| The mechanisms of brain injury caused by focal ischemic stroke arecomplicated. There are no other effective solutions except thrombolytictherapy. The only drug authorized by FDA is tPA, but its therapeutic timewindow is only4.5h. Due to those largely ineffective current therapeuticstrategies, the development of new and efficient therapeutic interventions isclearly needed to reduce the ischemic damage.The Wnt signaling pathwaycan increase the proliferation of stem cells to moduate the neurogenesis andangiogenesis process. The Wnt signaling pathway is actived after focalcerebral ischemia reperfusion. Morroniside can effectively promoteneurogenesis in SVZ area and increase the vessel density aroundperi-infarct after focal cerebral ischemia reperfusion. This study is toinvestigate the therapeutic time window of morroniside on focal cerebralischemia-reperfusion injury in rats and the time course changes of thefactors of Wnt signaling pathways after ischemia, on focal cerebralischemia-reperfusion injury in rats which supplied new ideas for clinicaltreatment of ischemic brain injury and the development of protective drugs.Purpose: To study the therapeutic time window of morroniside on focalcerebral ischemia-reperfusion injury and the changes of Wnt signalingpathway in rats.Methods: The animal model was made by the occlusion of middle cerebralartery(MCAO) stutre embolus, ischemia for30minutes. The rats wererandomly divided into sham operated group, model group,morroniside-30mg·kg-1,90mg·kg-1,270mg·kg-1and administeredintragastrically for7days after reperfusion for1d and3drespectively. Then the recovery of neurological function was examined by Zea Longa’smethod. TTC staining was used to examine the infarction volume in focalcerebral ischemia-reperfusion. The expression of neurogenic or angiogenicfactors were also detected by Western Blotting to determine the therapeuticwindow of morroniside on focal cerebral ischemia-reperfusion injury inrats. The time-course expression of Wnt3a and β-catenin was detected3h、6h、12h、24h、3d、7d、14d、28d respectively after MCAO was performed.Results:1. The recovery of neurological function: Compared with shamgroup, the neurological score of model group operated after8days and10days are significantly increased (P<0.001, P<0.001). The rats wereadministered intragastrically for7days after1d and3d reperfusionrespectively. Morroniside administrated at the concentration of270mg/kgat1day post-reperfusion, significantly decreased the Zea Longa scorescompared with model group(P<0.01); while no significant decrease wasobserved in30mg·kg-1,90mg·kg-1or270mg·kg-1morroniside-treatmentgroup compared with model group when morroniside was administered at3day post-reperfusion.2. The infarction volume in focal cerebral ischemia-reperfusion:Compared with sham group, the infarction volume of model group operatedafter8days and10days are significantly increased (P<0.001, P<0.001). Therats were administered intragastrically for7days at1d and3d afterreperfusion respectively. Morroniside administrated at the concentration of270mg·kg-1at1day post-reperfusion, significantly decreased the infarctionvolume compared with model group(P<0.05); while no significant decreasewas observed in30mg·kg-1,90mg·kg-1or270mg·kg-1morroniside-treatment group compared with model group whenmorroniside was administered at3day post-reperfusion.3. The expression of the factors related to neurogenesis: Both of theexpression of Wnt3a and β-catenin significantly increase (P<0.05，P<0.05) in the rats operated after8days compared with sham group, the same trandsas10days. The rats were administered intragastrically with morroniside for7days at1d and3d after reperfusion respectively. Morronisideadministrated at the concentration of270mg·kg-1at1day post-reperfusion,significantly increased the expression of Wnt3a and β-catenin comparedwith model group(P<0.01, P<0.01,); while no significant decrease wereobserved in30mg·kg-1,90mg·kg-1or270mg·kg-1morroniside-treatmentgroup compared with model group when morroniside were administeredat3day post-reperfusion.4. The expression of the factors related to angiogenesis: Both of theexpression of CD34and Ang-1significantly increase (P<0.05，P<0.05) inthe rats operated after8days compared with sham group, the same trands as10days.The rats were administered intragastrically with morroniside for7days at1d and3d after reperfusion respectively. Morroniside administratedat the concentration of270mg·kg-1at1day post-reperfusion, significantlyincreased the expression of CD34and Ang-1compared with modelgroup(P<0.05, P<0.01),and morroniside-90mg/kg is also effectively on theexpression of Ang-1(P<0.05); while no significant decrease was observedin30mg·kg-1,90mg·kg-1or270mg·kg-1morroniside-treatment groupcompared with model group when morroniside was administered at3daypost-reperfusion.5. The time-course expression of Wnt signaling pathway in rat afterfocal cerebral ischemia reperfusion: The expression of Wnt3a wasincreased at3h post-reperfusion, reached the peak at1day(P<0.001) andthen kept the high level at3d、7d and14d compared with the sham group（P<0.01、P<0.001、P<0.05). The expression of Wnt3a from14dpost-MCAO until28d were decreased compared with that of7d（P<0.01）.The expression of β-catenin was decreased at12h but increased at1d、3d、7d and14d (P<0.001、P<0.001、P<0.001、P<0.05), while there was nodifference between that of28d and the sham group. Conclusion: These results indicate that morroniside administered at1dpost-reperfusion could improve the neurological function, decrease theinfarction volume and increase the expression of factors related toneurogenesis and angiogenesis. The effects of morroniside on the abovefactors were not significant when morroniside was administered at3dpost-reperfusion. Conclusively, morroniside could prevent the brain frominjury and the therapeutic time window on focal cerebralischemia-reperfusion injury in rats is no more than3days. |
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