| Objective:Cerebral ischemia reperfusion injury（CIRI）is composed of the primary injury caused by cerebral ischemia and hypoxia and the secondary injury after restoring blood.CIRI injury stimulation can activate the degradation of vascular basement membrane through hypoxia inducible transcription factors（HIFs）,the proliferation and migration of brain microvascular endothelial cells（BMEC）,induce angiogenesis and play a ompensatory effect[1].Howener,this endogenous repair effect is limited,and the new blood vessels are prone to vascular leakage due to their immature structure and high permeability,which increases the risk of cerebral hemorrhage and cerebral edema[2].Therefore,promoting angiogenesis and maturation of new blood vessels after CIRI to improve the compensation and provide nutritional support for neurons is an important part of nerve repair after CIRI.Our previous research found that the p-methoxybenzyl alcohol,4-hydroxybenzaldehyde,3,4-dihydroxybenzaldehyde（hereinafter referred to as“3#、4#、8#）of Gastrodia elata Blume can increase the density and increase of cerebral cortex microvessel density and increase in middle cerebral artery occlusion/reperfusion（MCAO/R）injury model rats Cerebral blood flow improves nerve function,and 3#can up-regulate the expression of Smad-3 m RNA.Smad-3 is an important downstream protein of TGF-β1/Smad,wnich promotes the maturation of neovascularization[3].It is suggested that the phenolic component has the effect of promoting the maturation of new blood vessels,and its mechanism of action may be mediated by the TGF-β1/Smad signal pathway.TGF-β1 secreted by BMEC can stimulate smooth muscle cell,SMC)to secrete fibronectin and promote its proliferation through TGF-β1/Smad signaling pathway,and recruit mesenchymal stem cells,MSC)to gather in injured tissues,thus improving the coverage of pericytes（PC）,reducing vascular distortion and bleeding,and promoting the maturation of new blood vessels.To further clarify the mechanism of 3#,4#,8#,which promoting neovascularization was related to the regulation of TGF-β1/Smad signal pathway.This study replicated the rat MCAO/R model in vivo and replicated rat BMEC oxygen-glucose deprivation/reoxygenation（OGD/R）model in vitro,from animal,cell,gene,and protein levels revealed the regulation of 3#,4#and 8#on Smad-3 and related factors,in order to clarify the the mechanism of phenolic components of Gastrodia elata Blume promoting the maturation of neovascularization after CIRI.Method:1.The regulation of phenolic components of Gastrodia elata on the expression of TGF-β1,ALK5,Smad-2 and Smad-3 in brain tissue of MCAO/R model rats（1）The MCAO/R model of model rats was replicated by the thread embolization method,and the changes in cerebral blood flow（CBF）were monitored by laser speckle imaging（LSI）,and the neurological score（Longa 5 Sub-method）was used to evaluate the stability of the MCAO/R model.After 2 hours of ischemia,CBF decreased to 42～53%,CBF recovered to more than 70%after reperfusion,and neurological scores of 1-3 were included in the experiment.The successfully modeled rats were randomly divided into model group,Butylphthalidle（NBP）group and test substance 3#,4#,8#group.NBP group and test substance group were given NBP（80mg/kg）,3#（20mg/kg）,4#（20mg/kg）,8#（10mg/kg）for the first time 6 hours after MCAO/R,until reperfusion 7d,14d,28d after;sham operation group and model group were given intragastrically equal volume of solvent.The experiment was divided into three batches of 7d,14d,and 28d for index detection.