Erythropoietin Protects Neural Cell And Blood-brain Barrier From Ischemia-reperfusion Injury By Interfering In Endoplasmic Reticulum Stress

ObjectiveIn the case of erythropoietin (EPO), an established neuroprotectant againstischemic brain injury, its low BBB permeability requires such a high therapeuticdose that it can induce dangerous complications such as polycythmia andsecondary stroke. To improve this combination therapy, the present studyintended to establish a new strategy, and examine the effects of this strategy oncerebral ischemia and reperfusion injury and its underlying mechanism.Methods54Male Sprague–Dawley rats were divided into6groups: sham grop,vehicle group, EPO group, tPA group, EPO+tPA group and salubrinal group.Rats were subjected to middle cerebral artery occlusion and administrated withEPO (800IU/kg) via middle cerebral artery infusion. tPA was given via tale veinand salubrinal was given by peritoneal injection30min before MCAO. Vehiclewas given the some volume of saline in replace of EPO via MCAI. Reperfusion was induced2h after MCAO and the rats were executed after neurologicaldeficit evaluation. Infarct volume, brain edema were examined by TTC staining.The expression of AQP4, caspase-3, tight junction proteins and ERstress-associated proteins including CHOP, GRP78and ATF6α were assessedby western blot in both brain tissues and microvessels.Results1、Treatment with EPO plus tPA is superior to EPO or tPA alone inattenuating cerebral I/R injury：The advantage of EPO plus tPA over EPO or tPAalone in attenuating cerebral I/R injury was assessed in terms of infarct volume,brain edema, and neurological deficits. As expected, tPA alone had no effect onany insult examined after I/R, while EPO alone attenuated all the insults,compared to vehicle group (P<0.05). Importantly, EPO plus tPA revealed a moresignificant effect than tPA alone on the I/R-induced insults(P<0.05).2、Treatment with EPO plus tPA or EPO alone reduces ER stress-inducedAQP4expression, apoptosis and ERS relative protein expression following I/Rinjury: we first assessed the expression of AQP4and Caspase-3by western blot.The results showed a significant increase in AQP4and Caspase-3expression inboth the vehicle and tPA monotherapy groups at24h after I/R injury (P<0.05),as compared with sham group. Noticeably, EPO plus tPA or EPO alonetreatment attenuated cerebral I/R-enhanced AQP4expression (P<0.05). Wenext examined expression of GRP78and CHOP. Western blot revealed thatcerebral I/R provoked a significant increase in the expression of GRP78andCHOP(P<0.05). This I/R-induced upregulation of GRP78and CHOP expressionwas completely reversed by EPO plus tPA or EPO alone treatment(P<0.05).3、Treatment with EPO plus tPA or EPO alone prevents tight junctionproteins in brain microvessels from decrease after I/R: the expression anddistribution of tight junction proteins Claudin-5and Occludin in brain microvessels were determined. The western blot showed that vehicle and tPAgroups demonstrated a markedly decrease in Claudin-5and Occludin inisolated microvessels compared with sham operated animal(P<0.05). EPOalone or combination therapy with tPA significantly prevented the decrease ofClaudin-5and Occludin (P<0.05), whereas tPA alone did not show effect. Theimmunofluorescence staining demonstrated that the expression of Claudin-5and Occludin in endothelial cells markedly declined in vehicle and tPA groups,implying a significant disruption of the vascular endothelium, whereas thecombination treatment of tPA and EPO prevented the decrease of Claudin-5and Occludin after I/R.4、Combination of EPO with tPA reduces ER stress in brain microvesselsfollowing I/R injury: we examined the expression of GRP78and CHOP, the twoER stress related proteins, in cerebral microvessels by western blot. The resultsshowed that vehicle and tPA groups demonstrated a markedly increase inGRP78and CHOP in isolated microvessels compared with sham operatedanimal(P<0.05). While EPO alone or combination therapy with tPA significantlydecrease the expression of Claudin-5and Occludin (P<0.05).5、Treatment with EPO alone or EPO plus tPA depresses ER stress signalin brain tissue and isolated microvessels following I/R injury: the expression ofATF6, a ER stress signal that is upstream of CHOP, was determined in braintissue and isolated microvessels, respectively. The result showed that, in bothcases, the I/R markedly increased the expression of ER stress signal ATF-6,whereas EPO alone or EPO plus tPA treatment significantly down-regulated theexpression levels (P<0.05).Conclusions1、Combination treatment of EPO and tPA has neuroprotection againstcerebral ischemia reperfusion injury. 2、Combination treatment of EPO and tPA can not only decreasedischemia reperfusion induced apoptosis, but also reduced BBB disruption.3、ERS signaling of ATF6-CHOP pathway-mediated apoptosis maycontribute to the mechanisms for EPO neuroprotection.