The Effects And Mechanisms Of Nrf2 On Angiogenesis Of Brain Microvascular Endothelial Cells

Backgroud:Cerebrovascular disease is a clinical common disease,which has the characteristics of high prevalence,high fatality rate,high morbidity and high recurrence rate,greatly endangering people’s health.It has become the second cause of death and disability in the world.Ischemic cerebrovascular disease accounts for more than 80%.So it is an important task to explore its pathologic mechanism and effective treatment.Early cerebrovascular recanalization is the key of the treatment.Venous thrombolysis and endovascular treatment have already been applied to clinical treatment.While venous thrombolysis has the shortcoming of the lower rate of cerebrovascular recanalization(only 10%-30%)and short time window(4.5 hours)or less,only less than 3% of patients benefit.Patients have benefit from endovascular treatment represented by machinery thrombectomy,but the technique is only for large intracranial vascular lesions and only for three years.The development of this technique is limited by medical contions,.medical staff technical level and the time window and so on,which can’t benefit the most people.Either way,the aim is to cerebrovascular recanalization a,save the cells of the ischemic semidark zone,promote the regeneration of angiogenesis and the reconstruction of collateral circulation.Therefore,the mechanism and regulatory factors of angiogenesis after ischemic cerebrovascular disease have become hot spots.Angiogenesis means that the proliferation and differentiation of endothelial cells from the existing blood vessel to form new blood vessel in the form of buds or nonbuds.Under normal conditions,angiogenesis is in a state of equilibrium,and abnormal balance can lead to many pathological phenomena,such as cancer,macular degeneration,retinopathy,and ischemic diseases.Angiogenesis is a complex process,need a combination of multiple cells,including endothelial cells,parietal cells,inflammatory cells and blood cells,these cells are involved in cell adhesion,migration,proliferation and differentiation,but also need the stimulation of various cytokines and growth factors,such as vascular endothelial growth factor,placental growth factor,platelet-derived growth factor B,transforming growth factor B and angiogenin,etc.Angiogenesis is closely linked to the process of inflammation,which is the main source of ROS.The functional connection between the inflammation of ROS dependence and angiogenesis plays an important role in many diseases,and oxidative stress plays an important role in the positive feedback mechanism.Oxidative stress is defined as the imbalance of the body’s oxidation system and anti-oxidative system.It is related to the etiology and outcome of many vascular diseases,and regulating oxidative stress can promote angiogenesis and tissue repair.NF-E2 related factor 2(NF-E2-related factor 2,Nrf2)is an important transcription factor in cell oxidative stress,downstream variety of antioxidant genes and its mediated Ⅱ detoxifying enzyme gene transcription and expression,and has been confirmed in oxidative stress disorders especially selective expression in ischemic brain damage rise and play an important role in regulating expression.In addition,Nrf2 can mediate the neovascularization process of hypoxic induced cardiac microvascular endothelial cells,colon and glial carcinoma.Therapeutic angiogenesis plays a crucial role in improving blood supply and recovery in the brain.At present,although there are studies to clearly point out that Nrf2 regulates brain injury related diseases through oxidative stress,there is no report on the regulation effect of Nrf2 on the angiogenesis of cerebral vascular endothelial cells induced by hypoxia.Objective:To observe the effects of Nrf2 on the proliferation,migration and vascularization of cerebral microvascular endothelial cells under hypoxia condition,and further explore the mechanism of Nrf2 on angiogenesis of cerebral microvascular endothelial cells under hypoxia.condition.Methods:The mouse brain microvascular endothelial cell plants,b End.3 cells,was selected.The siRNA