The Effects And Mechanisms Of LXA4 On Endothelial Dysfunction And Cerebral Autoregulation After Subarachnoid Hemorrhage In Rats

Objective: Subarachnoid hemorrhage(SAH)is a lethal stroke with high mortality and disability,aneurysmal subarachnoid hemorrhage occupies the main position.A large number of patients with SAH suffer from secondary cerebral ischemia and delayed cerebral infarction,Delayed cerebral ischemic(DCI)after SAH was the main reason leading to high mortality and poor prognosis.Present studies suggest that the dysfunction of cerebral vascular autoregulation was regarded as a critical contributor to the delayed cerebral ischemia.As the basic structural and functional component of cerebral vascular neural network,Cerebrovascular endothelial cells is essentially responsible for stabilization of vascular tone and regulation of cerebral vasomotor function and cerebral blood flow.The integrity of cerebral vascular endothelial cells is a prerequisite for the automatic regulation of cerebral blood flow.Inflammatory response following SAH can disrupt the integrity of vascular endothelial cells.Lipoxin A4(LXA4)is an important endogenous lipid,and exerts potentanti-inflammatory actions via inhibition of neutrophil infiltration and suppression of pro-inflammatory cytokines after SAH.In this study,we explore the effect of LXA4 on cerebrovascular endothelial cells and cerebral vascular autoregulation after subarachnoid hemorrhage in rats,to explore the underlying mechanism,and shed new light on treatment of SAH.Methods:1?The SAH model was established by endovascular perforation of SD rats.Changes in cerebral blood flow(CBF)of each group were monitored by Laser Doppler flowmeter after SAH.Blood was collected in a sodium citrate tube from control or SAH rats.The levels of EMPs phenotype analysis was performed by flow cytometry.The level of LXA4 in cerebrovasculars after SAH was measured by a rat LXA4 ELISA kit.To identify the most severe time points of endothelial dysfunction and the difference in expression of LXA4 in blood vessels.2?Rat SAH model was induced,which were intracerebroventricular injection of LXA4 or saline at the most serious time point of endothelial injury after SAH.Neurological impairments were blindly evaluated using an 18-point score system known as the modified Garcia scale.Changes in permeability of blood brain barrier(BBB)and brain edema were detected in each group at 24 h after SAH by Evans Blue(EB)exudation and wet-dry weight method respectively.The levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6)were analyzed by ELISA.Neutrophil infiltration with endothelial cells was observed by double immunofluorescence.The level of NO was detected by Nitric Oxide Microplate Assay Kit.The basilar arteries and circle of Willis arteries were used to extract total protein.Western Blot was used to detect the expression of FPR2?NF-?B?MMP-9 ?MPO and ICAM-1.Microflow of the cerebral cortex following treatment of LXA4 was assayed by laser speckle contrast imaging.3 ? SAH rats were injected with interference RNA(si-FPR2)into lateral ventricle,downstream molecules FPR2,ERK1/2,NF-?B,MMP9 and the levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6),leukocyte adhesion molecule(ICAM-1)and MPO were measured either by Western blot or ELISA.Results:1 ? The marker of endothelial dysfunction(EMPs)was significantly increased at 6h after SAH and peaked at 24 h,and maintained at a high level at 48 h and 72 h,compared to sham grou.The results of ELISA showed that the level of LXA4 in cerebral arteries decreased from 6 hours after SAH and reached lowest at 24 hours therefore,subsequent studies were performed at 24 hours post-SAH.2?BBB rupture and severe cerebral edema were obvious in rats at 24 h after SAH,reduced cerebral blood flow and significant neurological disorders were observed.After administration of LXA4,it obviouslyreduced EB exudation and edema and promoted the recovery of CBF,improved the rats' behavioral function.The ameliorative symptoms mentioned above disappeared after the use of FPR2 siRNA.3?Immunofluorescence showed that neutrophil marker(MPO)was colocalized with specific marker of endothelial cells(VWF)in cerebral arteries at 24 hours after SAH,which is suggested that inflammation is involved in the process of endothelial injury.The expression levels of TNF-?,IL-1? and IL-6 in blood were significantly increased 24 h after SAH.LXA4 treatment suppressed the levels of pro-inflammatory cytokines and neutrophil infiltration in cerebral arteries.4?LXA4 reduced the level of EMPs in the blood,restored the reduced NO level,and ameliorate endothelial dysfunction.At the same time,it was found that LXA4 has protective effect on endothelial cell viability in hemoglobin-stimulated vascular endothelial model.5?LXA4 reduced the level of ICAM-1?MPO?NF-?B?MMP9 and p-ERK protein expression and upregulated the expression of FPR2.These effects of LXA4 were partially abolished by FPR2 siRNA intervention.Conclusions:1?Endothelial function was impaired after subarachnoid hemorrhage and the most serious injury occurred at 24 hours.Microflow in the cerebral cortex was decreased dramatically at 24 h after SAH.2?Exogenous Lipoxin A4 reduced EB exudation and edema,promotedthe recovery of CBF,improved the rats' behavioral function.3?Exogenous Lipoxin A4 could inhibit neutrophils infiltration,reduce inflammation,ameliorate endothelial dysfunction and recover the microflow after subarachnoid hemorrhage.4?The protective effect of Lipoxin A4 may involve FPR2/ERK1/2/NF-?B pathway.