Effect Of VEGF Gene Overexpression On Hypoxic Ischemic Brain Damage In Preterm Rats

Background: Previous studies have shown that vascular endothelial growth factor A(VEGFA)can improve hypoxic ischemic brain damage(HIBD)by promoting angiogenesis,but it is not clear whether VEGFA can treat HIBD in premature infants and recover long-term neurobehavioral symptoms.Additionally,Wnt/β-catenin axis is an important signaling pathway involved in HIBD,and our previous study also discovers that the Wnt/β-catenin axis is upregulated after VEGFA gene therapy.Hence,we aim to explore the therapeutic effects and possible mechanisms of VEGFA in a rat model of preterm ischemic brain injury.Objective: In this study,we explored the therapeutic effect of exogenous VEGF gene and its potential molecular mechanism in the hypoxic-ischemic brain injury model of premature SD rats.Methods: Lentivirus with overexpression of VEGF gene was injected into the ventricle of the preterm rats by stereotaxic localization.Immunofluorescence staining were performed to detect the protein expressions of VEGF,Wnt3 a,β-catenin,CD34,and neuron-specific nuclear protein(Neu N)in cortex and hippocampus to determine the damage of angiogenesis and neurons after VEGF gene therapy.TTC staining was used to observe the infarct area of brain tissue.In addition,Wnt/β-catenin pathway inhibitor LGK-974 was intraperitoneally injected,then,Immunofluorescence,Western blot and RT-QPCR were used to further detect the above indexes from protein level and m RNA level to determine whether the Wnt/β-catenin pathway is involved in the treatment of VEGF.Finally,primary neurons were cultured in vitro to establish cell glucose and oxygen deprivation(OGD)model.PI/Hoechst staining was used to observe the neuroprotective effect of VEGF gene therapy.Similarly,the protein expression of VEGF,Wnt3 a and β-catenin was detected by immunofluorescence staining and Western blot with LGK-974 intervention to determine the relationship between Wnt/β-catenin axis and VEGF gene therapy.Results: 1.VEGF gene therapy significantly reduced the behavioral score of HIBD in preterm mice(VEGF gene therapy group vs HIBD group,P < 0.001);2.Immunofluorescence results showed that VEGF gene therapy promoted angiogenesis(VEGF gene therapy group vs HIBD group,P <0.05)and improved neuronal injury(VEGF gene therapy group vs HIBD group,P < 0.001).3.TTC staining showed that the volume of cerebral infarction in the VEGF gene therapy group was 2 times less than that in the HIBD group(P < 0.01).4.VEGF gene therapy significantly upregulated the m RNA and protein expression of Wnt/β-catenin(VEGF gene therapy group vs HIBD group,P < 0.01);5.In the OGD model of primary neurons,PI/Hoechst staining showed that VEGF significantly reduced the death of neurons(VEGF group vs OGD group,P < 0.05)and upregulated the protein expression of Wnt/β-catenin.However,the use of Wnt/β-catenin pathway inhibitor LGK-974 significantly reduced the angiogenesis promoting and neuroprotective effects of VEGF.Conclusion: VEGF gene therapy promotes angiogenesis and neuroprotection by activating the Wnt/β-catenin axis,thereby improving neural function in preterm rat model with HIBD.