Effect Of Dl-3n-butylphtalid On The The Activity Of MMP-9and The Permeability Of The Blood-brain Barrier On Rats With Focal Cerebral Ischemia/Reperfusion Injury

Background and ObjectiveWith gradually entering the aging society, Ischemic cerebrovascular disease has gradually become a frequent and common diseases to human health.This disease Characterized by high morbidity, recurrence and morbidity rate brought heavy financial and emotional burden to society, families and individuals.So, to explore the effective drug therapy had became the focus of the modern medieal researeh. Cerebral artery occlusion caused by cerebral ischemia,which causes local cerebral blood decrease,cell dysfunction and the form of damage. Immediate resumption of blood flow is the key factor. However, with the restore blood flow reperfusion injury can not be ignored.The so-called ischemia-reperfusion injury which refered to the dysfunction of tissues and organs did not reduce even further aggravated the clinical symptoms after a certain amount of time.According to recent researches, the integrity of the blood-brain barrier play an important role in pathophysiologic process of ischemia reperfusion injury. The blood brain barrier is a complex system between the brain tissue and blood. The blood brain barrier consist of vascular endothelial cells of the central nervous system,which consists of capillary vascular endothelial cells and their tight junction, basement membrane and the astrocytes end foot.The blood brain barrier can control the mass transport between the brain tissue and blood to ensure a relatively stable environment in the central nervous system. Vascular basement membrane is mainly composed of extracellular matrix molecules (ECM),which include the collagen IV, laminin and fibronectin.The ECM is essential to maintain the blood-brain barrier (BBB) integrity.Recent studies substantited matrix metalloproteinases (MMPs), especially MMP-9, which plays a more important role in the opening mechanism of the blood-brain barrier. Given the exogenous inhibitor of MMP-9,the infarct volume of rat can reduce after cerebral ischemia.The over-active expression of MMP-9can damage the blood-brain barrier (BBB) by degradation of the neurovascular matrix, which causes edema and bleeding in brain injury.The main substrate of MMP-9is the collagenⅣ,which is also known as type IV collagenase.As a result,it is of great significance to find the MMP-9,collagen IV for the role of the target and reduces the permeability of the blood-brain barrierand reduce brain injury.Butylphthalide is the active ingredient which also isolated from the celery seeds.which was divided into the1-Butylphthalide(1-NBP),the dextral-Butylphthalide (d-NBP) and racemic-Butylphthalide (dl-NBP).dl-NBP is a yellow oily liquid with a celery flavor,which has been applied to the treatment of ischemic cerebrovascular disease.It can be used to improve the neurological symptoms and promote functional recovery of patients after stroke. However,the molecular mechanisms in the treatment of focal cerebral ischemia and reperfusion injury has not been fully elucidated. Previous studies focus on its role of oxygen free radicals, anti-apoptotic and anti-inflammatory response in the cerebral ischemia reperfusion injury mechanism.But it is not still clear that the activity express of MMP-9, collagen type IV and the permeability of the blood-brain barrier on the brain. In this study,they were observed that the activity of MMP-9, collagen type IV the permeability of BBB and the treatment of Butylphthalide by inserting a thread internal carotid artery.The neuroprotective effects of Butylphthalide were further explored in cerebral ischemia mechanism.Materials and methodsA total of90healthy male Wistar rats serve as subject, weighing240-280g. Reference and improvement ZeaLonga suture method serve as the middle cerebral artery ischemia and reperfusion model(MCAO/R).The rats were randomly divided into three groups which included sham operation group30(sham), ischemia-reperfusion injury group30(MCAO/R) and Butylphthalide treatment group30(NBP). Each group according to the time of reperfusion were devided into three subgrous:6h,12h,24h.each subgroup had10rats.Five of rats were used for immunohistochemical determination and the rest were used for blood-brain barrier permeability.The capsules Butylphthalide were made of10mg/ml with0.5%Tween80.The treatment group was received butylphthalide in5minutes after reperfusion by intraperitoneal injection of80mg/kg. The sham operation group was not administration, the model group was given the corresponding volume of saline (1ml/kg) by the same way.2%Evans blue was injected via the sublingual vein before30minutes at the booking time. The corresponding point the rats were treaed in time.In the damaged brain the expression of MMP-9and collagen IV activity were measured dy immunohistochemical method.The permeability of BBB was observed by the content of brain tissue EB from6h to24h.Statistical analyses were done by SPSS17.0software.All dates are presented as the mean±standard error. Multiple sets of data were compared using one-way ANOVA.Two groups were compared using LSD test. P<0.05was statistically significant.Results1The expression of MMP-9:the expression of MMP-9was lower at different time in the sham operation group. In ischemia-reperfusion group numbers of the MMP-9positive cells had a small amount of expression, constantly increased at12hour and significantly increased at24hour.There was a significant difference between in the sham and ischemia-reperfusion groups(p<0.05).The expression of MMP-9positive cells reduced at butylphthalide-treated6h,12h,24h which compared to the same point of the ischemia-reperfusion group(p<0.05).2The expression of collagenlV:the expression of the BBB and collagen IV were higher in the sham operation group at different time.In ischemia-reperfusion group numbers of the expression of collagen IV decreased slightly at6h,persistly decreased at12h and significantly decreased at24h.There was a significant difference between in the sham operation group and ischemia-reperfusion group(p<0.05).The expression of the collagen IV increased at butylphthalide-treated6h,12h,24h which compared to the same point of the ischemia-reperfusion group(p<0.05).3The content of EB:Rats were injected with Evans Blue,the body rapidly turns blue.In ischemia-reperfusion group brain tissue the content of EB began to increase at6h,continuly increased at12h and reached a peak at24h.The content of EB reduced at butylphthalide-treated6h,12h,24h which compared to the same point of the ischemia-reperfusion group(p<0.05).The permeability of the blood-brain barrier was lower in butylphthalide-treated(p<0.05).Conclusions1、After cerebral ischemia-reperfusion,the expression of MMP-9activity and the content of EB significantly increase while the collagen IV decreases.The conclusions illustrate that MMP-9,collagen IV and BBB relate to brain ischemia-reperfusion injury.2、Butylphthalide may reduce the permeability of BBB by reducing the expression of MMP-9and collagen IV,which may be one of neuroprotective mechanisms on cerebral ischemia-reperfusion injury.