| Objective: Intracerebral hemorrage(ICH)is a common type of stroke,accounting for10%-15%,refers to the intracranial vascular rupture,the blood into the parenchyma process,with the characteristics of high morbidity and high mortality.Hematomas after ICH can compress the healthy brain tissue surrounding,leading to mechanical and secondary injuries,including a variant response,neuronal cell death and neurological deficits.Over the past 10 years,the incidence of ICH has been on the rise,with an 18%increase in admission rates.About 20% of ICH patients experience varying degrees of neurological dysfunction even after surgery.Although efforts have been made clinically to alleviate post-ICH and ICH complications,the results are unsatisfactory.The monolayer of cells formed by brain microvascular endothelial cells(BMECs)is a component of highly differentiated BBB.BMECs,linked by connexins to the basement membrane and together with pericytes,smooth muscle cells,astrocytes,and circulating blood cells make up the so-called neurovascular unit(NVU).So far,many permanent brain endothelial cell lines have been established that can be used as BBB models in vitro.These cell lines have certain advantages over primary BMECs,they grow faster,and maintain stable characteristics over several generations.However,the brain endothelial cell line can not completely replace the primary cells because many important features are different,which lead to unreliable experimental results.Therefore,we explored the method of isolating primary BMECs according to Ruck's method.MiR-126 is encoded by an intron of the epidermal growth factor-like-domain 7(EGFL7)gene on chromosome 9 and has two mature forms,miR-126-3p and miR-126-5p.VCAM1 mediates the adhesion of leucocytes to endothelial cells.Studies have shown that endothelial cells regulate the expression of VCAM1 by regulating the expression of miR-126,which regulates vascular inflammation.There are also studies investigating the regulatory effect of miR-126 on the integrity of the blood-retina barrier(BRB).By acting on VCAM1 and Bcl2-like protein 11,it inhibits retinal endothelial cell apoptosis and reduces retinal vascular leakage and retinal permeability.PIK3R2 is a member of the PI3 K p85 subunit family that inhibits the activation of the PI3 K / Akt pathway and regulates cell proliferation,survival,invasion,metastasis and angiogenesis.PIK3R2 isthe target of miR-126-3p,which can promote the angiogenesis and inhibit the inflammatory reaction of spinal cord injuries in rat.VEGF and Ang-1 play an important role in angiogenesis.VEGF binds to its receptor VEGFR2,whereas Ang-1 promotes the ability of endothelial cells to survive,proliferate and migrate by binding to Tie-2.PI3 K /Akt path plays an important role in the proliferation,survival and migration of cells.In addition,our previous study has found that the expression of miR-126 in serum of ICH patients was significantly down-regulated compared to healthy controls and negatively correlated with the severity of edema around the hematoma.In recent years,the role of miR-126 in maintaining vascular integrity and endothelial cell barrier function has been demonstrated.But the role of miR-126-3p in ICH has not been reported.Although in previous experiments we have found a negative correlation between miR-126 and relative perihematomal edema(rPHE),is miR-126 directly involved in the development of complications such as edema and neuronal injury after ICH? The answer remains unclear.In summary,this study injects miR-126-3p mimic into the ventricle of the rats with ICH induced by collagenase and detects the expression level of target gene(VCAM1 and PIK3R2)to value its effect.We also detect the effect of miR-126-3p in vitro by knocking out miR-126-3p in BMECs.These ways we try to evaluate the potential therapeutic effect of miR-126-3p on ICH and explore its role mechanism.Methods: 1.Animal experiments: Healthy male adult Sprague-Dawley rats(n = 96,250-280g)were randomly divided into 4 groups according to random number table:(1)Sham group(n = 24);(2)ICH group(n = 24);(3)ICH + mi R-NC group(n = 24);(4)ICH + miR-126-3p group(n = 24).ICH rats model was established according to Rosenberg's technique.And then Type VII bacterial collagenase was injected into the right basal ganglia of ICH group,ICH + miR-NC group and ICH + miR-126-3p group(0.5 U in 2 ?l saline,0.4 ?l / Min,5min pump).Sham group animals were injected with the equal volume of saline using the same method in the same place.Twenty-four hours after the collagenase infusion,the rat brain tissue was dissected surgically.If the volume of the hematoma in the rat brain tissue was almost with the same volume after collagenase infusion,and no hematoma breaks into the ventricle,the ICH rat model establishment was considered successful,and with good repeatability.The ICH +miR-NC and ICH + mi R-126-3p groups were injected intraperitoneally with the transfection mixture: miR-NC and 10 ?M miR-126-3p mimic(dissolved in 5 ?l mixture,0.5 ?l / min speed).In the same way,Sham and ICH groups were given the same volume of transfection reagent only.Exam of paw placement and corner tests was used to detect the behavioral defects.The permeability of blood-brain barrier(BBB)was valued by evans blue leakage assay.Brain water content of the brain was measured.Detect the level of miR-126-3p by quantitative real-time PCR and the level of phosphoinositide-3-kinase regulatory subunit 2(PIK3R2),vascular cell adhesion molecule 1(VCAM1),p-Akt and Akt by Western blotting.Immunofluorescence staining,Fluoro-Jade B(FJB)staining and Immunohistochemistry were also involved.2.Cell experiment: Primary rat brain microvascular endothelial cells(BMECs)were isolated and identified by cell slide immunofluorescence staining.After identification of BMECs,they were randomly divided into three groups:(1)Control group;(2)Anti-miR-NC Group;(3)Anti-miR-126-3p group.Anti-mi R-126-3p(100pmol)and anti-miR-NC(100pmol)were introduced into the cells of Anti-miR-126-3p and Anti-miR-NC groups respectively by in vitro transfection reagent.After 24 h,the cells were harvested for real-time fluorescence quantitative PCR to detect miR-126-3p levels in the three groups of cells.BMEC permeability assay was adopted after the transfection.Apoptosis was detected by flow cytometry after treatment of vascular endothelial growth factor(VEGF)and angiopoietin-1(Ang-1).Results: 1.Animal experiments:(1)Intracerebroventricular administration of miR-126-3p mimic significantly alleviated behavioral defects 24 h after ICH.ICH led to increased BBB permeability and cerebral edema,both of which were attenuated by miR-126-3p mimic.(2)Treatment with mi R-126-3p mimic reduced the numbers of myeloperoxidase(MPO)-positive,OX42-positive,FJB-positive and NEUN/TUNEL double-positive cells around the hematoma,implying that miR-126-3p inhibited neutrophil infiltration,microglial activation and neuronal apoptosis following hemorrhage.(3)In addition,miR-126-3p mimic suppressed the upregulation of PIK3R2 and VCAM1 in the perihematomal area and maintained the activation of Akt.2.Cell experiment: Furthermore,in vitro assays confirmed upregulation of PIK3R2 and VCAM1 upon knockdown of miR-126-3p in rat BMECs,and silencing of miR-126-3presulted in impaired BMEC barrier permeability and reversed VEGF-and Ang-1-induced activation of Akt and inhibition of BMEC apoptosis.Conclusion: In summary,our results suggest that exogenous miR-126-3p may alleviate BBB disruption,cerebral edema and neuronal injury following ICH by targeting PIK3R2,VCAM1 and the Akt signaling pathway in brain vascular endothelium. |
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