The Regulation Of Ilexonin A On Activation Of Astrocyte And Microglia After Cerebral Ischemia-reperfusion In Rats

OBJECTIVE To explore the regulation of Ilexonin A on activation ofAstrocyte, microglia in the perienchyma of brain ischemic tissue and the CA1ofhippocampus and its possible mechanism of protection after cerebral ischemia-reperfusion in Rats.METHODS The model of Middle cerebral artery occlusion (MCAO) wasestablished to transient focal ischemia by placing an intraluminal filament at theorigin of left middle cerebral artery two hours. The rats were divided randomly on thebasis of random digits table into: control, sham operation, model and IlexoninA (IA20,40,80mg/kg) treatment groups. Neuronogical deficits were evaluated by Longa’sscoring method. TTC staining was applied to observe the infarct of cerebral ischemiain MCAO model group and IlexoninA treatment groups. Immunohistochemistry andwestern blotting were used to observe the expression of the number of GFAP positivecell, Iba-1positive cell and Nestin positive cell and protein in the perienchyma of brainischemic tissue and the CA1of hippocampus at different time of ischemia andreperfusion. Furthermore, compare the effects of IA intervention (20,40,80mg/kg) onneurological deficit score and the expression of astrocyte and microglia in theperienchyma of brain ischemic tissue and the CA1of hippocampus after ischemia andreperfusion.RESULTS⑴Neurological deficits of model group and treatment group weresignificant repaired at1d after ischemia and reperfusion. The Neurological deficitswere repaired progressively with the prolongation of time. Compare to model group,the neurological deficit score of the treatment group was much less. A significantdifference of the neurological deficit score at3d,7d and14d in IA40mg/kg andIA80mg/kg treatment group, and at3d,7d in IA20mg/kg group compared with modelgroup（P<0.05）.⑵T he expression of positive cellin the perienchyma of brainischemic tissue: In control and sham groups, there were scattered GFAP positive celland Iba-1positive cell (mainly in the cortex), and no Nestin positive cell.①Themodel group’s GFAP positive cell increased at1d and reached its peak at7d afterischemia and reperfusion, and it still slightly high expression at14d. With theprolongation of time, the soma of the GFAP positive cell was swelled progressively, the cytoplasm staining was darked progressively and the apophysises were grew andthicken progressively. There could intersection with each other at14d. The number ofGFAP positive cell was significant increased at1d,3d and7d, but decreased at14d intreatment group, with the morphology less chang. There was a significant differencebetween the treatment group and the model group at3d,7d and14d（P<0.05）.②Themodel group’s Iba-1positive cell increased at1d and reached its peak at7d afterischemia and reperfusion. It still has a certain amount at14d. The morphology ofstatic microglial cells with elliptic and small soma and more apophysises graduallychanged into the actived microglia and macrophages with round and big soma andless apophysise. The expression of Iba-1positive cell in the treatment group wassignificant reduced at each period, with the morphology less chang. There was asignificant difference between the treatment group and the model group at each period(P<0.05).③The model group’s Nestin positive cell increased at3d,7d,14d, andreached its peak at7d after ischemia and reperfusion. The expression of Nestinpositive cell in the treatment group was significant increased at3d,7d. There was asignificant difference between the treatment group and the model group at3d,7d(P<0.05). The change of the number of positive cells was most in40mg/kg treatmentgroup.⑶The expression of positive cell in the CA1of hippocampus: In control andsham groups, there were scattered GFAP positive cell, Iba-1positive cell and Nestinpositive cell. The model group’s GFAP positive cell and Nestin positive cellsignificant increased at3d and reached its peak at7d after ischemia and reperfusion.There was a certain amount at14d after reperfusion. The number of Iba-1positive cellin the model group was not changed compare with the control and sham group. Theexpression of GFAP positive cell, Iba-1positive cell and Nestin positive cell in thetreatment group were significant increased at3d,7d. There was a significantdifference between the treatment group and the model group at3d,7d (P<0.05). Thechange of the number of positive cells was most in80mg/kg treatment group.⑷Theprotein expression of GFAP, Iba-1and Nestin in western blotting has the similar resultwith the immunohistochemisty.⑸There was a certain amount of TNF-αand IL-1βin the control and sham group. Their expression was significant increased andincreased more and more with the prolongation of time in the model group. There was a significant difference between the control, sham group and the model group at eachperiod (P<0.05). The expression of TNF-αand IL-1βin hippocampus was significantdecreased in treatment group. It was much obviously at7d、14d with pronounced effctat dose of80mg/kg. There was a significant difference between the treatment groupand the model group at each period (P<0.05).CONCLUSION1. IlexoninA can significantly repair the Neurological deficits. It may be passthought enhance the activation of astrocyte in the early stage and inhibit the excessproliferation of astrocyte in the advanced stage, inhibit the excess activation ofmicroglia and promote the proliferation of neural stem cells after cerebral ischemiaand reperfusion. Then, provide an advantage condition for neuroregeneration.2. IlexoninA can enhance the activation of astrocyte in the early stage and inhibitthe excess proliferation of astrocyte in the advanced stage, active the microglia andpromote the proliferation of neural stem cells in CA1of hippocampus after focalcerebral ischemia and reperfusion. It may has a preventive and treatment effection ondelayed selective loss of vulnerable CA1pyramidal neurons in the hippocampusafter focal cerebral ischemia and reperfusion.