The Neuroprotective Effect And Mechanism Of Apolipoprotein E In Early Brain Injury After Subarachnoid Hemorrhage

Objective: The current study aimed to explore the neuroprotective effect of apolipoprotein E in early brain injury after subarachnoid hemorrhage and,to figure out whether apoE-based treatment strategy can attenuate the early brain injury after subarachnoid hemorrhage.This study also aimed to find new targets for subarachnoid hemorrhage treatment and,provide theory basis for the development of new pharmacological agent against subarachnoid hemorrhage.Methods:Firstly,to investigate the correlation between apolipoprotein E and early brain injury after subarachnoid hemorrhage.A total of 125 wild-type C57BL6/J mice(20±1.5g;8-10weeks)were randomly divided into control group(SHAM)and subarachnoid hemorrhage group.Subarachnoid hemorrhage group was further assigned to 4 subgroups including WT-6H,WT-24 H,WT-48 H and WT-72H(n = 25).The internationally recognized endovascular perforation method was used to establish the animal model of subarachnoid hemorrhage.Western blot(WB)and immunofluorescence staining techniques were used to detect the expression of apolipoprotein E after subarachnoid hemorrhage.Brain water content(BWC)and blood brain barrier(BBB)permeability detection,enzyme-linked immunosorbent assay(ELISA),neurological function scale,rotarod latency and magnetic resonance imaging(MRI)with T2 weighted scanning were used to assess the early brain injury after subarachnoid hemorrhage in mice.Statistical analysis was used to determine the correlation between apolipoprotein E and early brain injury after subarachnoid hemorrhage.Secondly,to study the effect and mechanism of apolipoprotein E on early brain injury after subarachnoid hemorrhage.A total of 50 wild-type C57BL6/J mice(19.6±1.4g;8-10weeks)and 50 apolipoprotein E gene knock-out mice(APOE-/-,KO)(19.8±1.8g;8-10weeks)were randomly divided into four groups,including WT-SHAM,WT-48 H,KO-SHAM and KO-48 H,(n = 25,respectively).The internationally recognized endovascular perforation method was used to establish the animal model of subarachnoid hemorrhage.Western blot(WB)and immunofluorescence staining techniques were used to detect the expression of apolipoprotein E after subarachnoid hemorrhage in each group.Brain water content(BWC)and blood brain barrier(BBB)permeability detection,enzyme-linked immunosorbent assay(ELISA),neurological function scale,rotarod latency and magnetic resonance imaging(MRI)with T2 weighted scanning were used to assess the early brain injury after subarachnoid hemorrhage in each group.Statistical analysis was used to detect the differences in early brain injury after subarachnoid hemorrhage between apolipoprotein E gene knockout mice and wild-type mice.Thirdly,to explore the therapeutic efficiency of apolipoprotein E based strategy on early brain injury after subarachnoid hemorrhage.A total of 105 wild type C57BL6/J mice(19.8±1.7g;8-10weeks)were randomly divided into control group(SHAM)and WT-W8 H and apolipoprotein E mimetic peptide treated group(WC-48H),(n= 25).Animal model of subarachnoid hemorrhage was conducted using the endovascular perforation method in wild-type C57BL6/J mice.In the apolipoprotein E mimetic peptide treated group,a dose of COG1410(1mg.kg-1.d.-1)was intravenously injected via the tail vein.The placebo group received intravenous injection of an equal amount of saline.Western blot(WB)and immunofluorescence staining techniques were used to detect the expression of apolipoprotein E after subarachnoid hemorrhage in each group.Brain water content(BWC)and blood brain barrier(BBB)permeability detection,enzyme-linked immunosorbent assay(ELISA),neurological function scale,rotarod latency and magnetic resonance imaging(MRI)with T2 weighted scanning were used to assess the early brain injury after subarachnoid hemorrhage in each group.Statistical analysis was used to detect the efficiency of apolipoprotein E mimetic peptide COG1410 on early brain injury after subarachnoid hemorrhage.Results:Firstly,after subarachnoid hemorrhage,apolipoprotein E expression was gradually increased and peaked at 48 hour after the bleeding and,began to decline at 72 hour after the bleeding.Using immunofluorescence staining,we found that apolipoprotein E is mainly secreted by astrocytes after subarachnoid hemorrhage.Moreover,the expression of apolipoprotein E around capillaries was significantly increased after subarachnoid hemorrhage.At the same time,the brain edema and blood brain barrier permeability,major markers that reflecting early brain injury,also peaked at 48 hours and then began to decline at 72 hour after injury.Therefore,these data indicate that apolipoprotein E is associated with the blood-brain barrier integrity in early brain injury after subarachnoid hemorrhage.Second,mice with apolipoprotein E gene knocked out showed significantly worse neurological outcome after subarachnoid hemorrhage.The blood brain barrier disruption was more apparent in APOE-/-mice when compared to the WT mice.Evans blue dye and immunoglobulin G(IgG)permeability and magnetic resonance T2 hyperintensity were all relatively increased in the APOE-/-mice over wild-type mice(p < 0.05).Further study showed that apolipoprotein E can alleviate neuroinflammation and thereby reduce the blood brain barrier disruption and early brain injury after subarachnoid hemorrhage.This was associated with the modulation of CypA-NFκB related signaling pathway.Third,after the treatment with apolipoprotein E mimetic peptide COG1410,blood brain barrier disruption was significantly decreased.When compared to the saline-treated mice,Evans blue and immunoglobulin G(IgG)leakage were significantly decreased,magnetic resonance T2 hyperintensity was reduced,and thereby the mice neurological function was significantly improved in the COG1410-treated mice(p< 0.05).Further studies revealed that apolipoprotein E mimetic peptide COG1410 can also attenuate early brain injury after subarachnoid hemorrhage through the regulation of CypA-NFκB related signaling pathway,thus to alleviate neuroinflammation and reduce the destruction of the blood brain barrier.Conclusion: Apolipoprotein E can alleviate neuroinflammation,reduce the destruction of the blood brain barrier and,thus to minimize the early brain injury after subarachnoid hemorrhage through regulating the CypA-NFκB related signaling pathway.Moreover,apolipoprotein E mimetic peptide COG1410 can also attenuate the early brain injury after subarachnoid hemorrhage in an apolipoprotein E-like manner that has great clinical transformation potential and worth further studying.