| Objectives To investigate the changes of micro RNA(miRNA)expression profile and function of miRNAs with differential expression during cerebral ischemic reperfusion injury with or without ischemic preconditioning.High throughput microarray technique was used to study the difference of miRNAs expression in hippocampus of rats in cerebral ischemia-reperfusion model group,ischemic preconditioning group and sham operation group,and to establish differential miRNA expression profile.The genes with significant difference were screened,and verified by PCR.A preliminary bioinformatics study was conducted with the gene of differential expression.This study may help to explain the pathogenesis of cerebral ischemic preconditioning and ischemia-reperfusion injury and the protective mechanism of Atto vastatin from the aspect of genomics,and furthermore to provide new ideas or approaches for the prevention and treatment of ischemic stroke.Methods The healthy male SD rats(3-4months old SPF)were selected,and randomly divided into the Sham group(Sham group)ischemia reperfusion group(I/R group),ischemic preconditioning group,the atorvastatin pretreatment group(Ator group),(33 rats/each group).I/R group: Normal saline was administered at 10mg/kg for 7d..The arterial occlusion model of rat right cerebral artery was made by the intraluminal middle cerebral artery occlusion method(MCAO),Right middle cerebral artery blocked for 2 hours and then restored to perfusion for 1 day.Establishment of CIP Model by twice Thread of Middle Cerebral artery:The same amount of physiological saline irrigated stomach for 7d.Before reperfusion,the right middle cerebral artery was blocked for 10 min.1d later MCA was occluded again for 2h followed by 1d reperfusion;Ator group:pretreatment with Ator 10mg/kg was given for 7d,then Right middle cerebral artery blocked for 2 hours and then restored to perfusion for 1 day.Sham group The same amount of physiological saline gavage for 7d without MCAO,Infarct volume(TTC staining)and neurological score were evaluated at 24 h after reperfusion..The paraffin sections of the hippocampal tissue were prepared and stained with HE.To observe the survival of neurons in CA1 region of rats.The total RNA of the hippocampal tissue was extracted and purified,then synthesized for c RNA.After gene chip preparation,the data was analyzed.The expression profiles of miRNAs with different variations were obtained by comparing the Sham group,the I/R group,and CIP group.Mi RNAs upregulated or downregulated for mutiple folds were selected and re-confirmed by q RT-PCR in real-time quantitative polymerase chain reaction(policy real-time polymerase chain reaction,q RT-PCR)detection.GO analysis was carried out with the differential expression of miRNAs which may be involved in ischemic preconditioning ischemia reperfusion differentially expressed miRNAs for acquisition of their individual of biological information and function description.The possible signaling pathways of these miRNA were obtained based on KEGG database for the purpose to speculate their individual mechanism.Results 1.In this experiment,male Sprague-Dawley rats were used to prepare cerebral ischemia-reperfusion model,ischemic preconditioning model,Ator vastatin calcium preconditioning model,the total success rate of model was 83.3%,Model preparation is ideal.2.The neurobehavioral scores of the CIP groups and the Atorgroups were significantly reduced compared with that of I/R group in I/R group(P=0.003,0.001).No significant difference in neurobehavioral score between the Ator group and CIP group(P=0.165).These indicate that the ischemic preconditioning(CIP)can significantly improve the motor dysfunction of rats induced by MCAO.Similarly,the volume of cerebral infarction was significantly reduced in CIP group and Ator group compared with that of I/R group(P=0.007,0.016).The cerebral infarction between the Ator group and CIP group was not obvious(P=0.649).The pretreatment of ischemic preconditioning(CIP)can significantly reduce the volume of cerebral infarction caused by MCAO.3.Through morphological observation,ischemic preconditioning and atorvastatin calcium pretreatment can significantly reduced neuronal injury indeced by MCAO in hippocampal CA1,reduce ischemic infarction volume,indicating the neuroprotective effect of CIP.4.Using high-throughput microarray chip technology,the expression profiles of miRNA in the hippocampus of the group I/R group CIP group and Sham group were analyzed,and miRNAs with different expressions were selected:In comparison with Sham group,29 miRNAs were up-regulated and 35 miRNAs were down-regulated in I/R group;64 miRNAs were up-regulated and 4 miRNAs were down-regulated in CIP group;Compared with CIP group,12 miRNAs were up-regulated and 67 miRNAs were down-regulated in I/R group.5.In the miRNAs spectra of the difference expression of the I/R group CIP group and Sham group,the homotropic screening was conducted,and there were 6 miRNAs with the up-regulated expression of homologue expression.We reported for the first time that four new miRNAs in these 3 groups had a homogenous high expression.By focusing on rno-miR-30b-5p,one of the four new miRNA,we also observed its expression in rat hippocampus of Ator group,and found Ator administration markedly increased the expression level of rno-miR-30b-5p.6.GO cluster analysis showed that the function of differentially expressed genes wre involved in the process of ischemic reperfusion in cerebral ischemia was associated with:Cytokine receptor activity,Calcium ion channel transmembrane signal receptor active neuron differentiation and projection,etc;KEGG data analysis results prompt:The difference expression of rno-mir-3068-3p may be involved in the AMP-activated protein kinase(AMKP)signaling pathway that regulates the cell energy homeostasis of the target gene.7.Using bioinformatics analysis technique,the network mode of rno-mir-3068-3p Transcriptional Factors(STRAD and TBC1D1)AMPK was constructed.8.By using q RT-PCR technology,miRNAs with differential expression were verified,which was consistent with the results of gene chips.It is suggested that these miRNAs participate in the pathological process of ischemia reperfusion in rats and play biological roles.Conclusion Cerebral ischemic precondition and ischemia reperfusion in the rat hippocampus cause significant alterations in miRNAs expression and prompt miRNAs may participate gene regulation in the occurrence and development of ischemic stroke atorvastatin pretreatment protects against cerebral ischemia reperfusion injury,which may also associate with the changes in miRNAs. |
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