Effects Of Exendin-4 Via GSK-3β/β-catenin Signaling Pathway On Reducing The Damage Of Hemorrhagic Transformation Induced By RtPA After Ischemic Stroke

Background: Ischemic stroke is one of the most common diseases of the central nervous system,which has the characteristics of high morbidity,high disability,high mortality and heavy economic burden.It severely harms patients’ healthy living standard,restricts the rapid development of the society and economy,and has become the first cause of death in our country.At present,tissue-type plasminogen activator(rt PA)is the only thrombolytic drug approved by the Food and Drug Administration for the treatment of acute ischemic stroke,but its effective therapeutic time window is limited within 4.5h of onset.Rt PA thrombolytic therapy beyond this time window will greatly increase the risk of hemorrhagic transformation(HT)and the mortality of patients.The specific mechanism of HT after rt PA thrombolysis is not yet clear,and clinical treatment still lacks safe and effective HT prevention and treatment measures.Studies have shown that activation of the wnt/β-catenin signaling pathway can protect the integrity of the structure and function of the blood-brain barrier after acute ischemic stroke.In addition,the glucagon-like peptide 1 receptor agonist exendin-4can increase the expression of phosphorylated GSK-3β protein and β-catenin protein in liver tissue.Therefore,we speculate whether exendin-4 reduces the risk of rt PA-induced HT by activating the wnt/β-catenin signaling pathway.Objective: To clarify the effect of exendin-4 on HT caused by rt PA thrombolysis in ischemic stroke;to study the effect of exendin-4 on the blood-brain barrier and the expression of related molecules in the GSK-3β/β-catenin signaling pathway.To elucidate the molecular mechanism of GSK-3β/β-catenin signaling pathway in prevention and treatment of rt PA-induced HT after ischemic stroke.Method: In this experiment,the middle cerebral artery occlusion(MCAO)model was used to induce hemorrhagic transformation with tissue-type plasminogen activator(rt PA)to study the effect of exendin-4(EX-4)on the blood-brain barrier and GSK-3β/β-catenin signaling pathway.Animals were 250-280 g male SD rats,randomly divided into 5 groups: sham operation group(Sham),control group(Vehicle),rt PA group,rt PA+EX-4 group,rt PA+EX-4+PRI-724 group.After removing the plug 4h after MCAO,rt PA(10mg/kg)and EX-4(100ug/kg)were injected intravenously,and PRI-724(10mg/kg)was injected intraperitoneally according to the group.the following experiments were performed 24 hours after MCAO:(1)neurological function score;(2)The degree of cerebral edema was measured by dry and wet weight method;(3)TTC method to detect the percentage of cerebral infarction;(4)Evans blue dye to detect blood-brain barrier permeability;(5)Hemoglobin detection kit to detect the degree of parenchymal hemorrhage on the infarcted side;(6)HE staining to detect the structure of the brain tissue on the infarcted side;(7)Immunohistochemistry to detect the content of β-catenin in the infarcted side of the brain;(8)Immunofluorescence to detect the expression level of MMP-9 in the infarcted side;(9)Western blot was used to detect the expression of BBB constituent proteins(Claudin-5,Occludin and ZO-1),GSK-3β/β-catenin signaling pathway proteins(GSK-3β,β-catenin and p-β-catenin),and MMP-9.Results: Compared with the control group,rt PA aggravated neurological symptoms of MCAO rats,increased cerebral water content of the infarcted side,the area of the cerebral infarction,the permeability of the blood-brain barrier and the amount of cerebral hemorrhage,activated GSK-3β/β-catenin signaling pathway,damaged the blood-brain barrier related proteins,including Claudin-5,Occludin and ZO-1,and increased the expression of MMP-9.Compared with the rt PA group,exendin-4 can significantly improve the neurological symptoms after rt PA treatment.Brain water content,infarction area,blood-brain barrier permeability and cerebral hemorrhage in infarct side were improved.GSK-3β/β-catenin signaling pathway was inhibited,the disruption of Claudin-5,Occludin,ZO-1 and the expression of MMP-9 was reduced.PRI-724 can reverse the protective effect of exendin-4 on MCAO rats treated with rt PA by inhibiting the GSK-3β/β-catenin signaling pathway.Conclusion: GSK-3β/β-catenin signaling pathway is involved in the process of cerebral ischemia and reperfusion.Inhibiting GSK-3β/β-catenin signaling pathway may reduce HT after rt-PA thrombolysis and improve the prognosis of neurological function by regulating MMP-9 activation and leukocyte infiltration to protect the integrity of the blood-brain barrier.Exendin-4 may improve the neurological function after rt PA thrombolysis by inhibiting the GSK-3β/β-catenin signaling pathway,and reduce the degree of cerebral edema and cerebral infarction in MCAO rats.