通脑饮通过PI3K/Akt/GSK-3β信号通路促进血管新生改善脑缺血损伤OA

Objective:To investigate the mechanisms of Tongnao decoction(TND)in mice with acute ischemic stroke(AIS).Methods:Fifty male C57BL/6J mice were randomly divided into a sham operation group,model group,TND low-dose group(1.86 g·kg-1),TND high-dose group(3.72 g·kg-1),and butylphthalide(NBP)group(10 mg·kg-1),with 10 mice in each group.A mouse model of cerebral ischemic injury was established using photochemical thrombosis(PT).The sham operation group and model group were administered an equal volume of normal saline by gavage.All five groups were treated once daily for 14 consecutive days.Behavioral tests were performed before modeling and at the end of administration.T2-weighted imaging(T2WI)was performed 3 days after modeling to evaluate the extent of injury.Hematoxylin-eosin(HE)staining was used to observe histological changes in the cerebral cortex,and Nissl staining was used to observe neuronal morphology.Cerebral blood flow in mice was detected using a laser speckle contrast imaging(LSCI)system.Immunofluorescence staining was used to detect the cell proliferation marker bromodeoxyuridine(BrdU)and the highly glycosylated type I transmembrane glycoprotein CD34.Western blot analysis was used to detect the expression levels of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),glycogen synthase kinase-3β(GSK-3β),and their phosphorylation levels,as well as tight junction-related proteins zonula occludens-1(ZO-1),Occludin,and Claudin-5 in the peri-infarct tissue.Thirty-five zebrafish were randomly divided into normal control group,model group,TND low and high dose groups(0.16,0.32 g·L-1)and NBP group(10 μmol·L-1),with 7 in each group.A stereoscopic fluorescence microscope was used to observe vascular growth in zebrafish.Results:Imaging showed that PT caused ischemia in the right cortical region.Behavioral tests indicated that,compared with the model group,the drug-treated groups reduced the error rate of irregular balance ladder climbing on the affected side and shortened the tape removal time(P<0.05).HE staining and Nissl staining showed that,compared with the model group,the drug-treated groups exhibited reduced brain tissue damage,fewer scars,and improved neuronal morphology.LSCI results showed that the drug-treated groups partially restored cerebral blood perfusion and promoted the establishment of collateral circulation compared with the model group.Immunofluorescence staining indicated that the drug-treated groups increased the positive rates of BrdU and CD34 compared with the model group(P<0.01),promoting angiogenesis.Meanwhile,compared with the model group,the drug-treated groups upregulated the expression levels of p-PI3K,p-Akt,p-GSK-3β,and tight junction proteins ZO-1,Occludin,and Claudin-5(P<0.05,P<0.01),and increased the number of intersegmental vessels in zebrafish(P<0.05,P<0.01).Conclusion:TND can promote angiogenesis around the infarct in PT model mice by regulating the PI3K/Akt/GSK-3β signaling pathway,thereby improving cerebral ischemic injury.