The Role And Mechanisim Of Pinocembrin In Hemorrhagic Transformation After Stroke

Objective Hemorrhagic transformation(HT)is a frequent complication of ischemic stroke,especially after thrombolytic therapy,which is associated with increased morbidity and mortality.Because of the pathomechanism and therapeutic target are not clear,there are no effective drugs to decrease the incidence of HT.Pinocembrin is a natural flavonoid compound and has neuroprotective effects in animal ischemic stroke models.However,the role of pinocembrin in HT was not investigated.In this study,we investigated the role and mechanism of pinocembrin in HT after stroke.Methods (1)Rat middle cerebral artery occlusion model preparation by electrocoagulation was used to evaluate the role of pinocembrin in t-PA induced HT.Rats were randomly divided into six groups as follows: sham operation group,model group,t-PA(10 mg/kg)group,t-PA+pinocembrin(5,10,20 mg/kg)group.t-PA was administrated at 6 h after ischemia and pinocembrin(5,10 and 20 mg/kg)was given 5min before t-PA administration.Infarct volume,neurological function and hemoglobin content were measured at 24 hours after ischemia.(2)Human brain microvascular endothelium cell(HBMVEC)damage model was induced by t-PA.The experiment was divided into the following groups: blank control group,normal group,t-PA(200 ug/ml)group,and t-PA+pinocembrin(10 μM)group.Cell viability was measured at 24 hours after treatment with t-PA and pinocembrin.(3)Rat middle cerebral artery occlusion model was used to evaluate the role of pinocembrin in hyperglycemic induced HT.Rats were randomly divided into four groups as follows: sham operation group,model group,50% high glucose group,50% high glucose+pinocembrin(10 mg/kg)group.Pinocembrin was administrated 5 min before reperfusion by tail vein.Infarct volume,neurological function and hemoglobin content were detected at 24 hours after ischemia.Pathological changes were observed by HE staining.The incidence of HT and the survival rate were counted.(4)Evans blue leakage was used to evaluate the integrity of blood-brain barrier(BBB).Ultrastructural changes of BBB were observed by transmission electron microscopy.The expression of tight junction protein and MMPs were detected by western-blot and immunohistochemistry.Results (1)In t-PA induced HT model,t-PA was administrated after 6 hours of ischemia significantly aggravated brain injury and promoted the occurrence of HT.The infarct volume and brain water content reached by 39% and 83.4%,respectively.Pre-administration of pinocembrin can decrease infarct volume and brain water content to 28.5% and 80.3%,respectively,which improved neurological function.In addition,the combination of pinocembrin with t-PA can reduce the hemoglobin content of brain tissue by 50%,indicated that pinocembrin decreased the occurrence of HT induced by t-PA.Among these doses,the effect of pinocembrin(10 mg/kg)is much better.(2)t-PA induced HBMVEC cell damage in a dose-and time-dependent manner,200μg/ml t-PA incubated 24 hours can induce suitable cell injury.Administration with 10 μM pinocembrin significantly increased cell viability.(3)In hyperglycemia-induced HT model,intraperitoneal injection of 50% glucose before 30 minutes significantly elevated blood glucose and maintained for 3.5 h.Compared with the normal blood glucose model,hyperglycemia can significantly aggravate brain injury.Pinocembrin(10 mg/kg)significantly lowered the behavioral score(9.5 VS 7.8),and infarct volume(37% VS 27%).The detection of hemoglobin content in brain tissue found that pinocembrin significantly reduced hemoglobin content in brain tissue from 43.7% to 30%,compared with the high glucose group.At the same time,the statistics of HT incidence and mortality indicated that the incidence of HT in high glucose group was as high as 95.5%,and reduced to 59.1% after treatment with pinocembrin.Compared with the high glucose group,pinocembrin increased the survival rate of rats from 42.7% to 85.2%.(4)The detection of BBB permeability showed that both t-PA and hyperglycemia significantly increased EB content in brain tissue,while pinocembrin reduced EB leakage and the inhibition rates to 40% and 47%,respectively,which suggested that pinocembrin protected BBB permeability.In a further study of t-PA-induced BBB injury,it was found that pinocembrin promoted the expression of tight junction protein ZO-1 and Occludin,inhibited the expression of MMP-2 and MMP-9,and thus protected the structural integrity of BBB.Conclusions Pinocembrin can attenuate hemorrhagic transformation induced by t-PA or hyperglycemia,reduce the incidence of HT and mortality.The mechanism is associated with inhibition of MMP-2 and MMP-9 and promotion of ZO-1 and Occludin expression,which protected the structure and permeability of BBB.Therefore,pinocembrin may be a potential therapeutic drug for HT.