Modulation Of Isosteviol Sodium In Oxygen-Glucose Deprived And Reoxygenation (OGDR)-Stressed Microglia

Stroke,an extremely dangerous neurological disease,is considered the second deadliest disease in the world.Restoring blood flow with thrombolytic agents in the early stages of stroke is the main method to save patients with cerebral ischemia.In addition to timely medical intervention,the patients subsequently need neurotherapeutic drugs to prevent the ischemic-reperfusion injury（I/R）resulted from thrombolytic agents’ administration.Isostevia sodium（STVNa）is a plant-derived compound that has proven neuroprotective effect in cerebral ischemia in vivo and in vitro models.Microglia,the brain immune cells that induce neuroinflammation events in cerebral ischemic brain,often aggravate ischemic injury and worsen the cerebral infarction.In this study,we established an in vitro ischemia-reperfusion injury（I/R）model with 4 hours of oxygen-glucose deprivation and 24 hours of reoxygenation（OGDR）on BV2 microglia to explore the biological dynamics of OGDR-stressed microglia and STVNa treatment.Methods and Results1.BV2 murine microglial cells were used to establish an in vitro ischemia-reperfusion injury（I/R）model in 4-hour oxygen glucose deprivation and 24-hour reoxygenated normal culture（OGDR）.Apoptotic detection by the Real Time-Glo ^TM Annexin V kit showed that five different concentrations of STVNa treatment reduce the expression of phosphatidylserine（PS）in OGDR-stressed microglia during the entire 24-hour reoxygenation process.However,no significant changes in caspase-9 expression were observed in all experimental groups,which suggests that OGDR-stressed BV2 microglia may in the acute phase of ischemia.This cell model can be used for the study of microglia in the early stage of cerebral ischemia.2.Activated microglia exacerbates the neuroinflammation during cerebral ischemia,we focused on the inflammatory factors and energy metabolism of OGDR-stressed microglia.The inflammatory cytokines level in OGDR-stressed microglia were determined by enzyme-linked immunofluorescence and luminescence.Two concentrations of STVNa（5,10μM）treatments significantly reduced protein levels of nitric oxide（NO）,tumor necrosis factor alpha（TNF-α）and active caspase-1.3.Total RNA was extracted from OGDR-stressed and STVNa treatment BV2 microglia to make an RNA-seq library.Differential gene expression analysis,KEGG-pathway and Gene Ontology enrichment analysis showed that the genetic differences of OGDR-stressed microglia are manifested in the biological regulation process,involving multiple pathways such as inflammation and apoptosis.A total of 112 differentially expressed genes were identified between OGDR-stressed and 10 μM STVNa treatment microglia.The key driver gene between OGDR-stressed microglia and STVNa treatment group were Nfkbiz,Il1 rn,Nlrp3 and Acod 1.4.The energy metabolisms of microglia were determined by monitoring the real time ATP,glycolysis,and mitochondrial respiration.OGDR-stressed microglia showed glycolysis reliance while STVNa reduced this aberrant glycolytic ATP production,which suggests that STVNa reduced the early apoptosis and inflammatory response of OGDR-stressed microglia by modulating microglia’s bioenergy profile.It proposes the relationship between anti-inflammatory and anti-apoptotic effect of STVNa and bioenergy metabolism in OGDR-stressed microglia.ConclusionIn this study,we established a feasible ischemic-reperfusion injury（I/R）model for microglia cell bioenergy detection.STVNa treatment showed a significant inhibition on microglial inflammatory responses and reverted the aberrant microglial bioenergy profile.It suggests that STVNa may exerts its neuroprotective effect via inhibiting inflammation and modulating bioenergy metabolism in microglia.This study can serve as a foundation to further validate the hypotheses in primary microglia and animal models.