Neuroprotective Effect And Mechanism Of Long Noncoding RNA ANRIL In Transient Cerebral Ischemia/Reperfusion

Accumulating evidence indicates that neuroinflammation plays a key role in the pathogenesis of ischemia stroke(IS).And BBB damage,edema and oxidative stress occurring secondary of inflammation to IS can also cause irreversible neuronal damage,with unrestrained neuroinflammation having the potential to exacerbate this damage.Neuroinflammation has become a target for therapeutic intervention.Nevertheless,the clinic protective of existing research on IS has limited effectiveness,therefore,it is extremely necessary to find effective therapeutic targets for IS.Long noncoding RNA over 200 nucleotides in length have been shown to play diverse roles in biological processes,controlling gene expression,m RNA splicing,and other epigenetic processes.Increasing evidence suggests that long noncoding RNAs can exert neuroprotective effects in cerebral ischemia-reperfusion injury.Long noncoding RNA ANRIL(ANRIL),which is encoded by the chromosome 9p21 region in humans.Previous studies have demonstrated that ANRIL was associated with metabolic disease,protected H9C2 cells against hypoxia-induced injure,could influence the imflammtion pathology of coronary artery disease owing to its ability to modulate micro RNA-181 b.Levels of ANRIL are reportedly altered in ischemic stroke(IS)patients,but its role in IS requires further clarification.This study was designed to explore the mechanistic function of ANRIL in IS,to provide a new therapeutic target and theoretical basis for the protection of neurons in IS patients.We planed to research the effect and mechanism of ANRIL on neuronal injury induced by cerebral ischemia and reperfusion.The hippocampus is rich in neurons and serves a critical function in memory,navigation,and cognition.Damaged the hippocampus can lead to neurological dysfunction,IS patients often have severe neurological dysfunction,therefore,we selected mouse hippocampus and mouse hippocampal neuronal cell line HT22 to research the effect and mechanism of ANRIL on neuronal injury in IS.ObjectiveThe present study aimed to investigate the neuroprotective effect and mechanism of ANRIL in transient cerebral ischemia/reperfusion,to provide a new therapeutic target and theoretical basis for the protection of neurons in IS patients.Methods1 Middle cerebral artery occlusion and reperfusion(MCAO/R)using the suture-occluded method in C57BL/6 mice was used as in vivo model of IS injury.Oxygen-glucose deprivation and reoxygenation(OGD/R)in a mouse hippocampal HT22 neuron line was used as in vitro models of IS injury.2 ANRIL levels in mice's hippocampus that were collected at MCAO1h/R 0,8,16,and 24 h post-MCAO were measured by q RT-PCR.ANRIL expression in HT22 cells was measured at OGD 2h/R 0,8,16,and 24 h post-OGD/R by q RT-PCR.MCAO /R 24 h and OGD/R 24 h was selected as the time point collecting samples.si-ANRIL,si-NC(negative control),pc DNA3.1-ANRIL,pc DNA3.1-NC,pc DNA3.1-NF-?B were transfected into different groups HT22 cells using the ribo FECT CP reagent at 24 h prior to OGD.si-ANRIL,si-NC,pc DNA3.1-NF-?B,or pc DNA3.1-NC were administered into the lateral ventricles of different groups mice at 72 h and 24 h prior to ischemia.3 miR-671-5p levels in mice hippocampal samples that were collected at MCAO 1h/R 0,8,16,and 24 h post-MCAO were measured by q RT-PCR.miR-671-5p expression in HT22 cells was measured at OGD2h/R 0,8,16,and 24 h post-OGD/R by q RT-PCR.MCAO /R 24 h and OGD/R 24 h was selected as the time point collecting samples.miR-671-5p agomir,agomir-NC,miR-671-5p antagomir,antagomir-NC,pc DNA3.1-NF-?B and pc DNA3.1-NC were transfected into different groups HT22 cells using the ribo FECT CP reagent at 24 h prior to OGD.miR-671-5p agomir,agomir-NC,pc DNA3.1-NF-?B,or pc DNA3.1-NC