The Impact Of MicroRNA-98 On The Ischemic/Reperfusion-Induced Injury In Rat

Stroke is a type of brain disease caused by cerebral angiogenesis ischemic or hemorrhagic lesions in vessels with high morbidity,mortality and disability.All over the world,there is still a high incidence of stroke in China.Stroke can be divided into ischemic stroke and hemorrhagic stroke.Ischemic stroke accounted for more than 80%of the incidence of stroke.Therefore,our research mainly focused on ischemic stroke.Up to now,the tissue plasminogen activator(tPA)is the only drug approved by the Food and Drug Administration(FDA)for thrombolysis in clinical treatment.However,there are shortcomings such as narrow treatment window and many adverse reactions in tPA,which limit its use in clinical practice.At present,there is still short of effective drug for stroke in clinical practice.As an important gene expression regulator,MicroRNAs(miRNAs)participate in a series of important processes of life process.In recent years,more and more research has been showed that miRNAs can participate in the pathological development of stroke.As a member of miRNAs family,microRNA-98 participates in many diseases such as colon cancer and myocarditis.Neuroinflammation is one of the pathological mechanisms of stroke injury.Microglial cells are immune effector cells in the central nervous system(CNS).They exert immunosurveillance in the brain’s microenvironment and respond to the damage in CNS.Therefore,microglia plays an extremely important role in the maintenance and repairation of the central nervous system homeostasis.Based on the previous discovery of the relationship between microRNA-98 and ischemic stroke,we futher studied the therapeutic effect and mechanisms of microRNA-98 on ischemic stroke rats.First,the present work evaluated the therapeutic effects of microRNA-98 on rat ischemic stroke models by preparing transient middle cerebral artery occlusion(tMCAO)rat model;Next,we used oxygen-glucose deprivation/reoxygenation(OGD/R)-induced microglial model to explore the mechanism of microRNA-98 on ischemic stroke.This study extends the new effect of microRNA-98 and provides the experimental evidence for developing clinical drug of stroke.AIM:To investigate the protective effect of microRNA-98 on focal cerebral ischemia-reperfusion injury in rats,and to clarify the involved mechanisms of microRNA-98 on neuroinflammatory injury.METHODS:1.Transient middle cerebral artery occlusion(tMCAO)was uesd to prepare model rats.2.2,3,5-triphenyl four azole nitrogen(TTC)staining was used to measure the volume of cerebral infarction;3.Longa behavioral test was used to evaluate the neurological dysfunction;4.Quantitative Real-time PCR(RT-qPCR)was used to detect the change of IL-1β,TNFα,HMGB1 in mRNA level.5.Western bloting(WB)was appiled to detect the expression of PAFR,iNOS,TGF-β,pP38 MAPK,P38 MAPK,pJNK,JNK in protein level.6.Immunohistochemical staining was used to analyze the amount,cellular morphology of NeuN-labeled neuron and GFAP-labeled astrocyte in the penumbra filed of ischemic brain tissue.7.Immunofluorescence(IF)was appiled to detect the phenotypes of microglia.8.The luciferase reporter gene method was used to detect the targeting regulation of microRNA-98 on PAFR.RESULTS:In vivo:1.There is a correlation between microRNA-98 and ischemic strokeIn clinical samples,we found that microRNA-98 of stroke patients were significantly less than those in healthy group;In the animal model of ischemic stroke,we also found that the level of microRNA-98 in model group was significantly lower than that in sham group in 24h to 3 days after ischemic reperfusion.2.MicroRNA-98 reduces cerebral infarct volume and neurological dysfunctionNeurological fuction test result showed that the motor dysfunction in the microRNA-98 treated group was significantly lower than that in the model group;Result of TTC staining showed that the infarction rate in the model rats was 24.59%.However,the volume of cerebral infarction in microRNA-98 group was only 14.54%.Compared with the model group,the infarction rate of microRNA-98 group reduced by 10.05%.3.MicroRNA-98 reduces neuronal damage,inhibit astrocyte activation and promote microglia polarize into M2 phenotypeImmunohistochemistry was used to analyze the amount,cellular morphology of NeuN-labeled neuron and GFAP-labeled astrocyte in the penumbra filed of ischemic brain.Immunofluorescence was uesed to detect the phenotypes of microglia in the penumbra filed of ischemic brain.NeuN-labeled neurons in the model group were obviously decreased with volume reduction and nuclear membrane contraction.GFAP-labeled astrocytes in model group were observed with abnormal proliferation and activation.At the same time,the M1-type marker of microglia in model group was significantly increased.The intervention of microRNA-98 can significantly reduce neuronal loss,alleviate nuclear condensation,inhibit the abnormal proliferation and activation of astrocytes,and promote microglia polarize into M2 phenotype.4.MicroRNA-98 reduces the neuroinflammatory response and the release of inflammatory factors(IL-1β,HMGB1,iNOS,TNF-α)Through qPCR and WB experiments,we found that the expression of various inflammatory factors were increased in model group while microRNA-98 significantly inhibited the increase in the expression of these inflammatory factors.5.MicroRNA-98 inhibits JNK/P38 MAPK signaling pathway in penumbra filed of ischemic brainWB results showed that the phosphorylation levels of JNK and P38 MAPK in model group were significantly increased compared with sham group.However,microRNA-98 significantly inhibited the phosphorylation of JNK and P38 MAPK.In vitro:1.MicroRNA-98 targetedly suppresses PAFR expression in primary microgliaThe luciferase gene assay result revealed that microRNA-98 can inhibit the expression of PAFR in gene level;OGD would up-regulate the expression of PAFR in primary microglia.However,the expression of PAFR was significantly reduced by microRNA-98.2.MicroRNA-98 promotes microglia polarize into M2 phenotype after OGDWB results showed that M1 marker was significantly up-regulated in primary microglial cells after OGD.However,compared with the model group,microRNA-98 decreased the expression of M1 marker and increased M2 maker significantly.3.MicroRNA-98 inhibits JNK/P38 MAPK signaling pathway in microglia after OGDWB results showed that phosphorylation levels of JNK and P38 MAPK in OGD group were significantly increased while microRNA-98 significantly inhibited the phosphorylation of JNK and P38 MAPK.CONCLUSION:1.MicroRNA-98 is associated with ischemic stroke;2.MicroRNA-98 can reduce neuronal damage,inhibit abnormal activation and proliferation of astrocytes,promote microglia polarize into M2 phenotype,inhibit the neuroinflammation reaction,and play a protective role in ischemic/reperfusion-induced injury in rat;3.MicroRNA-98 targetdly suppresses PAFR expression and then inhibits the downstream JNK/P3 8 MAPK inflammatory signaling pathways of PAFR.INNOVATION POINTS IN THIS PAPER1.There is a correlation between microRNA-98 and ischemic stroke;2.MicroRNA-98 has a protective effect in ischemic/reperfusion-induced injury;3.MicroRNA-98 exerts anti-neuroinflammatory function by inhibiting PAFR expression and relative inflammatory signaling pathway.