Study On Mechanism Of Ligustici And Astragali Active Ingredient On Blood-Brain Barrier And Protein Z0-1,OCLN After Thrombolysis Therapy In Rats

BackgroundScholars pay more attention about acute ischemic stroke because of its'high incidence,high morbidity,high mortality and neurological-deficit refractory.From the 1990s,after 10 years of multi-national,multi-center study,thrombolysis has been great advanced,but it is still the international discussion on key issues.Thrombolytic therapy which is an effective method for treatment of acute cerebral infarction confirmed by the only evidence-based medicine,but easily lead to many serious complications,this is greatly limiting its clinical application.Modern research has confirmed that the increased blood-brain barrier(BBB)permeability and destructed of the integrity can cause HT after thrombolytic therapy.Tight junctions(TJ)is an important factor in maintaining the BBB integrity,composed of transmembrane proteins and membrane-associated protein,whose function is to connect two adjacent cells,and closed the gap between the cells,blocking extracellular macromolecules and involved in cell growth and differentiation signal transfer through the intercellular space into the tissue.Molecular biology studies confirmed the TJ is the central link in the regulation of BBB permeability,ZO-1 and Occludin are the major structural protein of TJ,the expression level' s change closely related to the cerebral microvascular permeability and cerebral edema.Astragalus and Ligustici are often used Yiqihuoxue drug,modern studies have shown that they can have a significant protective effect on the blood-brain barrier,but in the role of TJ has not been reported.In this study,we explores the permeability changes of BBB at different times after thrombolysis and the effect of Ligustrazine Injection andAstragalus Injection in BBB permeability.Then apply the morphological,molecular biology,and other methods to prove the mechanism,and provide a theoretical basis for clinical prevention and treatment after thrombolysis.Methods1.To establish a simple method to improve the rat model of acute middle cerebral artery embolic stroke used in thrombolysis:Rats were anesthetized,and the right common carotid artery(CCA)?the right internal carotid artery(ICA)?the right external carotid artery(ECA)were exposed through a midline neck incision.Used a injector to draw 0.1ml arterial blood from the rat' s artery where can be easy hemostasis by ligation(such as the femoral artery),then inhaled 4U thrombin,blended them and set aside for 10 minutes.Injected to a 24G venous retention needle until the blood coagulation to make a about 1cm emboli,and the followed by a 1ml saline syringe.In the process of making emboli,taked the attention that does not make the venous retention needle into the air.Then the venous retention needle was inserted through an arteriotomy of the common carotid artery and gently advanced into the internal carotid artery to a point approximately 10 mm distal to the carotid bifurcation(forward medial to avoid pterygopalatine artery),pulled out the pin core,and pushed the emboli fast into ICA with a containing physiological saline syringe,after which the common carotid artery was ligated and the neck wound was rapidly closed.2.Animal grouping and Administration:rats were randomly divided into sham-operated group,thrombolysis group,Ligustici group,Astragalus group and combination group.Except the sham-operated group each time point according to the different execution(after thrombolysis 3,6,24 h)group.Except the sham-operated group groups femoral vein given rt-PA after building the model 3 hours,the sham-operated group was given equal volumes of saline.In addition,Ligustici group intraperitoneal injection the ligustrazine injection;Astragalus group intraperitoneal injection the Astragalus Injection;combination group injection the two injections.3.The BBB permeability was observed by measuring Evans blue(EB)transport across the BBB,and transmission electron microscopy to observe the changes of the blood-brain barrier permeability.4.Immunofluorescence method for the determination of protein expression and distribution of tight junction associated protein of Z0-1 and Occludin in ischemic brain tissue.5.Use western-blot to detect Z0-1 and Occludin.6.RT-PCR,immunohistochemisty,Western blot technique were used toexplore mRNA and protein expression and distribution of tight junction associated protein of Z0-1 and Occludin in ischemic brain tissue,with or without H7.ResultsPart 1:1.The comparison of EB at different time:The EB content of thrombolysis group was the highest,and it continued to increase with time pass by.Ligustrazine injection and Astragalus injection could decrease the EB content respectively at the 3h after thrombolysis.The combination of Ligustrazine injection and Astragalus injection could decrease the EB content at all time points after thrombolysis.There were no distinct statistical difference between Ligustici group and Astragalus group at any time point after thrombolysis.2.Immunofluorescence of Z0-1 and Occludin:The expression of Z0-1 and Occludin were normal and along the vascular endothelium in sham-operated group;The expression of Z0-1 and Occludin were decreased in thrombolysis group;the treatment groups' protein expression were better than thrombolysis group.3.The western-blot result of Z0-1 and Occludin:The Z0-1 and Occludin content of thrombolysis group was the lowest in each time point after thrombolysis,and it continued to decrease with time pass by.There were varying degrees of exaltation of Z0-1 and Occludin content in Ligustici group and Astragalus group.The Z0-1 and Occludin content in combined therapy group was higher than anyother groups at all time points after thrombolysis,the differences were significant.There were no distinct statistical difference between Ligustici group and Astragalus group at any time point after thrombolysis.Part 2:1.RT-PCR of PKCa,Z0-1 and Occludin:The Z0-1 mRNA content of thrombolysis group was the Highest;The PKCa mRNA content of thrombolysis group was the lowest;Occludin mRNA content of thrombolysis group is higher than cure and cure add H7 groups;PKC a mRNA content of cure add H7 is lower than H7 group and cure groups.2.Immunofluorescence of ZO-1 and Occludin and PKC?:The expression of ZO-1,Occludin and PKC? were normal and along the vascular endothelium in sham-operated group;The expression of ZO-1 and Occludin were decreased in thrombolysis group while PKC a is increased;the expression of ZO-1,Occludin in H7 group,cure group and H7 add cure group is increased while PKC? is decreased?3.The western-blot result of ZO-1 and Occludin:sham-operated group is different with other groups;The PKC ? content of thrombolysis group was the highest while ZO-1 and Occludin were lowest;PKC a content of thrombolysis group is lower than cure and cure add H7 groups.Conclusion1.Ligustrazine injection and Astragalus Injection can significantly reduce the blood-brain barrier permeability after thrombolysis,reduce thrombolysis damage on the tight junction ZO-1 and Occludin.2.Ligustrazine injection and Astragalus Injection can significantly reduce the blood-brain barrier permeability by restrain PKC pathway.