HMGB1-Binding Heptamer Peptide Ameliorates Neurovascular Complications Associated With Thrombolysis In Stroke Rats

BackgroundIschemic stroke is the second leading cause of death worldwide while the therapeutic approaches are limited.Tissue-type plasminogen activator(tPA)is the only FDA approved drug for ischemic stroke,but its use is finite due to the narrow therapeutic time window(within 3h or 4.5h)and the increased risk of severe neurovascular complications,such as hemorrhagic transformation(HT)and edema,resulting in the fact that more than 95%of stroke patients can not benefit from thrombolytic therapy.The leakage of the blood-brain barrier(BBB)is a critical factor causing tPA-associated HT and edema.Therefore,exploring a combination therapy that can preserve BBB integrity is a promising therapeutic strategy to reduce the risk of neurovasculature complications.Although the mechanisms underlying BBB breakdown and tPA-induced neurovascular complications are not fully understood,it has been suggested that they occur as a result of exaggeration of neuroinflammation.Danger signals,such as high-mobility group box 1(HMGB1)and ATP,were released into the environment from dying cells,leading to the activation of microglia and the expression of inflammatory factors in ischemic brain.HMGB1,a non-histone DNA-binding protein localized in the nucleus,can be either actively secreted by immune cells or passively released from necrotic cells in response to infections and tissue injuries.Studies conducted by us and others revealed that the binding of extracellular HMGB1 to pattern recognition receptors of microglia could induce a significant elevation of cytokines expression,thereby eliciting inflammatory responses during brain ischemia/reperfusion.Moreover,HMGB1 serves as an extracellular inflammatory cytokine and contributes to neuronal injury and BBB disruption.HMGB1 level was elevated in patients with ischemic stroke.Anti-HMGB1 monoclonal antibody reduces infarct volume by 90%and potently reduces BBB permeability in stroke animal models.Administration of low dose HMGB 1-binding heptamer peptide(HBHP)significantly suppressed HMGB 1-mediated neuronal damage.Given that neuroinflammation contributes to the BBB disruption and tPA's neurovascular complications,we logically hypothesize that blocking HMGB 1-induced inflammation could ameliorate neurovascular complications and protect BBB integrity,subsequently reducing tPA-induced hemorrhage during thrombolytic therapy for ischemic stroke.Purpose1.To confirm the dynamic change of serum HMGB1 in stroke patients and rats with thrombolytic therapy.2.To confirm the neuroprotective effect of HBHP,whether it could ameliorate neurovascular complications associated with thrombolysis in stroke rats.3.To identify whether the neuroprotective effect of HBHP is related to the decrease of the permeability of BBB and the reduction of inflammatory reaction.Method1.Collecting Blood samples from stroke patients and rats pre-and post-thrombolytic therapy.The serum level of HMGB1 was detected by enzyme-linked immunosorbent assay(ELISA)kit for self-comparison.2.MCAO model was employed to induce brain ischemia,and randomly divided into four groups,individuality administered saline,tPA only,tPA plus scramble peptide or tPA plus HBHP after MCAO.After 2h or 4.5h occlusion,evaluated the mortality,modified Neurological Severity Score(mNSS),brain swelling index,and hemorrhagic transformation.3.Detection of BBB disruption in stroke rats with or without tPA plus HBHP treatment.immunofluorescence staining to evaluate the leakage of IgG.Western blot analysis was adopted to dectect the expression of occludin protein.4.Detection of inflammatory response in stroke rats after tPA combined with HBHP treatment.immunofluorescence staining of Iba-1 to observe the activation of microglia.qPCR was performed to observe the gene expression of inflammatory factors,including IL-1?,IL-6,IL-12b and tumor necrosis factor-?(TNF-?).Western blot analysis was performed to dectect the protein expression of iNOS,COX-2 and IL-1? at 8h after ischemia.ELISA analysis were performed to observe the protein secretion of IL-1?.Results1.The serum HMGB1 level was significantly elevated in stroke patients at 2h post-tPA treatment.In stroke rats,HMGB1 level was not altered at 4h after saline injection,while in line with the data of human study,a significant increase of HMGB1 level was observed after tPA treatment.2.The tPA-treated rats with 4.5h MCAO had a much higher mortality,more severe neurological impairment and brain swelling than the vehicle saline group.Interesting,co-treatment of HBHP and 4.5h tPA significantly reduced the mortality,alleviated the neurological deficit and ischemia hemispheric enlargement.No statistical difference was noted in 2h-MCAO groups.3.tPA treatment at 4.5h resulted in evident HT in the ischemic brain,Notably,co-treatment of HBHP significantly reduced 4.5h tPA-induced hemorrhage,while scramble peptide did not.Early tPA infusion at 2h did not induce obvious HT.No statistical difference was noted in 2h-MCAO groups.4.24h after ischemic onset,Immunofluorescence staining assay showed that a large number of IgG permeated in ischemic ipsilateral hemispheres after tPA treatment,which was significantly decreased by HBHP treatment.Meanwhile,Western blot analysis showed that tPA-induced decomposition of occludin was rescued by HBHP.5.8h after ischemic onset,immunofluorescence staining assay showed that more Iba-1 positive cells permeated in peri-infarct area in tPA-treated group rats,which was ameliorated by HBHP treatment.qPCR showed HBHP decreased mRNA expressions of IL-1?,IL-6 and IL-12b.Meanwhile,HBHP treatment significantly down-regulated the protein expressions of iNOS,COX-2 and IL-1? in the ischemic hemisphere from tPA-treated rats,and the secretion of IL-1?.Conclusions1.Our research indicated that the serum HMGB1 levels were significantly elevated after treatment with tPA in stroke patients,and further confirmed in animal experiments.2.Co-treatment of HBHP could reduce the risk of tPA associated high-mortality,neurological impairment,brain swelling and cerebral hemorrhage.3.The neuroprotective effects of HBHP maybe relate to the protection of BBB integrality and the reduction of inflammatory reaction.These results further indicate that HMGB1 may potentiate the risk of tPA associated HT,and that blocking HMGB1 signaling would be helpful to prevent the complications brought by thrombolysis in ischemic stroke.