（2）Take the injured cerebral cortex tissue of the rat,and use Western Blot（WB）to detect the brain tissue TGF-β1,Activin receptor-like kinase 5（ALK5）,PAI1,Smad-2,the expression of Smad-3,observe the regulation effect of 3#,4#,8#on the TGF-β1/Smad signal pathway from the overall level.2.Study on the regulation effect of phenolic components of Gastrodia elata on the expression of m RNA of TGF-β1,ALK5,Smad-2 and Smad-3 in BMEC OGD/R modelAccording to the results of the first part,the intervention of 3#,4#on 7d,14d,28d can obviously promote the expression of ALK5,Smad-2 and Smad-3 in the brain tissue of MCAO/R model.In this part,the regulation of 3#and 4#on TGF-β1、ALK5、Smad-2、Smad-3 m RNA was further investigated from the cellular level.（1）OGD/R model replication:in vitro extraction,separation and purification to obtain primary BMEC.A three-gas incubator was used to replicate the BMEC hypoglycemia and hypoxia 6h compound glucose and reoxygenation 4h injury model.The CCK-8 method was used to check the cell viability,and the cell survival rate was reduced to 50%,which was the criterion for successful model building.（2）Divide BMEC into 8 groups randomly,namely:normal group,model group,test substance 3#high(5×10-4mol/L),medium(5×10-5mol/L),low(5×10-6mol/L)dose group,test substance 4#high(5×10-4mol/L),medium(5×10-5mol/L),low(5×10-6mol/L)dose group.After 6 hours of hypoglycemia and hypoxia,normal media containing different doses of test substances were given for 4 hours of reoxygenation and glucose recombination.（3）Collect the cells and use the Polymerase Chain Reaction（PCR）method to detect the m RNA expression of TGF-β1,ALK5,Smad-2,and Smad-3 in the cells,and observe the gastrodinol component 3#from the cell level,4#Regulates the TGF-β1/Smad signal pathway.Results:1.The regulation of phenolic components of Gastrodia elata on the expression of TGF-β1,ALK5,Smad-2 and Smad-3 in brain tissue of MCAO/R model rats（1）Compared with the sham operation group:7d model group TGF-β1 protein expression increased（P<0.05）,Smad-3 protein expression decreased（P<0.001）;14d model group TGF-β1 protein expression increased（P<0.05）,Smad-2,Smad-3 protein expression decreased（P<0.05,P<0.001）;28d model group TGF-β1 protein expression increased（P<0.001）,Smad-3 protein expression decreased（P<0.01）.（2）3#:Compared with the model group,the expression of Smad-3 protein in the brain of MCAO/R model rats increased（P<0.001）after 7 days of intervention;the expression of ALK5 protein in the brain of MCAO/R model rats after 14 days of intervention Increase（P<0.01）.（3）4#:Compared with the model group,the expression of Smad-2 and Smad-3protein in the brain of MCAO/R model rats increased for 7d after intervention（P<0.001,P<0.05）;after 14d intervention,The expression of ALK5,Smad-2 increased（P<0.05）;after 28 days of intervention,the expression of ALK5 and Smad-3 increased（P<0.05）.（4）8#:Intervention for 7d,14d,28d.Compared with the model group,there was no effect of increasing the brain TGF-β1,ALK5,Smad-2,Smad-3 protein of MCAO/R model rats.2.Study on the regulation effect of phenolic components of Gastrodia elata on the expression of m RNA of TGF-β1,ALK5,Smad-2 and Smad-3 in BMEC OGD/R model（1）Using CCK-8 method to detect the replication of OGD/R model:the survival rate of BMEC decreased after OGD 6h/R 4h of（P<0.01）.（2）Compared with the normal group:the expression of TGF-β1,ALK5,Smad-2,Smad-3 m RNA in the model group increased（P<0.05,P<0.01）.（3）3#:Compared with the model group,the expression of ALK5 m RNA in the middle dose(5×10-6mol/L)group（P<0.05）,and the Smad-2 in the high dose(5×10-4mol/L)group.The expression of Smad-3 m RNA increased（P<0.01）.（4）4#:Compared with the model group,the expression of Smad-3 m RNA in the low-dose(5×10-6mol/L)group increased（P<0.01）,and the middle-dose(5×10-5mol/L)group TGF-β1 m RNA expression decreased（P<0.01）,high dose(5×10-4mol/L)group TGF-β1 m RNA expression decreased（P<0.05）,Smad-3 m RNA expression increased（P<0.01）.Conclusion:The mechanism of action of p-methoxybenzyl alcohol、4-hydroxybenzaldehyde in promoting vascular maturation may be related to the up-regulation of ALK5,Smad-2,and Smad-3 factors in the TGF-β1/Smad signaling pathway mediated by BMEC. |
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