Effects Of EphA4 Receptor And Histone Deacetylase On BDNF Expression In Rat Hippocampal Neurons

Brain-derived neurotrophic factor(BDNF)is a member of the neurotrophin family.It can participates in the regulation of neurogenesis,neuronal differentiation,synaptic plasticity and neuron survival through its tyrosine kinase receptor signaling system process.BDNF plays an important role in the protection and repair of neuron damage after stroke.However,the regulatory mechanism of BDNF protein expression is unknown.Ephrin receptor is the most abundant receptor in tyrosine kinase receptor family.Ephrin ligand Eph receptor signaling system plays an important role in the process of organism development and homeostasis.EphA receptor is divided into two subtypes,EphA and EphB.EphA receptor is mainly involved in the formation of neural network,nerve regeneration and synaptic plasticity during the development of nervous system.It was found that the EphA4 receptor level of pyramidal neurons in CA1 area of hippocampus was up-regulated,and the function of EphA4 receptor was inhibited.Therefore,we hypothesized whether Eph receptor could enhance the intrinsic protective mechanism of damaged neurons by affecting the expression of BDNF protein.In this experiment,the expression of BDNF protein and different transcripts in primary neurons were observed after antagonizing the function of EphA4 receptor.Previous studies showed that the decrease of BDNF protein expression in CA1 region of hippocampus after ischemia-reperfusion was related to the change of BDNF transcript IV expression in CA1 region.In the present studies,we established a transient forebrain ischemia-reperfusion(I/R)model in rats,and used the histone deacetylase inhibitor SAHA to study the relationship among the differential expression of BDNF and histone acetylation.Part Ⅰ Effects of EphA4 receptor on BDNF protein and mRNA expression in cultured hippocampal neurons of rats Objective:BDNF plays an important role in the protection and repair of neuronal injury after stroke.However,the regulatory mechanism of BDNF protein expression is unclear.By culturing primary neurons in the hippocampal region of rats and observing the changes in the expression of BDNF protein and various mRNA transcripts after antagonizing EphA4 receptor function.Methods:1.Experimental grouping: 1-3d SD rat suckling rats were used to isolate hippocampal tissue and culture primary hippocampal neurons.The cultured rat hippocampal brain neurons were randomly divided into a normal control group(control group)and a treatment group(EphA4-Fc group).The cultured cells were used for experiments on the seventh day.2.Apply Western blot and RT-qPCR detection technology,to analyze the changes of BDNF protein and mRNA expression in hippocampal neurons after administration of EphA4-Fc to antagonize EphA4 receptor function.Results:1.Western blot results showed: compared with the normal control group,the expression of BDNF protein in the hippocampal neurons of the EphA4-Fc group was increased.2.RT-qPCR results show that compared with the control group,the BDNF transcript IIc expression in hippocampal brain neurons in the EphA4-Fc group was upregulated,and the expression of BDNF transcripts I,III,IV,VI,and X1 did not change significantly.Conclusion:After administration of EphA4-Fc,increased translation of BDNF protein in rat hippocampal neurons was associated with increased expression of mRNA transcript IIc.Part Ⅱ SAHA affects BDNF transcript expression in hippocampus of cerebral ischemia rats by inhibiting histone deacetylase functionObjective:The decrease of BDNF protein expression in hippocampal CA1 region of cerebral ischemia rats is related to the change of BDNF transcript expression in CA1 region.However,the mechanism by which BDNF transcript expression is altered is unknown.A transient forebrain ischemia-reperfusion(I / R)model was established in rats,and the histone deacetylase inhibitor SAHA was injected intraperitoneally to observe the changes of BDNF transcript expression in various hippocampal regions of rats.Methods:1.Experimental groups: Sham group(sham),Transient forebrain ischemiareperfusion group(I/R),I/R + DMSO group(DMSO)and I/R + SAHA group(SAHA).2.Transient forebrain ischemia-reperfusion model preparation:I/R group: pentobarbital sodium(5mg/100 g,i.p.)was used to anesthetize male adults SD rats(240-260g),and rats were fixed on the stereotactic instrument by ear.After electrocoagulation of bilateral vertebral arteries and separation of bilateral common carotid arteries,the perfusion of bilateral common carotid arteries was resumed 15 minutes later,resulting in transient forebrain ischemia in rats.In the DMSO group,rats were injected with DMSO(50mg / kg,i.p.)1 hour before and 24 hours after I/R.In the SAHA group,rats were treated with SAHA(50mg / kg,i.p.)1 hour before and 24 hours after I/R.In sham group,except vertebral artery was not electrocoagulated and common carotid artery was not clamped,the other operation was the same as that in I/R group.3.RT-qPCR was used to determine the expression level of different BDNF transcripts in hippocampus of different experimental groups.Results:RT-qPCR results showed that: compared with the I/R group or I/R + DMSO group,SAHA increased expression of BDNF transcripts IV,VI,and X1 in the CA1 region,while transcripts I,IIc,and III did not change significantly.At the same time,SAHA can increase the expression of BDNF transcript X1 in the DG region.SAHA had no significant change in BDNF expression in CA3 region.Conclusion:SAHA increases the expression of BDNF transcripts IV,VI and X1 in the CA1 region by regulating protein acetylation levels.This will provide a new way to prevent and treat stroke.