and negative control of the Nrf2 gene were synthesized by chemical synthesis of Nrf2 gene,and the effect of interference was detected by Western blot and RT-PCR.Anoxic incubation method was used to establish the anoxic model of the b End.3.cells.The experiment was divided into three groups: anoxic group,Control siRNA group,Nrf2 siRNA group.The wound healing experiment was done to observe the migrate ability of each group b End.3.cells.Cell Counting Kit-8 experiment was done to observe the proliferation ability of each group b End.3.cells.Tube formation experiment was done to observe the blood vessel formation ability of each group b End.3.cells.The expression of VEGF、p-Akt、Akt、ERK、p-ERK、HO-1 and ACTIN protein in each group was detected by Western blot.Using the Graphpad Prism 4.0 software for statistical analysis,all datas were represented by means of the mean number plus or minus standard deviation,and the difference between the Bonferroni post-test analysis group was performed after the single-factor analysis of variance analysis(one-way ANOVA).Results:1.The expression level of Nrf2 was evaluated under hypoxia condition by Western blot and RT-PCR.Nrf2 protein expression in b End.3 cells under hypoxia condition significantly raised on the time 48 h than 24h(P<0.01).Nrf2 m RNA in b End.3 cells increased almost three times on the time 48 h more than 24h(P<0.01).The protein expression and m RNA of Nrf2 were not up-regulated during prolonged exposure to hypoxia.The largest expression appears in 48h(P<0.01).Therefore,Nrf2 expression is expressed in endothelial cells to adapt to the early increase of hypoxia and decrease in the long-term hypoxia response.2.The experimental design and synthesis 3 Nrf2 siRNA sequences(siRNA-1,siRNA-2,siRNA-3)and 1 negative control siRNA sequences,the results show that Nrf2 siRNA-1 inhibit Nrf2 protein and m RNA in the lowest levels,compared with siRNA NC,the inhibition rate reached four times than others(P <0.01).The inhibitory effect of Nrf2 siRNA-1 was confirmed again by Western blot and RT-PCR.Therefore Nrf2 siRNA-1 is used to study the role of Nrf2 in the b End.3 cell.We also found that the protein expression of Nrf2 in Nrf2 siRNA group was significantly lower than that of the siRNA NC group(P<0.01).3.Nrf2 siRNA or siRNA NC was transfected to b End.3 cells and b End.3 cells mobility was measured.Results show that in the Nrf2 siRNA group b End.3 cells migration rate induced under hypoxia condition was significantly reduced compared with siRNA NC group(P<0.01),so Nrf2 can promote brain microvascular endothelial migration.4.Nrf2 siRNA or siRNA NC was transfected to b End.3 cells and b End.3 cells proliferation ability was measured.Results showed that the survival rate of bend.3 cells in Nrf2 siRNA group was significantly reduced,compared with that of the siRNA NC group(P <0.01),so Nrf2 could promote the proliferation of microvascular endothelial cells.5.Nrf2 siRNA or siRNA NC was transfected to b End.3 cells and introduce the Matrigel into a small tube.Results show that Nrf2 siRNA transfection significantly inhibit the formation of tubules,compared with siRNA NC(P < 0.01),suggesting Nrf2 can promote tubule formation.6.In order to clarify the downstream effect of Nrf2,the expression level of VEGF、p-Akt、Akt、ERK、p-ERK、HO-1and ACTIN protein was studied by Western blot.We found that Nrf2 siRNA significantly reduced the expression of VEGF、p-Akt、Akt、 HO-1 protein,compared with siRNA NC(P < 0.01),ERK and P-ERK expression were not affected.It was shown that Nrf2 was in the low-oxygen induced b End.3 cells,through the P13K/AKT signaling pathway,affected the expression of VEGF and HO-1.Conclusion:1.The siRNA transfection effectively inhibits the expression of the b End.3 cell Nrf2.2.The Nrf2 siRNA inhibits the migration ability,proliferation ability and the ability to form blood vessels under hypoxic conditions.These results indicate that Nrf2 has the effect to promote angiogenesis.3.Hypoxia induced the expression of Nrf2 by activating PI3K/Akt signaling pathway,while Nrf2 activated and transferred to the nucleus to promote HO-1 and VEGF transcription and protein expression.