Neuroprotective Effects And Mechanism Of Lipoxin A4 Methyl Ester On Intracerebral Hemorrhage

Background:Intracerebral hemorrhage(ICH)is the most severe acute cerebral vascular disease,and is prone to multiple dysfunction,which causes great pain to patients and adds to the social burden.Although many studies have been conducted on the pathogenesis of cerebral hemorrhage,the progress has been limited and has not been translated into obvious clinical benefits.To explore the early pathological changes and influencing factors of cerebral hemorrhage,and to seek an active and effective treatment plan,which may seize the treatment time window to improve the curative effect.Recent studies have found that increased blood-brain barrier(BBB)permeability in early stage of cerebral hemorrhage leads to secondary brain injury such as cerebral edema,which is the main reason for poor prognosis of cerebral hemorrhage.In this process,with the activation of inflammation,oxidative stress and apoptosis factors,they interact with each other in a cascade reaction mode,aggravating the structural and functional damage of brain tissue.Therefore,strengthening the research on the above influencing factors after cerebral hemorrhage may find a potential new direction for the treatment of cerebral hemorrhage.Lipoxins(LXs)are endogenous active mediators with anti-inflammatory and pro-inflammatory effects,which are widely involved in the regulation of inflammation in vivo.In pathological conditions,lipid oxygen element by inhibiting neutrophil recruitment and reactive oxygen species generation,adjust the microglia activity,lower matrix metalloproteinases-9(MMP-9)expression way,such as in the chronic neural degenerative diseases,ischemia-reperfusion in the nervous system diseases such as brain injury,brain trauma,plays a very important role.At present,there are few studies on the neuroprotective effect of lipoxygenin on the secondary brain injury after cerebral hemorrhage,especially on the effect of early cerebral hemorrhage and the related regulatory mechanism are still unclear.Objective: To investigate the neuroprotective effect of Lipoxin A4 methyl ester on the early secondary injury of cerebral hemorrhage in rats and its related regulatory mechanism.Methods:1.The ICH model of rats was established by autologous blood,and LXA4 ME(Low dose group:10ng/ul,High dose group:100ng/ul)was injected into the lateral ventricle for treatment.The neurological function recovery of rats 1-5 days after surgery was evaluated by modified neurological severity score(MNSS)and rotarod test.HE staining and nissl staining were used to observe the pathological morphological changes of brain tissue and the survival of nerve cells.nissl corpuscle number of neurons of around hematoma on 24 h after ICH was observed by nissl staining.Cerebral tissue water content and evans blue assay were used to evaluate the BBB permeability at 1-3 days after ICH.2.Apoptosis was detected by TUNEL assay mediated by terminal deoxynucleotide transferase.The expressions of IL-1,IL-6,TNF-α,NF-κBp65,MMP-9,Occludin,and ZO-1 in the brain tissue of the hematoma were detected by western-blot.The activity of MMP-9 was detected by gel enzyme spectrum electrophoresis.To evaluate the mechanism of Lipoxin A4 methyl ester in cerebral hemorrhage.3.Phorbol-12-myristate-13-acetate(PMA)intervention was given,and neurological dysfunction was assessed by MNSS and rotarod test.Terminal deoxynucleotide transferase-mediated TUNEL assay was used to detect neuronal apoptosis.The protein expression levels of NF-κBp65,adenine nucleotide translocator-1(ANT1),mitofusin 2(MFN2),cytochrome C(CytoC),Bcl-2/Bax,caspase-3 in the damaged brain tissue around hematoma after ICH were determined by western-blot and RT-PCR.At the same time,the mitochondrial reactive oxygen species(ROS),adenosine triphosphate(ATP)content and mitochondrial membrane potential were detected.Results:1.After ICH,the nerve cells were swollen,the number of neurons was decreased,the blood-brain barrier permeability and brain water content were increased,and the nerve motor function was impaired.LXA4 ME treatment can improve the morphological changes of neurons,reduce cerebral edema,improve the neurological dysfunction of ICH rats,and play a neuroprotective role,which is dose-dependent.2.After ICH in rats,the apoptosis of neurons in the area around hematoma increased,and the expression of IL-1,IL-6,TNF-α,NF-κBp65 and MMP-9 increased significantly,while the expression of Occludin and ZO-1 decreased,and the activity of mmp-9 increased significantly.LXA4 ME can inhibit the inflammatory response,reduce the apoptosis of nerve cells and reduce the blood-brain barrier permeability through the NF-κB/MMP-9 pathway.3.Activation of NF-κB by PMA promotes nerve function injury and increases neuronal apoptosis.The protein and mRNA expression of ANT1,MFN2 and Bcl-2 were significantly decreased,and the expression of mitochondrial NF-κBp65,CytoC,Bax,caspase-3 and mRNA were significantly increased.Meanwhile,the content of mitochondrial ROS was increased and the mitochondrial ATP and mitochondria membrane potential were decreased.Conclusion: LXA4 ME can reduce the neurological damage after early cerebral hemorrhage by inhibiting the inflammatory reaction and cell apoptosis,and this neuroprotective effect may be achieved by inhibiting the activation of NF-κB/MMP-9 pathway.