Study On The Mechanism Of Forebrain Ischemia-reperfusion Affecting The Acetylation Of Bdnf In The Hippocampus Of Rats

Objective:Stroke is already a neurological disease that has a serious impact on public health.brain-derived neurotrophic factor(BDNF)is one kind of neurotrophic factors.This project establishes a transient forebrain ischemia-reperfusion(I/R)model to explore the neuronal survival in various regions of the hippocampus after transient ischemia and the regulatory mechanism of BDNF expression.Provide breakthroughs and potential treatment methods for better treatment of stroke.Methods:(1)I/R model: select male,healthy Sprague-Dawley(SD)rats(260-280g).Divide the rats into the two group(I/R and the sham group).After the rats in the I/R group were anesthetized with chloral hydrate,the heads were settled by a stereotaxic device,the first cervical pterygoid foramen was found,the artery running below it was intercepted,and the arteria carotis communis was separated.On the next day,the arteria carotis communis isolated the day before was clamped with a microarterial clip and timed for15 min to observe whether the model was successful.The sham group was in contrast with the I/R group,but there was no need to coagulate the vertebral arteries,nor to clamp the bilateral common carotid arteries.Then the rats were decapitated and their brains were removed.(2)Nissl staining: The successfully modeled rats are anesthetized with chloral hydrate and reperfused from the ascending aorta,and then treated with saline and paraformaldehyde.When the lungs of the rats turn white and the limbs are stiff,the heads are removed.The brain was fixed,then sectioned with a cryostat,and stained with the kit.(3)Chromatin Immunoprecipitation(Ch IP): 48 h after the successful modeling,the rats were decapitated and their brains were removed,and the hippocampal CA1,CA3,and DG areas were separated under a stereomicroscope.Extract DNA from different tissues with Ch IP kits,and detect the binding of histone deacetylase 3(HDAC3)or histone deacetylase 4(HDAC4)to Bdnf promoter p1,p2,p4,and p6 regions.(4)Bioinformatics analysis: use the tool RNAInter to predict the mi RNAs related to the regulation of rat Bdnf,and then use DIANA Lnc Base Predicted v.2 to predict the lnc RNAs related to the regulation of rat Bdnf,and find the lnc RNAs related to Bdnf in combination with literature.(5)Quantitative reverse transcription PCR(RT-qPCR): The hippocampus CA1,CA3,and DG were isolated,and RT-qPCR was used to examine the expression of long non-coding RNA antisense brain-derived neurotrophic factor(BDNF-AS)and taurine-upregulated gene 1(TUG1)expression.Result:(1)According to the consequence of Nissl staining,compared with the sham group,the neurons in the CA1 area of the hippocampus of SD rats in the I/R group were observably reduced,while the neurons in the CA3 and DG areas did not change significantly.(2)Ch IP results showed that compared with the sham group,in the I/R group,the CA1 area,HDAC3 binding to Bdnf-p1 and Bdnf-p2 decreased,and HDAC4 binding to Bdnf-p2 and Bdnf-p6 decreased;In CA3 area,the binding of HDAC3 to Bdnf-p1 and Bdnf-p2 increases,and the binding to Bdnf-p6 decreases;the binding of HDAC4 to Bdnf-p1,Bdnf-p2 and Bdnf-p4 decreases;In DG region,the binding of HDAC3,HDAC4 to Bdnf-p1,Bdnf-p2,Bdnf-p4 and Bdnf-p6 did not change significantly.(3)The results of further bio-information analysis revealed a diversity of lnc RNAs related to Bdnf gene expression.(4)RT-qPCR results displayed that,compared with the sham group,in the I/R group of SD rats,the expression of BDNF-AS in the CA1 area was decreased,and the expression of TUG1 was increased;the expression of BDNF-AS and TUG1 in the CA3 area increased;In the DG area,the expression of BDNF-AS increased,but the change of TUG1 was not statistically significant.Conclusion:After transient cerebral ischemia and reperfusion,the expression of BDNF-AS in the CA1 area of the rat hippocampus decreased,which reduced the binding degree of HDAC3 and HDAC4 with Bdnf-p1,Bdnf-p2 and Bdnf-p6;the expression of BDNF-AS and TUG1 in the CA3 area increased,which increased the degree of binding of HDAC3 to Bdnf-p1 and Bdnf-p2,and decreased the degree of binding of HDAC4 to Bdnf-p4;There was no significant difference in the expression of TUG1 in the DG region,so that HDAC3 and HDAC4 have no significant difference in binding with Bdnf-p1,Bdnf-p2,Bdnf-p4 and Bdnf-p6.