| Objective :Ischemia-reperfusion(I/R) injury, which can damage endothelium and cause vascular endothelial dysfunction, is an important pathogenesis of ischemic stroke. Endothelial progenitor cells(EPCs) can differentiate into mature vascular endothelium, which is involved in the repair of damaged blood vessels and angiogenesis. Hemopexin(HPX) plays a neuroprotective role via specifically binding and removing excess free heme. And heme oxygenase-1(HO-1) is the rate-limiting enzyme in degradation of free heme. With focal I/R model in rats, our previous research has found that HPX could narrow the area of cerebral infarction in rats after focal I/R injury. However, the specific mechanism is unclear. This study was carried out to explore whether HO-1- EPCs signaling is involved in hemopexin alleviating the cognitive dysfunction in rats subjected to focal I/R injury. Methods:Focal cerebral I/R injury model was induced by middle cerebral artery occlusion(MCAO) with male Sprague–Dawley(SD) rats, 7 to 8 weeks old, weighting 250-280 g. Rats were randomly divided into five groups,which were Sham group, MCAO group, MCAO+Vehicle group, MCAO+HPX group and MCAO+HPX +ZnppIX group. All the reagents including saline?vehicle?HPX and ZnppIX were given via lateral cerebroventricular injection with the location of brain solid positioned once right at the moment after ischemia-reperfusion. 10?l saline(0.9%)?sodium azide(0.1%)?HPX(1.86?g/?l) and HPX+ sodium azide(1.86?g/?l+0.1%) were given to rats in MCAO group?MCAO+Vehicle group, MCAO+HPX group and MCAO+HPX+ZnppIX group respectively.Part one: The effect of HPX on behavioral performance in rats after focal I/R injury. Rats were randomly divided into four groups(20 per group): Sham group, MCAO group, MCAO+Vehicle group, MCAO+HPX group and MCAO+HPX +ZnppIX group. Neurological functional outcome determined by mNSS was assessted before ischemia, 12h?24h?3d?5d and 7d after ischemia-reperfusion. Assessment of escape latency, the time percentage in target quadrant and platform-crossing number was carried out by Morris water maze assay equipped with acquisition system of data and vedio image at 2 to 7 days respectively after ischemia-reperfusion.Part two: The effect of HO-1-EPCs signaling on HPX alleviating cognitive dysfunctuion in rats after focal I/R injury. First of all, the rats were randomly divided into sham group, MCAO group, MCAO+Vehicle group and MCAO+HPX group, the modeling method was the same as part one above. Six rats per group were sacrificed at 2h, 6h, 12 h, 24 h and 7d after reperfusion to obtain the ipsilateral ischemic penumbra brain tissues. RT-PCR was used to analyze the mRNA levels of HO-1, followed by western blot to detect HO-1 protein expression; Then rats were randomly divided into sham group, MCAO group, MCAO+Vehicle group, MCAO+HPX group and MCAO+HPX+ZnppIX group, the modeling method was the same as part one above.The peripheral blood was obtained from inner canthus retrobulbar venous plexus with glass capillary. The circulating EPCs were counted by flow cytometry identification and quantitative analysis technology combined with double-immunofluorescence staining of CD34 and CD133 antibodies.Part three: The influence of HPX on the angiogenesis at ischemia penumbra area in rats after focal I/R injury.The modeling was the same as part one above. Rats were randomly divided into Sham group, MCAO group, MCAO+Vehicle group, MCAO+HPX group and MCAO+HPX+ZnppIX group. Rats were sacrificed to obtain their brain tissues for coronal frozen sections at 7 day after reperfusion.Angiogenesis at cortex, hippocampus and striatum of ischemia penumbra in rats was observed by immunohistochemical technique combined with immunofluorescent chromogenic detection to detect differentiation of EPCs into vascular endothelium. Results :Part one: Compared with Sham group, the focal ischemia-reperfusion injury caused severe neuorological function deficit in rats(p<0.05), increased their escape latency(p<0.05) and decreased their quadrant time percentage and platform-crossing number(p<0.05); HPX given by lateral i.v. alleviated their neurological function deficit(p<0.05), reduced their escape latency(p<0.05),increased their quadrant time percentage and the number of platform crossing(p<0.05), improving their learning and memory ability. The effect of sodium azide given via lateral i.v. on the neurological deficit, learning and memory ability in rats was slight-no statistical difference(p > 0.05); Cmpared with HPX group, the inhibitor of HO-1, ZnppIX, could blocked the effect of HPX on alleviating the neurological function deficit, decreasing the escape latency, increasing their quadrant time percentage and platform-crossing number in Morris water maze experiement(p<0.05).Part two: Compared with sham group, focal I/R injury obviously induced the transcription and expression of HO-1 at ischemic penumbra area at different target time points(p<0.05), which peaked at 24 h after I/R and substantially increased the peripheral circulating count of EPCs(p<0.05); HPX injection can notably up-regulate those of HO-1 at different times respectively(p<0.05); Sodium azide given via lateral i.v. hardly had effect on those of the HO-1 and circulating EPCs count at different time points respectively(p>0.05). Compared with the circulating EPCs count in HPX group, the inhibitor of HO-1, ZnppIX, could suppressed the effect of HPX on mobilization of EPCs from bone marrow to peripheral blood and incresing the peraphral circulating EPCs count after focal cerebral I/R(p<0.05).Part three: Compared with sham group, focal I/R injury could obviously induce the angiogenesis at cortex?hippocampus and striatum zone of ischemia penumbra area in rats after 7 day after focal I/R injury(p<0.05); HPX given via lateral i.v. obviously enhanced the angiogenesis at cortex, hippocampus and striatum zone of ischemia penumbra in rats( p<0.05); Sodium azide had slight effect on the angiogenesis at all cerebral zones of ischemia penumbra(p>0.05); ZnppIX can inhibit and remove the effect of HPX on enhancing angiogenesis at all cerebral ischemia penumbra areas(p>0.05).Conclusion:HPX alleviated the neurological function deficit after focal cerebral I/R injury in rats. The underlying molecular mechanism may be associated with the up-regulation of HO-1, which promotes mobilization of EPCs from bone marrow to peripheral blood and their differentiation and maturation to endothelium to participate in vascular repair and angiogenesis. |
PDF Full Text Request