular mechanism involved is also complex.Neuroinflammation is a well-recognized consequence of SAH and may be responsible for EBI.TLR4-mediated neuroinflammation plays an important role in EBI.Depending on its unique TIR domains,TLR4 can then bind to the specific adaptor proteins transmitting extracellular signals to the downstream modules,which initiates the activation of transcription factors that regulate expression of pro-inflammatory cytokine genes,mainly via the MyD88-dependent(Mal/TIRAP,MyD88)and MyD88-independent(TRAM,TRIF)signaling pathways.(+)-Naloxone is a structural analogue of morphine.In recent years,it has been found that in addition to his ability to rescue acute poisoning of narcotic analgesics and acute alcohol poisoning,and to antagonize the respiratory depression and wake-up effects of these drugs,it can also act as an inhibitor of TLR4 and play an anti-inflammatory role.However,there is no research about whether naloxone can be used for early brain injury treatment after SAH.Therefore,this study intends to explore the anti-inflammatory and neuroprotective effects of naloxone on early brain injury after SAH by constructing a SAH rat model.Objective: The purpose of this study is to explore the mechanism of TLR4 in the early brain injury of SAH and the anti-inflammatory and neuroprotective effects of naloxone through bioinformatics analysis combined with basic experiments.Methods: In the present study,we firstly downloaded and reanalysed the gene expression dataset of SAH rats by using bioinformatics analysis.The differentially expressed genes(DEGs)and molecular pathways related to early brain injury of SAH were identified.Next,the results of bioinformatics analysis were validated by RT-q PCR,western blot,immunohistochemistry,and Immunofluorescence.Then,in the SAH cell model,si RNA(si-MyD88 and si-TRIF)was used to interfere with the expression of MyD88 and TRIF,and experimental methods such as RT-q PCR,Western blot,and flow cytometry were used to explore the specific molecular pathway of TLR4 for NF-?B activation.Finally,experimental methods such as Western blot,immunofluorescence,and Tunel staining were used to detect TLR4 pathway-related proteins to explore the mechanism of naloxone's anti-neuroinflammation and neuroprotection in SAH rat model.Results: 1.After re-analysing the gene expression dataset of SAH rats by bioinformatics analysis,a total of 173 DEGs were identified in the SAH group including 153 up-regulated genes and 20 down-regulated genes.The enrichment analysis of the identified DEGs showed that the Toll-like receptor signaling pathway plays an important role in SAH.The results of bioinformatics analysis were verified by RT-q PCR and Western blot,and it was found that the expression of TLR4 and NF-?B increased significantly after SAH,which reached the peak at 48 h.Immunofluorescence results showed that after SAH,TLR4 was mainly activated and strongly expressed in microglia and neurons,but rarely expressed in astrocytes.2.In our study,the BV2,N9,and primary neurons/microglia were used to construct the SAH model in vitro and si RNAs were used to interfere with the expression of MyD88 and TRIF.Our results showed that compared with SAH group,the knockdown of MyD88 reduced the expression of NF-?B and release of inflammatory factors such as IL-1?,IL-6,TNF-?,and also inhibited neuronal apoptosis.Although the expression of TRIF increased after SAH,it did not affect the expression of NF-?B and inflammatory factors.These results proved that the release of IL-1?,IL-6,and TNF-? in early brain injury after SAH is mediated by the TLR4-MyD88-NF-?B pathway,not the TRIF pathway.3.Our results show that compared with the SAH group,(+)-naloxone treatment can significantly improve neurological dysfunction and reduce brain tissue edema in SAH rats.At the same time,(+)-naloxone treatment can significantly reduce the expression of TLR4,MyD88 and NF-?Bp65,inhibit the release of IL-1?,IL-6 and TNF-?,and decreased neuronal apoptosis in brain tissue after SAH.The above results confirm that(+)-naloxone has a neuroprotective effect in early brain injury after SAH.Conclusions: 1.After SAH,TLR4 is mainly activated and strongly expressed in microglia in EBI after SAH.2.TLR4/NF-?B signaling pathway plays an important role in early brain injury after SAH.3.TLR4 activates NF-?B and promotes the release of inflammatory factors through the MyD88-dependent pathway during the early brain injury of SAH.4.(+)-Naloxone can inhibit neuroinflammation mediated by TLR4-MyD88 pathway in the EBI after subarachnoid hemorrhage,thereby playing a neuroprotective role.