| BackgroundStroke is one of the major diseases affecting human health,with the characteristics of high incidence,high fatality rate,high disability rate and so on.In China,ischemic stroke accounts for about 70%of stroke.Vascular recanalization and neuroprotection are the main clinical treatment strategies for ischemic stroke.Due to the narrow time window and complex course of the disease,the current treatment methods have their advantages but still have disadvantages.Therefore,a variety of treatment strategies for complex clinical needs are necessary and urgent.It was found that neovascularization was positively correlated with the prognosis of ischemic stroke,indicating that neovascularization plays an important role in the treatment of ischemic stroke.Through angiogenesis and vascular remodeling to increase blood supply and improve ischemic and hypoxic injury in ischemic stroke.Blood flow recovery and revascularization run through the acute,subacute and chronic recovery stages of ischemic stroke.The process of angiogenesis and neovascularization in the acute phase of ischemic stroke can reduce the injury of ischemic penumbra,and promote the recovery of neurological function in the subacute and chronic phase of ischemic stroke.Therefore,angiogenesis and revascularization are the keys to rehabilitation after ischemic strokePinocembrin(PCB)is a natural dihydroflavonoid with good therapeutic effect on ischemic stroke.Through the screening of multiple targets in vivo and in vitro,it was found that PCB has a good effect of dilating blood vessels,improving cerebral blood flow and strong neuroprotective effect.It has a good protective effect on brain tissue injury caused by experimental cerebral ischemia,can reduce the infarct area of brain tissue,improve the behavioral disorder of cerebral ischemia animals,and promote the recovery of brain function in cerebral ischemia animals.It has an extensive inhibitory effect on the injury cascade reaction between vascular system and nerve tissue after cerebral ischemia.It has been approved as a new drug for the treatment of cerebral ischemia by CFDA and is currently in phase Ⅱ clinical trials.Although some studies have found that PCB has a positive effect on the cerebrovascular system,there is no clear conclusion on whether PCB has the effect of promoting angiogenesis.Objective:Based on the above research background,this study took ischemic stroke as the research object to establish in vitro and in vivo models to explore whether PCB has the effect of promoting angiogenesis and to explore its possible mechanismMethods:Firstly,MTT test,scratch test,tubule formation test and oxygen-glucose deprivation/reoxygenation(OGD/R)model were applied to evaluate the effects of PCB on proliferation,migration,tubule formation and anti-hypoxia-hypoglycemia ability of human brain microvascular endothelial cells(hBMECs).Then the chick embryo chorioallantoic membrane model was used to investigate whether PCB could promote angiogenesis in vivo.Then the middle artery embolization model(MCAO)was used to evaluate the effects of PCB on neurobehavioral score,microvessel density in ischemic penumbra and cerebral microblood flow in rats,to investigate whether PCB had an angiogenic effect on the disease model.Finally,the effect of PCB on the expression of vascular growth-related factors was determined by Western Blot technique,to explore its possible mechanism.Results:1.Pinocembrin has the effect of promoting angiogenesis in vitro.The results of MTT experiment showed that PCB 5 to 150 μM could significantly promote the proliferation of hBMECs in a dose-dependent and time-dependent manner for 24 hours and 48 hours.Scratch test results showed that PCB 5 to 80 μM can significantly promote hBMECs migration for 24 hours,and the migration rate is about 40%to 50%,in a dose-dependent manner.The migration-promoting effect of various concentrations of PCB is equal to the positive drug(bFGF 20 μg/mL).The results of tubule formation experiment showed that the values of tubule branches and the total length of tubule branches in PCB 80 μM group(202±37 and 12239±2425)were significantly higher than those in control group(130±23 and 8048±2000).It showed that PCB could promote tubule formation in hBNECs.The OGD/R model was applied to simulate the ischemia-reperfusion process.The results showed that after 2 hours of oxygen and glucose deprivation,PCB intervention for 12 hours could significantly improve the survival rate of hBMECs,and in a dose-dependent manner.The effect of PCB 80 μM is equal to the positive drug(bFGF 20 μg/mL).These above results show that PCB 5 to 80μM can improve the proliferation,migration,and tubule formation of hBMECs,and enhance the anti-hypoxia and hypoglycemia ability of