Clinical And Basic Research Of LTB4-BLT1 Axis-mediated Neutrophil Infiltration In Subarachnoid Hemorrhage

BACKGROUND:Aneurysmal subarachnoid hemorrhage(SAH)is an acute and deadly emergency of the central nervous system with high morbidity and mortality,and is an extremely great threat to human beings' life and health.In recent years,a large number of studies confirm that early brain injury(EBI)after SAH is a major cause of poor prognosis in patients with SAH.EBI has complicated pathophysiologic process,mainly including the acute mechanical damage of arterial blood spilling from the artery,the increased intracranial pressure,decreased cerebral blood flow,and the decreased cerebral perfusion pressure,as well as the subsequent toxic effect of the blood's components,oxidative stress,inflammatory reaction,blood-brain barrier damage,cerebral edema and neural injuries.It's difficult to achieve satisfying curative effect for patients with SAH through targeting a single progress.Hence,it is becoming more and more important to find factors that can regulate multiple mechanisms of EBI.Activated neutrophils could enhance the local vascular permeability,inflammatory response and oxidative stress reaction when they recruit.to and infiltrate the tissue,leading to tissue damage.Meanwhile,leukotriene B4(LTB4)is proved to be a highly potent chemotactic agents for neutrophils and acts through its receptors,LTB4 receptor 1(BLT1).It is reported that marked neutrophil infiltration occurs in the rat brain tissue accompanied with microvascular injury,therefore,we speculated that neutrophil infiltration may be closely associated with the EBI after SAH and LTB4-BLT1 axis may also involve in the process.However,at present,there is no any related report on the effects of LTB4-BLT1 axis-mediated neutrophil infiltration on the EBI after SAH.Hence,this study aimed at comprehensively elaborating the significance of LTB4-BLT1 axis-mediated neutrophil infiltration in EBI after SAH both clinically and basically.METHODS:In this study,firstly,the levels of LTB4,neutrophil marker myeloperoxidase(MPO)and related inflammatory factors in the cerebrospinal fluid(CSF)of patients with SAH and their clinical significance in SAH were investigated.Secondly,expressions and cell distribution of LTB4 and BLT1 at different time points after SAH were analyzed by using Western blotting,quantitative real-time polymerase chain reaction(real-time PCR),immunohistochemistry staining and immunofluorescent double staining,and the correlation of BLT1 and nuclear factor-?B(NF-?B)expression levels was also analyzed.Finally,up-regulated and down-regulated experiments using in vivo model were performed to investigate the effects of LTB4-BLT1 axis-mediated neutrophil infiltration on the NF-?B-mediated inflammatory response,oxidative stress reaction,neuronal apoptosis and death,blood-brain barrier disruption and neurological injury after SAH.RESULTS:Clinical research demonstrated that,level of LTB4 in the CSF of patients with SAH elevated significantly in the early period of SAH insult,and closely correlated with the disease severity and unfavorable prognosis of SAH patients.In vivo research showed that,levels of LTB4 and BLT1 in the rat brain cortex significantly increased early after SAH,and BLT1 mainly expressed in neurons microglia and endothelial cells in the brain,and its expression level positively correlated with the expression level of nuclear NF-?B p65.Further intervention research showed that,up-regulation of LTB4 early after SAH could exacerbate brain edema and neurological dysfunction of rats,and promoted the infiltration of neutrophils,activation of microglia and nuclear translation of NF-?B p65,and increased the degradation of blood-brain barrier tight junction protein ZO-1,while simultaneous use of BLT1 specific inhibitor U75302 reversed these effects of LTB4 up-regulation.Inhibitory research results showed that blockade of LTB4-BLT1 axis with U75302 early after SAH could reduce the infiltration of neutrophils in the rat brain parenchyma,alleviate the NF-KB-mediated inflammatory response and oxidative stress reaction,and reduced the degradation of blood-brain barrier-related proteins and the death of neurons.Finally,blockade of LTB4-BLT1 axis with U75302 early after SAH in rats could ameliorate brain edema and improve neurological function at day 1 but not at day 3.CONCLUSIONS:This study provides clinical and basic evidence that,elevation of LTB4 level in the CSF of patients with SAH correlates with the inflammatory factors and.unfavorable prognosis,meanwhile,LTB4-BLT1 axis-mediated neutrophil infiltration participates in the EBI process,including the NF-KB-mediated inflammatory response,oxidative stress reaction,blood-brain barrier disruption and neuronal injury in the early period of SAH,suggesting the potential curative value of intervention of LTB4-BLT1 axis-mediated neutrophil infiltration in SAH.