HIF-1? Repression Of PTEN Transcription Mediates Protective Effects Of BMSCs On Neurons During Hypoxia

Background:Neonatal hypoxic-ischemic brain damage is a disease caused by hypoxia and/or ischemia in fetal or neonatal brain,Stem cells have become a focus point in treatment.In the previous experiment we found that HIF-1?was involved in the cognitive repair process and neuronal synaptic plasticity by the regulation of mesenchymal stem cells in HIBD rats under hypoxic conditions of CoCl₂ pretreatment,PTEN has also been proved widely involved in the pathophysiological process of ischemic brain injury and synaptic regulation of AMPA receptors,but the specific mechanism is still unclear.Objective:To investigate the role of HIF-1?and PTEN in the protective paracrine effect of BMSCs on hippocampal neurons against hypoxia.Methods:1.BMSCs were not directly contacted with hippocampal neurons by nesting and separating co-cultures,but their secretory factors could play a role.The experiment was divided into(?1?normal oxygen culturegroup?Control?,?2?oxygen-glucosedeprivationgroup?Oxygen-glucose deprivation,OGD?,?3?oxygen-glucose deprivation separation co-culture group?OGD+OGD-BMSCs?,?4?oxygen-glucose deprivation hippocampal neurons and BMSCs pretreated with HIF-1?agonist CoCl₂ were co-cultured?OGD+CoCl₂-BMSCs?,?5?oxygen-glucose deprivation hippocampal neurons and BMSCs pretreated with HIF-1?agonistCoCl₂andHIF-1?inhibitor2ME-2wereco-cultured?OGD+2ME-2-CoCl₂-BMSCs?and?6?PTEN inhibitor bpV pretreated oxygen-glucose deprivation hippocampal neuron group?OGD+bpV?.After24 hours,LDH and Annexin V-FITC/PI assay were used to detect neuronal cell damage and apoptosis to verify the protective effect of BMSCs on hippocampal neurons after OGD injury and the role of HIF-1?and PTEN played in this protective process.?3?BMSCs and hippocampal neuronal proteins were extracted and the expression levels of HIF-1?protein,PTEN protein and p-PTEN protein in each group were determined by Western blot.?4?ELISA detection of VEGF protein levels in each group of BMSCs verified the paracrine effect of BMSCs.?5?Co-immunoprecipitation and ChIP-qPCR confirmed the interaction between HIF-1?and PTEN in BMSCs and hippocampal neurons.Results:?1?LDH and AnnexinV-FITC/PI results showed that The degrees of hippocampal neuronal cell injury?P<0.01?and apoptosis?P<0.001?significantly decreased co-cultured with BMSCs after oxygen-glucose deprivation and increased after HIF-1?inhibitor 2ME-2pretreatment?P<0.001?,while LDH levels?P<0.01?and apoptosis?P<0.001?decreased after PTEN inhibitor bpV pretreated.?2?Western blot analysis showed that HIF-1?protein levels?P<0.01?and PTEN protein levels?P<0.05?were increased in BMSCs and hippocampal neurons after oxygen-glucose deprivation,and p-PTEN protein expression was decreased?P<0.01?;After pretreatment of BMSCs with HIF-1?inhibitor 2ME-2,PTEN protein level?P<0.05?increased and p-PTEN protein expression decreased?P<0.05?;HIF-1?protein levels?P<0.01?and PTEN protein levels?P<0.001?decreased in neurons when co-cultured with BMSCs,and the level of PTEN protein?P<0.05?and p-PTEN protein?P<0.001?decreased after the pretreatment of HIF-1?inhibitor 2ME-2,p-PTEN protein expression?P<0.05?increased after PTEN inhibitor bpV pretreated oxygenated-glucose deprived hippocampal neurons.?3?The secretion of growth factors in BMSCs culture medium was investigated by ELISA:The level of VEGF in OGD group was increased compared with Control group?P<0.001?,and decreased after the addition of HIF-1?inhibitor?P<0.001?,There was no significant difference between the CoCl₂ group and OGD group.?4?Co-immunoprecipitation showed that HIF-1?and PTEN were not directly cross-linked at the protein level in BMSCs and hippocampal neurons.?5?ChIP-qPCR results indicated that HIF-1?protein could enter the nucleus and enriched on the promoter region of PTEN gene in BMSCs and hippocampal neurons,and the enrichment factor of OGD group was significantly higher than that of Control group?P<0.001?.Conclusion:?1?BMSCs can protect neurons after OGD injury and reduce neuronal cell damage and apoptosis in hypoxia;?2?HIF-1?activation,PTEN inhibition are involved in the protective effect of BMSCs on OGD injured neurons and PTEN protein expression increased when HIF-1?was inhibited;?3?HIF-1?protein can enter the nucleus and enrich on the promoter region of PTEN gene to regulate its transcription process negatively in BMSCs and hippocampal neurons.