The Protective Effect And Mechanism Of Kudiezi Injection On The Blood-brain Barrier Of MCAO Model Rats

BackgroundIschemic stroke and post-ischemia/reperfusion(I/R)induced by thrombolytic therapy result in the blood-brain barrier(BBB)disruption,leading to the development of brain edema and hemorrhagic conversion.Despite the advances in the overall management of acute stroke,the propensity of ischemic brain tissue to develop edema remains the major cause of death within the first few days of stroke in patients with serious herniation.Thus,drugs protected the BBB may be a promising management strategy for treatment of ischemic stroke.The endothelial cells of the BBB are the first-line of the defense between the blood and the brain.The permeability of the endothelial barrier is regulated by two different routes,one is the paracellular pathway,which is through interendothelial junctions,and the other is transcellular pathway which is via caveolae-mediated vesicular transport.Tight junction(TJ)between adjacent endothelial cells plays critical role in the BBB disruption during ischemic stroke.The TJ consists of three types of integral transmembrane proteins:claudins,occludin,and junction adhesion molecules(JAMs),and several cytoplasm accessory proteins including zonula occludens(ZOs),among which claudin-5,occludin,JAM-1 and ZO-1 play a pivotal role in the ischemic BBB injury.Caveolae,50-100 nm plasmalemmal vesicles in the cytoplasm,are important for regulation of a range of endothelial cell functions.Caveolin proteins represent the principal structural proteins of the caveolae.As the principal marker of caveolae,cavelin-1 is required for the caveolae-mediated transcytosis of albumin.Under pathological condition the breakdown of BBB causes the increase in albumin leakage via both paracellular and transcellular pathway.The Kudiezi injection had been widely used in China for treatment dizziness associated with cerebrovascular diseases.Our previous studies have demonstrated that KDZ can reduce cerebral microcirculation disorder and brain injury.However,the effect of KDZ on BBB injury elicited by focal cerebral I/R has not been well explored.ObjectiveThis study was designed to investigate the role of KDZ injedction in BBB disruption after I/R,with particularly addressing the involvement of tight junction and caveolae.Methods1 Establish middle cerebral artery occlusion(MCAO)model:male SD rats,280g,1 hours after middle cerebral artery occlusion for Kudiezi injection 4 ml/Kg,micro-injection pump intravenous administration.24 hours after reperfusion,observing the index.2 ZeaLonga category five scoring method is used to nerve function injury score of model rats,observe the model and drug intervention after the score changes in rats.3 TTC staining observe the change of the area of infarction rats.4 Laser doppler blood flow imaging system to observe the blood flow changes in rats.5 Calculate the dry wet rate,detect the degree of brain edema.6 Albumin leakage was observed using an upright fluorescence microscope7 Evans blue staining to observe the Evans blue leakage rate.8 Western-blot detection is closely connected to the expression of the related proteins and caveolin-1.Results1 Middle cerebral artery occlusion model rats have been varying degrees of nerve function injury symptoms,after KDZ injection administration,the nerve function injury score decreased significantly(P<0.01).2 Middle cerebral artery occlusion model rats with Kudiezi injection after intervention,reduce the infarction area(P<0.01).3 Middle cerebral artery occlusion model rats reduced blood flow,application of Kudiezi injection after intervention,the apparent increase in the rat brain blood(P<0.01).4 Middle cerebral artery occlusion model rats edema is aggravating,Kudiezi injection was used to after the intervention,edema,reduce rats(P<0.01).5 Middle cerebral artery occlusion model rats,the Evans blue leakage increase,but after Kudiezi injection intervention significantly reduced(P<0.01).6 Middle cerebral artery occlusion model rats,the albumin leakage increase,but after Kudiezi injection intervention significantly reduced(P<0.01).7 Western blot analysis showed that protein claudin close connection between endothelial cells after ischemia claudin-5,JAM-1,occludin and ZO-1 obvious degradation,alleviate after Kudiezi injection treatment.ConclusionThe post-treatment with Kudiezi injection significantly reduced BBB permeability and brain edema,which were correlated with preventing the degradation of the tight junction proteins and inhibiting the expression of caveolin-1 in the endothelial cells.These findings provide a novel approach to the treatment of ischemic stroke.