STVNa Plays A Neuroprotective Role Through Inhibiting MAPK/NF-κB And Up-regulation Of Akt/HIF-1α Pathway

Cerebral ischemia is a leading cause of adult death and disability worldwide.It seriously threatens human health for reducing oxygen concentration in microenvironment of the brain.Isostevia sodium（STVNa）extracted from plants has been proved to protect nerve cells from cerebral ischemia injury in mice.But its mechanism is still unclear.Therefore,this thesis studied the protective effect and mechanism of STVNa to cerebral ischemia injury on mouse neuroblastoma cell line N2a cells and in vivo C57BL/6 mice model.Cerebral ischemia attack can cause severe complications,a series of inflammatory factors and apoptotic factors are activated,which further aggravate condition.NF-κB subunit p65 is phosphorylated after cerebral ischemia,transferring into the nucleus,binding relative to DNA-binding sites,regulating the transcription of pro-inflammatory mediator genes at last.Besides,MAPKs activation can further activate transcription factors,induce excessive synthesis of pro-inflammatory cytokines,and subsequently trigger an inflammatory response and form an apoptosis cascade reaction.In normoxic conditions,cellular HIF-1 is degraded by the ubiquitin-proteasome pathway,resulting a rather short half-life.In hypoxic conditions,the level of HIF-1αwas increased,entering the nucleus,binding to HIF-1βon specific DNA fragments,and regulating the expression of its target genes.HIF-1 is a nuclear transcription factor with numerous upstream pathways.However,the main upstream regulatory pathways are MAPK pathway and PI3k-Akt pathway.ObjectiveTo explore whether STVNa plays a neuroprotective role by inhibiting the phosphorylation of MAPKs,NF-κB and upregulating Akt/mTOR/HIF-1.Methods1.Cell viability and apoptotic rate were measured using LDH,CCK8,and flow cytometry to determine the concentration of CoCl₂ and STVNa.2.Ca²⁺concentration and reactive oxygen species（ROS）of N2a cells,which was treated with chemical ischemia and hypoxia through STVNa,was determined by fluo-4am and ROS respectively.The mRNA changes of TNF-α,iL-6,iL-1β and iNOS were detected by RT-PCR,and the ratio of Bcl2/Bax protein and Caspase 3 were detected by Western Blot.3.The mRNA and protein changes of the upstream and downstream genes were detected after treated by STVNa on the chemical hypoxia of nerve cells.Searching the protective effect of pretreatment through Akt/HIF-1 and MAPK as well as the NF-κB pathway.4.The mRNA and protein changes of MAPK,NF-κB and Akt/HIF-1 signaling pathways in the neuroprotective effect of 30mg/kg STVNa on MCAO were detected.Conclusion1.STVNa inhibits cell inflammation and apoptosis by inhibiting the activation of the MAPK and NF-κB pathways induced by CoCl₂.2.STVNa plays a neuroprotective role by up-regulating the Akt/mTOR/HIF-1 signaling pathway.