| Objective: 1.To investigate the influences of HIF-1α augment on angiogenesis and endothelial cell tight conjuction in the premature infant brain injury model by measuring the expression of VEGFR2 and Claudin-5.2.To investigate the relativity between the TGF-β1 and HIF-1α on microvascular barrier in the premature infant brain injury model.Methods: To establish a premature brain injury model(white matter damage,WMD),3-day-old postnatal SD rats were subjected to left common carotid artery ligation and hypoxic ventilation(6.0% O2,2h).The model rats were randomly divided into three groups:(a)normal saline(NS)group(group Ⅰ)(b)HIF-1α group(group Ⅱ)(c)HIF-1α + TGF-β1 inhibitor group(group Ⅲ).In the HIF-1α group,FG-4592 was injected into the lateral ventricle 24 hours,which is a prolyl hydroxylase inhibitor(PHI)that inhibits the degradation of HIF-1α and increases the expression of HIF-1α in vivo.HIF-1α +TGF-β1 inhibitor group was injected with PHI and TGF-β1 inhibitor.The NS group was injected with the same amount of normal saline.Brain tissues were taken at day 1,3,7 and 14 after lateral cerebral ventricle injection.q RT-PCR and western-blot were used to detect the expression of VEGFR2,claudin-5 and TGF-β1 in brain tissue at each time point.Immunofluorescence staining of brain tissue sections was performed to observe the expression of VEGFR2.Results: 1.The effect of HIF-1α on the VEGFR2,Claudin-5 m RNA and protein expression in the premature brain injury model.The m RNA expression levels of VEGFR2 were compared in group Ⅱ and group Ⅰ,and on day 3 after the injection,group Ⅱ was higher than group Ⅰ(P<0.001).On day 7 and 14,group Ⅱ was significantly higher than group Ⅰ(P<0.0001).On day 7 and 14,the m RNA expression of Claudin-5 in group Ⅱ was significantly higher than that in group Ⅰ(P<0.0001).On day 3 and 14,the expression of VEGFR2 protein in group Ⅱ was higher than that in group Ⅰ(P<0.01).On day 3,7 and 14,the expression of Claudin-5 protein in group Ⅱ was higher than that in group Ⅰ(P<0.05).The results of immunofluorescence show that VEGFR2 expression in HIF-1α group is higher than that in NS group.2.Role of TGF-β1 in the effect of HIF-1α on microvascular barrier after brain injury in preterm infants(a)The expression of TGF-β1 in brain tissue of each group at different time points: The relative expression levels of TGF-β1 m RNA in group Ⅲ and II were compared.On day 1,group Ⅲ was slightly higher than group Ⅱ(P<0.05).On the 7th day,group Ⅲ was significantly lower than group Ⅱ(P<0.0001);on the 14 th day,group Ⅲ was still lower than group Ⅱ(P<0.01).The expression of TGF-β1 protein were compared in group Ⅲ and group Ⅱ,on the 3rd and 14 th day,group Ⅲ was lower than that of group Ⅱ(P<0.01).(b)The effect of TGF-β1 inhibition on the expression of VEGFR2 and Claudin-5 in brain tissue The m RNA expression level of VEGFR2 in group Ⅲ was higher than that in group Ⅱ on the first day(P<0.01).On day 3,group Ⅲ was still slightly higher than group Ⅱ(P<0.05).On day 7,the m RNA expression of VEGFR2 in group Ⅲ was lower than that in group Ⅱ(P<0.01).On the 14 th day,group Ⅲ was significantly lower than group Ⅱ(P<0.0001).The relative m RNA expression of Claudin-5 in group Ⅲ was lower than that in group Ⅱ on the 7th and 14 th day(on day 7,P<0.01.on day 14,P<0.0001).The expression of VEGFR2 protein in group Ⅲ was lower than that in group Ⅱ on the 7th and 14 th days(P<0.01).Compared with Group II,Claudin-5 protein expression was lower in Group III than Group II on Days 3,7,and 14(P<0.05 on day 3,P<0.01 on day 7,P<0.001 on day 14).On day 1,the fluorescence intensity of VEGFR2 in group Ⅲ was stronger than that in group Ⅱ.On the 3rd,7th,and 14 th days,the fluorescence intensity of VEGFR2 in group Ⅲ was lower than that of the group Ⅱ.Conclusion: 1.In the premature brain injury model,increasing the expression of HIF-1α in the brain tissue after injury can increase the VEGFR2 and Claudin-5 exprssion,promoting angiogenesis and endothelial cell tight junction in brain.2.In the premature brain injury model,the expression of TGF-β1 in the brain tissue after hypoxic-ischemic has an upward trend,suggesting that TGF-β1 signaling pathway may be activated after injury.3.After inhibiting the expression of TGF-β1,the expression of VEGFR2 and Claudin-5 in the brain tissue was lower than that in the HIF-1α group,indicating that the role of HIF-1α in promoting angiogenesis and tight junction was also inhibited.This indicates that HIF-1α may exert its effect on the microvascular barrier in the premature brain injury model through TGF-β1 signaling pathway. |
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