A Study On The Inhibitory Role Of Quercetin In Microglial Cell-Derived Oxidative Stress And Inflammatory Response And Effect Of Quercetin On Hypoxic-Ischemic Brain Injury Neurobehavior In Neonatal Mice

Part 1 Inhibitory role of quercetin in microglial cell-derived oxidative stress and inflammatory responseBackground : Neonatal hypoxic-ischemic encephalopathy(HIE)means cerebral hypoperfusion in newborn babies(fetuses)caused by perinatal asphyxia,which is regarded as a clinical challenge as it is accompanied by acute brain injury attributed to cerebral anoxia and usually induces a series of nervous system sequelae.With microglial cell-mediated neuroinflammation(MCMN)being a crucial player in HIE development and progression,it offers a potential direction to resolve HIE through attenuation of MCMN by regulating the process of microglial cell activation.Quercetin(3,3’,4’,5,7-pentahydroxyflavone)is a natural flavonoid that has been proven to benefit patients with nervous system disease.However,it has not been fully clarified the application and protective mechanism of quercetin as a treatment for HIE.Objectives:To investigate the effect of quercetin at different concentrations on the oxidative stress and inflammatory response of microglial BV2 cells subject to in vitro oxygen glucose deprivation(OGD)and confirm whether the toll-like receptor 4(TLR4)signaling pathway is involved in this process.Methods:The mouse microglial cell line BV2 was exposed to OGD to mimic hypoxic ischemia(HI),and the cells were assigned to a control group,an OGD+DMSO group,and an OGD+QUE group.In the OGD+QUE group,quercetin was given at varying concentrations.The Cell Counting Kit-8(CCK-8)was employed to determine cell viability.Secretion of lactate dehydrogenase(LDH)was measured to assess cell damage.The q RT-PCR and ELISA were conducted to measure proinflammatory cytokine(including IL-1β,IL-6 and TNF-α)m RNA and protein expression.Levels of cellular malondialdehyde(MDA)and reactive oxygen species(ROS)were determined.The Western blot(WB)test was performed to detect TLR4,My D88,and NF-κB expression.Results:Quercetin,with negligible cytotoxicity to BV2 cells,protects BV2 cells from OGD and reverses OGD-induced BV2 cell damage and morphological changes.At the concentrations of 20 μM and 40 μM,quercetin significantly suppressed OGD-induced ROS and MDA in BV2 cells,reserved the OGD-induced decreases in superoxide dismutase 1(SOD1),superoxide dismutase 2(SOD2),glutathione peroxidase-1(GPX-1),and catalase(CAT)expression and substantially downregulated IL-1β,IL-6 and TNF-α expression in post-OGD BV2 cells or supernatant.In short,quercetin reversed changes in oxidative stress-related molecules in microglial BV2 cells;additionally,the WB test results showed that quercetin inhibited the expression of relevant molecules related to the iba1 and TLR4/My D88/NF-κB signaling pathways in post-OGD BV2 cells.Conclusions:1.Quercetin reverses molecular attenuation associated with the oxidative stress on microglial BV2 cells;2.quercetin can alleviate OGD-induced BV2 cell damage and suppress the subsequent inflammatory response,which is partly attributed to its inhibitory effect on OGD-induced cellular oxidative stress and TLR4-mediated inflammatory response;3.quercetin inhibits the expression of iba1 and TLR4/My D88/NF-κB signaling pathway-related molecules in BV2 cells exposed to OGD.Part 2 Effect of quercetin on post-hypoxic-ischemic brain injury neurobehavior in neonatal miceBackground : HIE often leads to serious nervous system sequelae such as cerebral palsy,epilepsy,and cognitive impairment,which can result in neurobehavioral disorders(including movement,cognition,emotion)in pediatric patients.Currently,HIE animal models(e.g.,HIBI mouse model)are constructed by ligation and hypoxic treatment of the right common carotid artery.The efficacy of a treatment method can be determined through observation and evaluation of long-term neurobehavioral changes in such HIBI mice.Objective:To investigate the protective effect of quercetin against brain injury through cerebral infarction volume measurement and neurobehavioral assessment following in vivo quercetin intervention of HIBI mice.Methods : A classic Rice–Vannucci,neonatal mouse HIBI model was constructed,and the mice were divided into three groups,namely a sham group,a HI+PBS group,and a HI+QUE group.Cerebral infarction volume was measured and neurobehavioral assessment was undertaken: Open field,Morris water maze(WMW)and cylinder tests were carried out to assess the neuroprotective effect of quercetin.Results:Place navigation in the MWM showed that quercetin reversed the serious impairment of spatial learning ability in the HI+PBS group;results of the spatial probe suggested that the three groups did not differ greatly in swim speed;yet,quercetin was shown to improve HI-induced memory disorders after removal of the platform.In the open field test,there was no significant difference among the three groups in total swim distance or average swim speed;a greater number of mice in the HI+QUE group entered and spent more time in the center zone.According to the cylinder test results,the HI mice tended to use the unaffected(right)forelegs,while their left forelegs had motor dysfunction;quercetin helped improve the asymmetric motions of the forelegs.In terms of brain morphology,the HI+PBS group exhibited severe infarction,and the cerebral infarction volume was reduced after treatment with quercetin.Conclusion:Quercetin can effectively reduce the cerebral infarction volume in HIBI mice and alleviate cognitive and movement deficits.