were administered into the lateral ventricles of different groups mice at 72 h and 24 h prior to ischemia..4 HT22 cells were treated with OGD/R 24 h,si-ANRIL and miR-671-5p antagomir were transfected into different groups HT22 cells using the ribo FECT CP reagent at 24 h prior to OGD.Established MCAO/R 24 h mice model,si-ANRIL and miR-671-5p antagomir were administered into the lateral ventricles of different groups mice at 72 h and24 h prior to ischemia.5 The neurological functions of each group mice were assessed by neurological scores system and rotarod test,TTC was used to measure the area of infarction,nissl staining was used to visualize the injured cells,immunofluorescent staining was used to analysis the Neu N of mice's hippocampal tissue.MTT assay was used to assess HT22 cells viability,LDH leakage assay was used to assess HT22 cell cytotoxicity,immunofluorescent staining was used to analysis the expression of P65 in HT22 cells.The expression of ANRIL,miR-671-5p and m RNA NF-?B were quantified via q RT-PCR.The protein level of p-P65 and P65 were measured via western blot.The expression level of IL-1?,IL-6,TNF-?,and i NOS levels were measured via ELISA.Results1 Neurological deficit scores were increased in the MCAO/R mice group as compared with the sham group;fall latency was reduced in the MCAO/R model animals as compared to the sham group in a rotarod test.The synapsis and form of HT22 cells shrank after OGD/R treated;HT22cell viability was reduced after OGD/R as compared to the untreated control;HT22 cell cytotoxicity was increased after OGD/R as compared to the untreated control.2 ANRIL expression increased both in MCAO/R mice and in OGD/R HT22 cell.In vitro,successfully downregulating or overexpress ANRIL by transfecting si-ANRIL or pc DNA3.1-ANRIL into different groups HT22cells;si-ANRIL reduced OGD/R-induced cell injured and neuroinflammation,which reversed by overexpressed NF-?B expression.In vivo,intracerebroventricular injection si-ANRIL reduced ANRIL expression successfully in MCAO/R model;si-ANRIL protected MCAO/R mice against cerebral ischemia-reperfusion injury and neuroinflammation,which reversed by overexpressed NF-?B expression.3 miR-671-5p expression reduced both in MCAO/R mice and in OGD/R HT22 cell.In vitro,successfully upregulated or dowregulated miR-671-5p by transfecting miR-671-5p agomir or miR-671-5p antagomir into different groups HT22 cells;Target Scan 7.2 predicted that NF-?B was a target gene of miR-671-5p;a dual-luciferase reporter show that NF-?B was a direct target of miR-671-5p;miR-671-5p agmir reduced OGD/R-induced cell injured and neuroinflammation,which reversed by overexpressed NF-?B expression.In vivo,intracerebroventricular injection miR-671-5p agomir reduced miR-671-5p expression successfully in MCAO/R model;miR-671-5p agomir protected MCAO/R mice against cerebral ischemia-reperfusion injury and neuroinflammation,which reversed by overexpressed NF-?B expression.4 Negative correlation between the expression of ANRIL and miR-671-5p both in MCAO/R mice and in OGD/R HT22 cell.In vitro,si-ANRIL reduced OGD/R-induced cell injured and neuroinflammation,which reversed by miR-671-5p antagomir.In vivo,si-ANRIL protected MCAO/R mice against cerebral ischemia-reperfusion injury and neuroinflammation,which reversed by miR-671-5p antagomir.ConclusionsWe showed that the expression of ANRIL increased in the pathology of cerebral ischemia reperfusion injury.ANRIL downregulating sufficiently reduced neuroinflammation via suppression of NF-?B expression both in vitro and in vivo models of IS,and ANRIL downregulating sufficiently reduced neuroinflammation via regulatle miR-671-5p/ NF-?B both in vitro and in vivo models of IS;which suggesting that ANRIL might be a potentially viable therapeutic target to diminish neuroinflammation in IS patients.