Effect Of HXSLF On Angiogenesis Of Focal Cerebral Ischemia Reperfusion Injury Rats And The Mechanisms Study

The Study showed that Vascular proliferation emerge in ischemic region after vascular occlusion, the scope and extent of hyperplasia was related to the improving of semidarkness blood current and the impacting on physiological functions of neurons recovery. Accordingly, it requires to promote therapeutic angiogenesis to save the impaired brain tissue, in addition to the protection of ischemic region capillary vessel. Angiogenesis can be regulated by a series of vascular growth factor after cerebral ischemia ,which includes specific endothelial cell vascular ndothelial growth factor (VEGF) family, angiopoietin (Ang) family and so on.VEGF/VEGFR system may play a key role in switching on angiogenesis,and be the central link in the regulation angiogenesis of the whole cerebral ischemia, yet Ang and its receptor family is considered the most important vascular stability factor,they restrain the multiplication of the vascular endothelial system (ECS).Cerebral ischemia and reperfusion can lead to cerebral microvascular endothelial cell function impaired, its permeability significantly increased, blood brain barrier is seriously damaged,which is one of the most common reason caused by cerebral edema.So the study of drugs and methods play a protective role on cerebral microvascular endothelial cells has important clinical significance.Ischemic cerebrovaseular diseases belong to"apoplexy,"'stroke',in Traditional Chinese Medicine theories, We consider the pathogenesis of ischemic cerebrovascular disease is impatency of the arteries and veins ,which inducing the blood lost in the nutrition,thus we put forward hexueshengluo Method, an opinion of stroke treatment. On the basis of Yang Rong Tang Ginseng, hexueshengluofang( HXSLF) achieved good results in the clinical treatment of ischemic cerebrovascular disease .Objectives:the aim was to study the effect of Hexueshengluofaing on angiogenesis of focal cerebral ischemia Reperfusion Injury rats and its mechanism of angiogenesis,the expression of VEGF,Ang-1 neurologieal functional deficiency score and water content of brain tissue were observed. At the same time, we study the effect of HXSLF on activity and expression of VEGFmRNA cultured rat cerebral microvascular endothelial cell with hypoxia to investigate the mechanisms of Hexueshengluo Method,so we could offer some theorical grounds for the treatment of cerebral infarction.Methods:The content of this study were divided into four partsPart 1:The ffect of HXSLF on the Expression of MVD and VEGF in Rat with Focal Cerebral Ischemia and Reperfusion InjuryHealthy rats in Wistar,the weight of 250-300 gs, were randomly divided into normal group, sham-operated group, model group, HXSLF group.The latter two groups would be prepared MCAO/IR model by using of suture.Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (350mg/kg body weight) , fixed in the operating table in supine, the neck slices exact center,separating and exposing the right common carotid artery (CCA)and external carotid artery(ECA), the root and branch of ECA and CCA were ligated,the CCA of the right side were sheared,inserting the nylon fish line head to burn a bluntness that diameter is the nylon fish line of the 0.2 mms,entering the line length about 18±0.5 mms. then the right CCA and the nylon fish line were ligated together,sewed up the skin. Then pull out nylon fish line after 2h, the IR model were set up. The rats of sham operation group were set separate and exposed the vessel only, not be ligated the total carotid artery and outside carotid artery, not be inserted the nylon fish line.The neurological scores were made on Zea Longa 5-point scale·: 0 points :No symptoms of nerve injury;1 points the rat Can not fully extend the contralateral forepaw 2 points the rat turns circle to the flank3 points the rat Inclines to the opposite side4 points the rat c annot walk spontaneously, consciousness losesThe rat which has 1~3 points was qualified,and entered the experiment. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate , the chest was opened, the cardiac was poured into 0.9% physiological saline and perfusion fixed by 4% paraformaldehyde. Brain tissue was stripped ,coronal slice line was done from the optic chiasm to the department .The paraffin sections, conventional dewaxing, ethanol gradient dehydration, hematoxylin - eosin staining, xylene transparent, neutral rubber mount, light microscopy.Immunohistochemistry steps: using PV staining, the staining steps carried out according to specification, 8 factor, VEGF concentrations were 1:100, DAB color.The cytoplasm of positive cell had brown or tan particles.Determination of MVD:Any brown stained endothelial cell or a single family of endothelial cells was considered a vascular count.We calculate the number of microvessels in each of 1 mm2 area in accordance with Weidener count method,that is microvessel density, and then calculate the average.The count data was expressed by means±standard deviation(±s),and it were statisticed using the SPSS for Windows 10.0 software processing,The"q"examination was adoptted when comparing between two groups.part 2 The ffect of HXSLF on the Expression of Ang1 and its reception in Rat with Focal Cerebral Ischemia and Reperfusion InjuryThe dividing groups,the method of model replications and the steps of Immunohistochemistry were same to above.Part 3 The effects of HXSLF on activity and expression of VEGFmRNA cultured rat cerebral microvascular endothelial cell with hypoxiaThe cultivation of Rat cerebral microvascular endothelial cell (CMVEC) The brain of 1 week SD rats were removed under sterile conditions,then added it to D hank's cooling fluid, strip leptomeningeal , large vessels and the brain medulla, the cerebral cortex was cut into small pieces of 1mm3 with ophthalmic shear.Then Homogenate it in the homogenizer , the homogenate was filtered through 100 mesh and 200 mesh nylon net filter, Collected the capillary vessel on the 200 mesh nylon net.The capillary