The Effect And Mechanism Of Wnt Signaling In The Hemorrhagic Transformation Induced By Thrombolytic Therapy In Acute Ischemic Stroke Patients

Acute ischemic stroke is caused by occlusion of brain arteries by a thrombus and subsequent cerebral ischemia.It has the characteristics of high incidence,high mortality and high disability rate,which brings heavy mental and economic burden to the family and society.Recombinant tissue-type plasminogen activator(rt-PA)is the only FDA approved drug for the treatment of acute ischemic stroke(AIS).Intravenous rt-PA thrombolysis increases the risk of hemorrhagic transformation(HT),which is a major complication of acute ischemic stroke and seriously threatens the life of patients.It is necessary to study the pathogenesis of HT and explore potential treatment methods to reduce the risk of HT in AIS patients and improve the prognosis.The canonical Wnt/?-catenin pathway plays an important role in the blood-brain barrier(BBB)integrity and cerebral hemorrhage in stroke.However,the association of the canonical Wnt/?-catenin signaling pathway in the breakdown of the blood-brain barrier in HT after stroke has not been thoroughly studied and conclusive evidence has been lacking.Therefore,this study investigated the effect and mechanism of Wnt signaling in the hemorrhagic transformation induced by thrombolytic therapy in patients with ischemic stroke at the clinical,molecular and cellular levels.The main research contents include the following two aspects:1.The association between Wnt signal and hemorrhagic transformation after thrombolysis in ischemic stroke.With the rapid progress of gene sequencing technology,gene detection is leading cerebrovascular diseases into the era of precision therapy.Through gene diagnosis,patients can be known as early as possible before the onset of disease,early prevention,especially cerebrovascular disease needs the guidance of genetic testing and auxiliary diagnosis.The purpose of this study was to explore the relationship between blood gene sequence variation associated with Wnt signaling pathway and HT in AIS patients receiving rt-PA venous thrombolysis.A total of 355 AIS patients receiving rt-PA intravenous thrombolysis(IVT)were enrolled.Inclusion criteria were similar National Institute of Health Stroke Scale(NIHSS)baseline scores;time from onset to start of treatment(OTT):2-4 hours;computered tomography(CT)scan was performed at admission and 24 hours after thrombolysis;Blood samples were collected on admission before rt-PA administration.The exons of 126 single nucleotide polymorphisms(SNPs)and 4 core Wnt signaling elements(GPR124,RECK,FZD4 and CTNNB1)of 28 Wnt signaling genes were determined using a customized sequencing chip.Results show that compared with non-p H patients,5 gene variants belonging to WNT7 A and GPR124 were selectively enriched in PH patients,including WNT7 A SNPs(rs2163910,P = 0.001,OR 2.727;rs 1124480,P = 0.002,OR 2.404)and GPR124 SNPs(rs61738775,P = 0.012,OR 4.883;rs146016051 P < 0.001,OR 7.607;rs75336000,P = 0.044,OR 2.503).The two SNPS of RECK were enriched in PH patients,but did not reach statistical significance(rs12235235,P = 0.090,OR 1.882;rs12235235,P = 0.090,OR 1.882).2.The effect of GPR124 c.3587 G > A mutation on Wnt signaling activity.According to the genetic test results of clinical patients,GPR124 c.3587 G > A mutation was significantly enriched in PH patients.G protein-coupled receptor 124(GPR124)is an orphan receptor in the GPCRs adhesion family.It was found that GPR124 exerted the specific synergistic activator action of WNT7A/7B through the unclear direct or indirect mechanism,and played a role in the Canonical Wnt/?-catenin pathway.GPR124 regulates the Wnt signaling of endothelial cells in the pathologic state of stroke in adult mice,thereby regulating the integrity of the blood-brain barrier.This finding demonstrates the potential of GPR124 as a potential therapeutic target for human central nervous system diseases in which the blood-brain barrier is broken.Study investigate the effect of GPR124 c.3587 G > A mutation on protein expression and localization by western blot and immunofluorescence assay.The effect of this mutation on the activity of Wnt/?-catenin signal was detected by q PCR and Top Flash.The combination of GPR124 and DVL was detected by immunoprecipitation.Results show that the mutation did not affect the expression and localization of the protein.GPR124 c.3587 G > A mutation has functional significance and significantly affects the activity of Wnt/?-catenin pathway.DVL1 is combined with GPR124 Wild-type(WT),while DVL2 and DVL3 have no interaction.GPR124 c.3587 G > A mutation can significantly reduce the interaction with DVL1.DIX,PDZ and DEP domains of DVL1 all interact with GPR124,among which the PDZ domain plays a more prominent role,while DIX/DEP domain plays a smaller role.In summary,this study demonstrated that Wnt signaling plays an important role in the hemorrhagic transformation induced by thrombolytic therapy in patients with ischemic stroke.Five Wnt signal-related gene variants were selectively enriched in patients with hemorrhagic transformation,among which GPR124 c.3587 G > A mutation affected the functional activity of Wnt signal.The results of this study broaden our understanding of the hemorrhagic transformation caused by thrombolysis in ischemic stroke and provide new targets and theoretical guidance for the prediction of hemorrhagic transformation in clinical practice.