The Protective Mechanism Of KP-4 Through MiR-219a-5p And Its Target Gene Signaling Pathway In Cerebral Ischemia Injury

Cerebral stroke is an acute cerebrovascular disease with high morbidity and disability.And cerebral stroke divided into hemorrhagic and ischemic stroke which occupied about 80%cerebral stroke.Ischemic stroke is caused by a short or permanent interruption of local cerebral blood flow,resulting in cerebral infarction and brain impairment such as consciousness,language,movement or even life.Thrombolysis or thrombectomy is used as early as possible to recanalize the infarcted vessels which will restores oxygen and energy supply to the brain are the most effective means to treat cerebral ischemia.However,the recovery of blood oxygen supply will cause secondary cerebral ischemia injury which is called cerebral ischemia reperfusion(I/R)injury.I/R damage process is a complex cascade reaction,involving a variety of damage mechanisms,such as intracellular Ga2+overload,oxidative stress,excitatory amino acid toxicity,impaired mitochondrial energy supply,formation of inflammatory cortices,activation of inflammatory pathways and various membrane receptors in injury-related molecular patterns,and various factors leading to cell apoptosis and autophagy.Currently,there are very few effective methods and drugs for the treatment of I/R injury,so the research on its pathogenesis and prevention measures is still an urgent scientific problem to be solved.MicroRNA is a class of non-coding small RNA with a length of about 22 nucleotides.MiRNA plays an important regulatory role in a variety of physiological and pathological processes such as ontogeny,cell proliferation,cell differentiation,cell aging and tumor formation.Recent studies have indicated that miRNA is involved in the occurrence and development of stroke.Previous studies have confirmed that miRNAs lead to mRNA degradation or translation inhibition by binding to the 3’UTR of target gene mRNA.The post-transcriptional effect of miRNA is faster,and many downstream target genes can be regulated simultaneously.Therefore,miRNA may be a potential target for the treatment of stroke.KP-4,a new drug of plant monomer developed by our team,is a derivative of Isosteviol,which has relatively ideal water solubility and bioavailability.KP-4 has been shown to have a neuroprotective effect on MCAO/R mice,and the studies have also shown that kp-4 can exert a neuroprotective effect on mice with focal cerebral ischemia through miRNA-181b/CYLD.However,KP-4 is a multi-target drug,and the mechanism of its protective effect has not been fully clarified.Therefore,the significance of this study is to further explore the effect of KP-4 on miRNAs in cerebral ischemia/reperfusion(I/R)injury and its potential mechanism of action.Thus,it will brings a new theoretical and experimental basis for KP-4 clinical administration,and also provides a new idea for finding cerebral ischemia markers and potential drug targets.ObjectiveWe aimed to investigate the effect of KP-4 on abnormal versions of miRNA and miRNA target genes in I/R injury,and to explore the potential molecular mechanism.Methods1.Through the volume of cerebral infarction,pathological changes and behavioral changes,we constructed the MCAO/R injury model of mice and determined the drug concentration of KP-4 in vivo.Meanwhile,through the experiment of CCK8 and flow cytometry,we constructed the OGD/R injury model of N2a cells and determined the drug concentration of KP-4 in vitro.2.The influence of KP-4 on miRNA and its effect on miRNA were investigated by miRNA sequencing,WB,RT-PCR,flow cytometry,etc.3.The target genes of miR-219a-5p were screened and verified by bioinformatics analysis,cell transfection,Western Blot and luciferase reporter gene experiments;4.Western Blot,RT-PCR and flow cytometry were used to analyze the role of miR-219a-5p/PDE4D signaling pathway and the influence of KP-4 on this signaling pathway.Results1.In vivo and vitro,KP-4 was found to have a protective effect on hypoxic/ischemia-reperfusion injury.2.In the process of anoxic/ischemia-reperfusion injury,KP-4 can play a protective role on anoxic/ischemia-reperfusion injury in vivo and vitro by upregulating the expression of miR-219a-5p.3.MiR-219a-5p can target regulate the expression of PDE4D by combining on the target sequence of PDE4D-3’UTR;4.KP-4 can play a neuroprotective role on OGD/R N2a cells through the miR-219a-5p/PDE4D signaling pathway.