vessel was digested by 0.2% collagenase 20minute at 37℃, was suspension using DMEM after centrifugal precipitation , was vaccinated in culture bottle with gelatin pre-coated ,then was static cultured in incubator Containing 5% CO2This experimental used 3rd generation of cell, The cells were divided into normal group and hypoxia group, The cells were incubated at anaerobic incubator to establish the hypoxia models. The cytoactive of RCMEC was determined by MTT. The level of expression of VEGF mRNA was measured by reverse ranscriptase Polymerase chainreaction(RT-PCR)Part 4: The effect of HXSLF on neurological scores and cerebral water volum in Rat with Focal Cerebral Ischemia and Reperfusion InjuryThe neurological scores were made on Zea Longa 5-point scale·.After the models were made,we did the nerve function mark by Longa 5point.Determination of brain water content: the rat brain was taken before cross-lateral organizations on operated side, the wet weight was determinated,then dry weight were measured after oven at 90℃to constant weight. Brain water content was caculated by(wet weight-dry weight)/wet weight×100%Results:Part 1:The ffect of HXSLF on the Expression of MVD and VEGF in Rat with Focal Cerebral Ischemia and Reperfusion Injury1 The effect of HXSLF on the Expression of MVD in Rat with Focal Cerebral Ischemia and Reperfusion InjuryThere was no diference on the expression of MVD between normal group and sham-operated group . A lot of microvessel proliferations in the HXSLF group , the MVD in the HXSLF group was significantly higher than that of the model group.2 The ffect of HXSLF on the expression of VEGF in Rat with Focal Cerebral Ischemia and Reperfusion InjuryThere were little expression of VEGF in normal group and Sham-operated group.There were little expression of VEGF in model group at 1d.Compared with the normal group, the number of VEGF-positive cells gradually increased after 3d,the positive expression in HXSLF group was remarkable higher than that of model group.The number of positive cells reached a peak on 7d, the most marked upregulation occurred in gliocytes,neurons and vascular endothelial cells in the infarct and the border zones in HXSLF group. The positive expression of VEGF in model group and HXSLF group began to decline on 14d, and had not resume to normal levels on 21d.Part 2: The effect of HXSLF on Ang1 and Tie-2 in Rat with Focal Cerebral Ischemia and Reperfusion InjuryThere were little expression of Ang1 in normal group and Sham-operated group.The expression of Ang-1 was weak in model group,in the first day.Compared with the normal group, the number of Ang-1-positive cells in model group gradually increased after 3ds.Compared with the model group, the number of Ang-1-positive cells HXSLF group obviously increased ,reaching peak at 14ds.The expression features of Tie-2 was similar to Ang-1.Part 3: The effects of HXSLF on activity and expression of VEGFmRNA cultured rat cerebral microvascular endothelial cell with hypox1 The Effect of HXSLF on cytoactive in Cultivated Rat Brain Microvessel Endothelia cell with hypoxiaThe activity of CMVEC in the model groups are obviously lower than that in the normal groups (P<0.01).The activity of CMVEC in HXSLF low and moderrate dosage groups is higher than that in the model groups(P<0.05). The activity of CMVEC in the high dosage group of HXSLF was higher than control group remarkedly (P<0.01).2 The Effect of HXSLF on VEGFmRNA in cultured rat cerebralm icrovascular endothelial cell with hypoxiaCompared with the normal groups, the expression of VEGFmRNA augmented obviously in the model groups;compared with the model groups, the expression of VEGFmRNA augmented in the hexueshengluo low dosage, moderrate dosage and high dosage .Part 4: The effect of HXSLF on neurological scores and cerebral water volum in Rat with Focal Cerebral Ischemia and Reperfusion Injury1 The effect of HXSLF on General condition of focal cerebral ischemia ratsHXSLF method could improve the general condition of focal cerebral ischemia mouse obviously ;It could improve their condition obviously and so on mouse's activity,superficial glossiness,diet.2 The effect of HXSLF on neurologieal functional deficiency score of focal cerebral ischemia ratsThe neurological scores in normal groups and sham operated groups were normal (0 mark),and the neurological scores in model groups were higher than 2 marks.The neurological scores in HXSLF groups decreased on 7 days compared with sham operated group.Water content of brain in Model group was significantly increased at 1 day, compared with sham-operated group and the normal group.(P<0.05,P<0.01)3 The effect of HXSLF on water content of focal cerebral ischemia ratsThe water content was the highest on the 3 day, it had not returned to normal on the 7day.The brain water content decreased significantly in HXSLF Group at 3d and 7d, compared with the model group(P<.05, P <0.01).Compared with the normal group there was no significant difference of the brain water content in the sham operated group, indicating that brain surgery itself had no significant impact on water content.Conclusions:1 HXSLF could increase the number of blood vessels and promote expression of VEGF in cerebral ischemia in the surrounding area , the number of positive cells reached a peak on 7d and the most marked upregulation occurred in gliocytes,neurons and vascular endothelial cells in the ischemic area2 HXSLF could increase expression of Ang-1 and Tie-2 in ischemic area , the strongest expression appears at 14 days which play an important role in the late angiogenesis .3 HXSLF could increase activity and expression of VEGFmRNA of hypoxic cells ,Which may be one of mechanisms of neuroprotective effects of HXSLF on cerebral ischemia一reperfusion injury.4 HXSLF could improve nerve function score, reduce the brain water content in brain tissue of Focal cerebral ischemia rats,inhibite the peak of the formation of brain edema.and reduce ischemic brain injury in effect. The results showed HXSLF possessed protective effect against cerebral ischemia-reperfusion injury.