hBMECs,which indicates that PCB can promote angiogenesis in vitro.2.Pinocembrin can promote angiogenesis on chick embryo chorioallantoic membrane.The chick embryo chorioallantoic membrane model was used to investigate whether PCB can promote angiogenesis in vivo.After fenestration administration of 6-day-old chicken embryos,PCB 50 and 100 μM intervention for 3 days could significantly increase the number of blood vessels on the chorioallantoic membrane and promote vascular branches in a dose-dependent manner.These results suggest that PCB can promote angiogenesis in vivo.3.Pinocembrin can promote neovascularization of brain tissue in rats with acute cerebral ischemia-reperfusion.The MCAO model of rats was made by thread occlusion method.After 1.5 hours of ischemia,the thread was removed and the cerebral blood flow was reperfused.2 hours after the operation,PCB(5mg/kg)was injected into the tail vein of the administration groups,and the same volume of normal saline was injected into the tail vein of the model groups and the sham operation groups,once a day until sampling.The neurobehavioral scores of rats were evaluated by Zea-Longa’s score on the 3rd,7th,and 14th day after the operation.The results showed that PCB could significantly reduce the neurobehavioral score of rats on the 3rd,7th and 14th day after the operation,and in a certain time-dependent manner,indicating that PCB can reduce the injury of the nervous system and promote the recovery of the nervous system of rats.The percentage of cerebral infarction volume was determined by TTC staining on the 7th day after the operation.The results showed that PCB could significantly reduce the percentage of cerebral infarction volume,increase the body weight of rats and promote the functional recovery of rats.The microvessel density in the ischemic penumbra of rats was detected by immunofluorescence staining on the 3rd,7th and 14th day after the operation.The results showed that the intervention of PCB for 7 and 14 days could significantly promote the microvascular neovascularization and maturation in the ischemic penumbra,increase the microvessel density and save the function of the ischemic penumbra in a certain time-dependent manner.The changes of cerebral microblood flow in rats were measured by laser Doppler flow meter before the operation,immediately after the operation,3rd,7th and 14th day after the operation.The results showed that PCB could significantly increase the cerebral blood flow on the 7th and 14th day after cerebral infarction and restore the blood supply in some areas.These above results suggest that PCB can promote the neovascularization of cerebral tissue and increase the blood supply of cerebral infarction in MCAO rats.4.Pinocembrin may promote cerebral angiogenesis by activating HIF-1α/Ang-1/Tie-2 and HIF-1α/VEGFA/VEGFR2 signal axes.Western Blot technique was adopted to determine the effect of PCB on the expression of angiogenesis-related proteins in cells and cerebral cortex of rats.The results showed that PCB 40 and 80 μM intervention for 12 hours significantly increased the expression of Ang-1 and Tie-2 protein in OGD/R model of hBMECs.In animal experiments,the expression of Ang-1 protein was significantly up-regulated by PCB on the 3rd day after the operation,indicating that PCB could up-regulate the expression of some angiogenesis-related proteins at the early stage of ischemic stroke.PCB significantly up-regulated the expression of VEGFR2,Tie-2,Ang-1 protein and the expression of upstream regulatory factor Hypoxia-inducible factor-1α(HIF-1α)on the 7th and 14th day after the operation.It is suggested that PCB still has an effect on the expression of angiogenesis related proteins in subacute and chronic recovery stages of ischemic stroke.PCB may play a role in promoting angiogenesis by activating HIF-1α/Ang-1/Tie-2 and HIF-1α/VEGFA/VEGFR2 signal axes.Conclusion:Overall,PCB can promote angiogenesis in vivo and in vitro,improve the proliferation,migration,and tubule formation of hBMECs,improve the survival rate of endothelial cells after glucose and oxygen deprivation injury,and promote angiogenesis on the chick embryo chorioallantoic membrane.In rat MCAO model,PCB can promote angiogenesis by upregulating the expression of VEGFA,VEGFR2,Ang-1,and Tie-2 protein through activating HIF-1α protein.Thus,increase the microvessel density in the ischemic penumbra of MCAO rats,increase the blood supply of cerebral infarction area,and reduce the volume of cerebral infarction.This study systematically confirmed that PCB can promote angiogenesis in vivo and in vitro,and preliminarily explored its mechanism,which provides a more theoretical basis for its clinical trials. |
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