The Role And Mechanism Of CyPA/CD147 In Hemorrhagic Transformation After Rt-PA Thrombolysis In Ischemic Stroke

Objective:Hemorrhagic transformation(HT)is a serious complication after rtPA thrombolysis in patients with acute ischemic stroke.The blood-brain barrier(BBB)disruption is the critical process leading to HT after rt-PA thrombolysis.Previous studies indicated that the interaction between CyPA and CD147 may be closely related to BBB disruption.This study is aimed to explore the clinical role of CyPA/CD147 in HT after rt-PA thrombolysis,as well as the possible mechanism of mediating HT after thrombolysis.Methods:1.Association between serum CyPA/CD147 and hemorrhagic transformation after rt-PA thrombolysis in ischemic stroke(1)Patients with acute ischemic stroke(AIS)who received rt-PA thrombolysis from April 2021 to December 2022 and healthy controls who underwent physical examination in the health management center of our hospital during the same period were continuously enrolled.(2)Clinical data and peripheral blood samples from patients with AIS and healthy controls were collected.ELISA detected the levels of CyPA or CD147 in serum.(3)The univariate and multivariate regression analyses were performed to evaluate the association of serum CyPA or CD147 levels with ischemic stroke and HT after rt-PA intravenous thrombolysis.2.Relationship between CyPA and CD147 expression and HT in brain tissue of MCAO rat after rt-PA thrombolysis(1)The thromboembolic middle cerebral artery occlusion(MCAO)model was established in SD rats and divided into Sham,MCAO,and MCAO + rt-PA(IVT)groups based on different interventions.(2)The modified neurological impairment score(m NSS)evaluated the neurological impairment of rats.Infarct volume was measured in the TTC-stained coronal section.Hemoglobin levels were measured with the Drabkin reagent.The blood-brain barrier permeability was evaluated by Evans blue staining.The expression and distribution of CyPA and CD147 in brain tissue were detected by Western blotting(WB)and Immunofluorescence(IF).3.Rt-PA activates ERK/NF-κB signaling pathway to mediate BBB disruption by up-regulating the expression of CyPA/CD147.(1)Human cerebral microvascular endothelial cells(hCMEC/D3)were used to construct the oxygen-glucose deprivation(OGD)model in vitro,which was treated with rt-PA when reoxygenated.The interaction between CyPA and CD147 was assessed by using co-immunoprecipitation and immunofluorescence(IF).Using Western blotting(WB)to detect the expression of CyPA,CD147,ERK1/2 and its phosphorylation protein(p ERK1/2),NF-κB and its phosphorylation protein(p NF-κB),MMP-9,and tight-junction protein.(2)The expression of CyPA or CD147 was silenced using small interfering RNA.WB was used to detect the expression of CyPA,CD147,ERK1/2 and p ERK1/2,NF-κB and p NF-κB,MMP-9,and tight-junction protein.IF was used to observe the distribution of p ERK1/2 and p NF-κB in cells.4.Inhibiting CyPA/CD147 alleviated the occurrence of HT after rtPA thrombolysis in MCAO rat(1)The MCAO model was established in SD rats and divided into Sham,IVT + normal saline,IVT + CsA,IVT + Taurine,and IVT + CsA +Taurine group based on different interventions.(2)The m NSS evaluated the neurological impairment of rats.Infarct volume was measured in the TTC-stained coronal section.Hemoglobin levels were measured with the Drabkin reagent.The blood-brain barrier permeability was evaluated by Evans blue staining.WB was used to detect the expression of CyPA,CD147,ERK1/2 and p ERK1/2,NF-κB and p NF-κB,MMP-9,and tight-junction protein in ischemic brain tissue.Immunohistochemical or IF was used to observe the distribution of p ERK1/2,p NF-κB,MMP-9,and tight-junction protein in brain tissue.Results:1.Association between serum CyPA/CD147 and hemorrhagic transformation after rt-PA thrombolysis in ischemic stroke(1)Compared with healthy controls,serum CyPA and CD147 levels were increased in AIS patients.(2)Compared with non-HT patients,serum CyPA and CD147 levels in HT patients were increased after thrombolysis.Multivariate logistic regression analysis showed that high serum CyPA and CD147 levels were significantly associated with HT after rt-PA intravenous thrombolysis in patients with ischemic stroke.(3)Compared with non-SICH patients,serum CyPA and CD147 levels in SICH patients were increased after thrombolysis.Multivariate logistic regression analysis showed high serum CyPA level was significantly associated with SICH after rt-PA intravenous thrombolysis in patients with ischemic stroke.2.Relationship between CyPA and CD147 expression and HT in brain tissue of MCAO rat after rt-PA thrombolysis(1)In the MCAO model of rats,delayed rt-PA therapy can aggravate BBB disruption and intracranial hemorrhage.(2)In the MCAO model of rats,rt-PA can significantly up-regulate the expression of CyPA and CD147 in ischemia brain tissue.3.Rt-PA activates ERK/NF-κB signaling pathway to mediate BBB disruption by up-regulating the expression of CyPA/CD147(1)In the OGD model of hCMEC/D3 cells,rt-PA can promote the expression CyPA,CD147,p ERK1/2,p NF-κB,and MMP-9,the degradation of tight-junction protein,and the interaction between CyPA and CD147.(2)Specific knockout CyPA or CD147 can inhibit the expression of p ERK1/2,p NF-κB,and MMP-9,as well as the degradation of tightjunction protein.4.Inhibiting CyPA/CD147 alleviated the occurrence of HT after rtPA thrombolysis in MCAO rats.(1)Compared with the IVT group,CsA or Taurine can improve neurological defects and reduce cerebral infarction volume,BBB disruption,and HT after rt-PA thrombolysis.(2)Compared with the IVT group,CsA or Taurine can down-regulate the expression of CyPA,CD147,p ERK1/2,p NF-κB,and MMP-9 in ischemic brain tissue,as well as the degradation of tight-junction proteins.Conclusion:(1)High serum CyPA and CD147 levels were correlated with HT after rt-PA thrombolysis in patients with acute ischemic stroke.(2)The expression of CyPA and CD147 increased in ischemia brain tissue of MCAO rats after rt-PA thrombolysis,which may be participated in the occurrence of HT after rt-PA thrombolysis.(3)Rt-PA can activate the ERK/NF-κB/MMP-9 signaling pathway to degrade the tight-junction protein by increasing the expression and interaction of CyPA and CD147 in hCMEC/D3 cells.Knock-down of CyPA or CD147 can inhibit the activation of the ERK/NF-κB/MMP-9signaling pathway by mediating rt-PA and reducing BBB disruption.(4)Inhibition of CyPA/CD147 expression significantly reduces the activation of ERK/NF-κB/MMP-9 signaling pathway in ischemia brain tissue,alleviating rt-PA thrombolysis-related BBB disruption and HT.