Study On The Effects Of MicroRNA-146a On Cerebrall Ischemia-reperfusion Injury In Mice

BackgroundStroke is a brain system disease which has high mortality and disability.Ischemic stroke(IS)comprises approximately 80%of stroke patients Increasing evidence confirmed that microRNAs(miRNAs)are a class of non-coding small RNAs that play an important role in cerebral ischemia-reperfusion injury by regulating the expression of target genes.MiRNAs,as important candidate biomarker substances,play an important role in the development of cardiovascular diseases,especially in the development of stroke.Previous studies reported that miR-146a plays a crucial role in the regulation of multiple ischemia reperfusion.However,the effects of miR-146a in brain ischemia reperfusion.In this study,we will explore the effects and underlying mechanism of miR-146a in brain ischemia reperfusion of mice.Thus,the brain ischemia reperfusion mouse model was established,and the real-time fluorescent quantitative PCR(qRT-PCR),five level of grading method,2,3,5-triphenyltetrazolium chloride(TTC),evans blue(EB)staining,enzyme linked immunosorbent assay(ELISA),and western blotting were employed in this study.Part 1Amis The purpose of this study was to investigate the effects of miR-146a on I/R injury in the brain of mice.MethodsThe healthy male C57BL/6J mice(22-25 g),were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.,were anesthetized with 10%chloral hydrate and fixed.The right median line of the neck was selected in the right side of the neck.The incision was cut about 1 cm by ophthalmic scissors.The fascia and muscle were separated by layer of eye forceps.The eyelid opener ensures a clear and open surgical field,separating the common carotid artery.Then the common carotid artery was ligated and loosened with silk thread.Then the external carotid artery of mice was separated,and the external carotid artery was cut off at the distal end of the ligation site by electrocoagulation knife;then the external carotid artery was clamped by the artery,and a small opening was cut by iris shears,from which the thread bolt entered,and the loosening knot was used to fix the thread.Then the clamp was opened,and the end of the artery was pulled to a straight line with the internal stiff artery,then the embolism was pushed forward slowly,and finally the embolism went up from the external carotid artery to the internal carotid artery until it stopped at about 23 mm of the mouse brain.After the insertion of the emboli,the time of blood flow cut-off was controlled at about 60 mins.After that,the emboli was quickly removed and ligated near the bifurcation of the external carotid artery.The above-mentioned knots were removed to make the cerebral blood flow in mice reperfusion and complete the establishment of the model of ischemia-reperfusion.Finally,the skin of the mouse brain was sutured,the wound was disinfected with iodophor,and 1 mL penicillin was injected into the abdominal cavity of the mice.The sham-operated group was the same as the above steps,but no ischemic model was established and suture was performed.Mice were randomly divided into 5 groups:pro-operation group,sham group,MCAO 6 h group,MCAO 24 h group,MCAO 72 h group.The expression of miR-146a was measured by using qRT-PCR.Results The results of qRT-PCR showed that no obvious changes on the expression levels of miR-146a were observed in sham group and MCAO 6 h group compared with pro-operation group while significantly increased in MCAO 24 h group and MCAO 72 h group.Conclusion MiR-146 was significantly elevated in I/R,and might serve as a crucial regulator in I/R.Part 2Aims The purpose of this study was to investigate the underlying mechansism that contributes to the functional role of miR-146a on I/R injury in the brain of mice.Methods All mice were anesthetized and fixed in a stereotaxic apparatus.24 h before MCAO operation,the miR-146a inhibitor was injected into the right lateral ventricle at a rate of 0.2 ?l/min with a microsyringe to decrease the expression of miR-146a,and the mice were divided into sham group,MCAO group,MCAO+NC group,MCAO+miR-146a inhibitor group.The expression of miR-146a was measured by qRT-PCR.Neurobehavioral impairment was measured by five level of grading method.Cerebral infarction volume was measured by TTC.The degree of BBB damage was determined by EB staining.The levels of inflammatory factors were measured by ELISA.The protein levels of IRAK1/NF-?B pathway were analyzed by western blotting.Results(1)The expression of miR-146a was downregulated by intra-cerebral injection of miR-146a inhibitor at 24 h before MCAO or sham.Compared to sham group,the neurological impairment was injured in MCAO group.The injury of neurological impairment and the cerebral infarction volume was increased in MCAO group by knockdown of miR-146a.Moreover,cerebral edema was occurred by brain I/R damage.The exudation amount of EB in brain I/R damage mice by knockdown of miR-146a.(2)Compared to sham group,the levels of TNF-a,IL-1? and IL-6 were increased in MCAO group.And the levels of TNF-a,IL-1? and IL-6 were significantly increased by knockdown of miR-146a in MCAO group.(3)Compared to sham group,the protein levels of p-I?B? and NF-?B of nucleus were increased,and the I?B? and NF-?B of cytoplasm were decreased in MCAO group,the levels of proteins were increased by knockdown of miR-146a.(4)IRAK1 was predicated as the downstream target gene of miR-146a.The expression of IRAKI was increased in MCAO group,the mRNA and protein of IRAKI was increased in brain I/R damage mice.Conclusion(1)The downregulation of miR-146a promotes brain I/R damage in mice.(2)Knockdown of miR-146a promotes brain I/R damage-mediated inflammatory response.(3)Knockdown of miR-146a activates brain I/R damage-mediated NF-?B pathway and promotes expression of IRAKI.Innovative pointsCompared to the research status of the subject,the innovation of this paper as following:(1)cerebral apoplexy is one of the main causes of high disability rate and mortality in adults.Tissue plasminogen activator is the only effective treatment drug for cerebral apoplexy,but it has not been widely used.In the present study,we demonstrated that miR-146a promotes cerebral ischemia-reperfusion injury in mice through accelerating the inflammatory response.It has significance to further understand cerebral ischemia-reperfusion injury.(2)In this study,the cerebral ischemia-reperfusion injury model was established to confirm the effects of miR-146a in the process of cerebral ischemia-reperfusion injury,and knockdown of miR-146a results in cerebral ischemia-reperfusion injury.Our findings will provide a novel therapeutic target for ischemia-reperfusion injury.(3)miR-146a inhibitor promotes IRAK1 by I/R injury-mediated NK-?B pathway activation to increase ischemia-reperfusion injury in mice.The study confirmed the mechanism of miR-146a in cerebral ischemia-reperfusion injury,providing a theoretical basis for future studies on miR-146a as a target for the prevention and treatment of cerebral ischemia-reperfusion injury.