The Roles Of Edaravone And Gpx4 In Depressive-like Behavior Induced By Chronic Social Defeat Stress Of Mice

PART 1 THE ROLE OF EDARAVONE IN DEPRESSIVE-LIKE BEHAVIOR INDUCED BY CHRONIC SOCIAL DEFEAT STRESS OF MICEObjectiveDepression is a common neuropsychiatric disease that seriously harms human health.Its pathogenesis is complex and diverse.Oxidative stress and inflammation are one of the important pathological mechanisms of depression.Edaravone(EDA)processes strong biological activities including antioxidant,anti-inflammatory and neuroprotective effects.It is commonly used to treat acute ischemic stroke.Some studies have shown that patients using EDA can relieve depression.However,the molecular mechanisms of EDA remain unclear.This study aims to investigate the effects of EDA on chronic social defeat stress(CSDS)-induced depressive-like behavior in mice.Methods1.Depressive-like behavior was induced by CSDS model of mice.Depression,anxiety and learning and memory abilities were evaluated by social interaction(SI),sucrose preference test(SPT),open field test(OFT),elevated plus maze(EPM),tail suspension test(TST),forced swimming test(FST),and novel object recognition(NOR).2.Hippocampus(Hip)and medial prefrontal cortex(m PFC)tissues of mice were collected for Nissl staining,immunofluorescence staining,targeted energy metabolomics,enzyme-linked immunosorbent assay(ELISA),superoxide dismutase(SOD),glutathione(GSH),glutathione peroxidase(GSH-PX),total antioxidant capacity(T-AOC)and transmission electron microscopy(TEM).3.Western blotting(WB)and real-time quantitative PCR(RT-q PCR)were used to detect silent information regulator 2 homolog 1(Sirt1),nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1)and glutathione peroxidase 4(Gpx4)expressions.4.EDA and Sirt1 inhibitor EX527 or Nrf2 inhibitor ML385 were injected intraperitoneally to detect their effects on depressive-like behavior of mice.Gpx4 in the Hip was silenced by adeno-associated virus to detect its effects on depressive-like behavior of mice.Results1.Intraperitoneal injection of EDA reversed the anxiety and depressive-like behaviors in CSDS-induced mice,which indicated by increased social ratio(p<0.05),sucrose preference(p<0.05),time in the central area of OFT(p<0.01),time in open arms of EPM(p<0.05)and decreased immobility time in TST and FST(p<0.05).2.EDA attenuated CSDS-induced neuronal loss,microglia activation,astrocyte dysfunction,oxidative stress injury,energy metabolism and pro-inflammatory cytokines activation in the Hip and m PFC regions of mice.3.The protein expression levels of Sirt1(p<0.05),Nrf2(p<0.05),HO-1(p<0.001)and Gpx4(p<0.001)in the Hip regions of CSDS mice were significantly decreased,these changes were reversed by EDA administration.4.Sirt1 inhibitor EX527 decreased the protein levels of Sirt1(p<0.01),Nrf2(p<0.01),HO-1(p<0.001)and Gpx4(p<0.01)and abolished the antidepressant effect of EDA.5.Nrf2 inhibitor ML385 reversed the antidepressant and anxiolytic effects of EDA and reduced the protein expression levels of Nrf2(p<0.001),HO-1(p<0.01)and Gpx4(p<0.01)in Hip,but had no effect on the protein expression of Sirt1.6.Silencing the Gpx4 gene in the Hip region abolished the antidepressant and anxiolytic effects of EDA.ConclusionsEDA has anxiolytic and antidepressant effects and can alleviate anxiety and depressive-like behaviors in mice through Sirt1/Nrf2/HO-1/Gpx4 signaling pathway.PART 2 THE ROLE OF GPX4 IN DEPRESSIVE-LIKE BEHAVIOR INDUCED BY CHRONIC SOCIAL DEFEAT STRESS OF MICEObjectiveGpx4,also known as phospholipid hydroperoxide glutathione peroxidase(PHGPx),is an antioxidant enzyme belonging to the glutathione peroxidase(GSH-PX)family(The fourth of GSH-PX family).It reduces the conversion of lipid hydroperoxides to lipid alcohols,which are important for maintaining lipid homeostasis in cells,and prevent the accumulation of toxic lipid peroxides,which in turn prevents ferroptosis.It has been confirmed that ferroptosis is related to various neurological diseases,including Alzheimer disease(AD),Parkinson’s disease(PD),traumatic brain injury(TBI),epilepsy and stroke.However,there is still lack of researches on the involvement of ferroptosis in psychiatric diseases,especially depression.The absence of Gpx4 is an important feature of ferroptosis,so this part aims to explore the role and mechanism of Gpx4 in depression.Methods1.Gpx4 levels in serum of major depressive disorder(MDD)and CSDS mice were measured by ELISA.2.Depressive-like behavior was induced by CSDS model.The protein level of Gpx4 in depression-related brain regions including Hip,m PFC,nucleus accumbens(NAc),amygdala and hypothalamus was detected by WB.3.Gpx4 overexpression virus with adeno-associated virus(AAV)vector was injected into the Hip region.We then detected depressive-like behavior and ferroptosis-related indicators including malondialdehyde(MDA),SOD and GSH-PX.4.Gpx4 knockdown virus was injected into the Hip brain region of mice and the subthreshold social defeat stress(SSDS)model was used to detect the effect on stress susceptibility of mice.5.Transcriptomics detection was performed on mice injected with Gpx4 knockdown virus and control mice,and bioinformatics were used to analyze differentially expressed genes and predict the downstream targets of Gpx4.Results1.The serum levels of Gpx4 in MDD and CSDS susceptible mice were significantly decreased.2.The protein expression of Gpx4 in the Hip was significantly decreased(p<0.001),and the expression levels of Gpx4 in m PFC,NAc,amygdala and hypothalamus were not changed.3.Injection of Gpx4 overexpression virus into the Hip abolished CSDS-induced depressive-like behaviors in mice,including significantly increased social ratio(p<0.01),sucrose intake(p<0.05),and decreased immobility time in FST(p<0.05).After the CSDS model,the MDA level in the Hip of mice was significantly increased(p<0.001).Injection Gpx4 overexpression virus in the Hip of mice decreased MDA level(p<0.05)and increased SOD(p<0.01)and GSH-PX activities(p<0.05).4.Gpx4 knockdown virus enhanced stress susceptibility of mice,indicated by social avoidance behavior(p<0.01),decreasing sucrose preference rate(p<0.01)and increasing immobility time in FST(p<0.05).5.Bioinformatics analysis showed endoplasmic reticulum stress-related proteins may be the downstream targets of Gpx4 in depression.ConclusionsGpx4,as an essential factor of ferroptosis,plays an important role in CSDS-induced depressive-like behavior,and endoplasmic reticulum stress-related proteins may be the downstream targets of Gpx4 in depression.