A Study On The Inhibition Of OGD/R Inflammatory Activation In BV-2 Cells By Fluoxetine And The Mechanism

Objective:The NF-κB inflammatory signaling pathway is closely associated with the inflammatory activation of microglia(MG)induced by cerebral ischemia and reperfusion.Microglia,after activation,will secrete inflammatory factors and thus injure neurons.The regulation of the NF-κB pathway,therefore,is of significance for treatment of patients with cerebral ischemia.The development of anti-inflammatory drugs targeting proteins regulated by NF-κB has always been a research hotspot in the development of drugs for treatment of cerebral ischemia.IκB,as one of three chief links of the NF-κB pathway,is among the targets of studies on anti-inflammatory drugs.fluoxetine,a monoamine,is mainly used for improving injured nerve.In the study on the repair of neurons,it has been found that fluoxetine can protect the neuron by inhibiting inflammatory response in MG,but there has been no in-depth study on its anti-inflammatory action.Most inflammatory inducers,like lipopolysaccharide,activate NF-κB through inhibiting the expression of IκBα or increasing its degradation,so inhibition of the degradation of IκBα can effectively suppress the activation of inflammatory response.In the present study,we established an oxygen glucose deprivation/reoxygenation(OGD/R)model in the murine microglial cell line BV-2,to explore the anti-inflammatory role of fluoxetine and the mechanism,to provide a foundation for the development of drugs for treatment of cerebral ischemia.Contents:1.The inhibition of OGD/R inflammatory activation in BV-2 cells by fluoxetine was confirmed.Methods: BV-2 cells in logarithmic phase were seeded to 96-well plates,cultured overnight,added with fluoxetine dissolved in dimethyl sulfoxide(DMOS)to final concentrations of 0,1,2.5,5,10 and 20 μM,and cultured for 24 hours,followed by cell viability assays using Cell Counting Kit 8(CCK-8).The proliferation of all groups was compared,and the highest concentration without obvious cytotoxicity was used as the high dose for following experiments.BV-2 cells pretreated with fluoxetine(1,2.5 or 10 μM)for 2 h were subjected to OGD/R treatment and supernatants were collected 24 h after reoxygenation for examination of TNF α,IL-1β and IL-6 through enzyme linked immunosorbent assay(ELISA).Cells were collected and total RNA and protein were extracted separately for measurement of TNF α,IL-1β and IL-6 mRNA by real-time PCR and the phosphorylation of p65 and p50,two subunits of NF-κB,by western blotting.Results: According to the results of CCK-8 assays,treatment of 20 μM fluoxetine for 24 hours significantly decreased the proliferation of BV-2 cells(P < 0.05),and other concentrations had no detectable effect on BV-2 cells(P > 0.05).OGD/R treatment significantly enhanced the contents of TNF α,IL-1β and IL-6 in the supernatant of BV-2 cells(P < 0.01),and fluoxetine pretreatment impaired the increase,high dose of fluoxetine significantly reducing the increase caused by OGD/R(P < 0.05).And the decrease of the inflammatory factors was dependent on the dose of fluoxetine.There was no obvious difference in the inflammatory factors between the OGD/R + solvent group and the OGD/R group(P > 0.05).The mRNA levels of TNF α,IL-1β and IL-6 were in agreement with the level of the inflammatory factors in the supernatant.Western blotting showed that OGD/R treatment significantly increased the phosphorylation of p65 and p50 in BV-2 cells(P < 0.05),and pretreatment of fluoxetine suppressed the increase,the high dose of fluoxetine significantly reducing the increase caused by OGD/R(P < 0.05).The increase in fluoxetine dose corresponded to a decrease in the phosphorylation of p65 and p50.There was no obvious difference in phos-p65 and phos-p50 between the OGD/R + solvent group and the OGD/R group(P > 0.05).2.The binding of fluoxetine to IκBα was confirmed.Methods: molecular simulation refers to the approach for obtaining the structural information on the potential binding sites of the protein by site prediction and molecular probe scanning and to calculate the binding capacity and intermolecular interaction of specific ligands.We employed bioinformatics software to predict and analyze the binding sites of IκBα to fluoxetine.Based on the prediction of binding sites,we created an experimental system to confirm the binding through DARTS experiment.How will the binding of fluoxetine to IκBα affect the IκBα level in cells? We utilized G132,a proteasome inhibitor,and CHX to treat BV-2 cells to suppress protein degradation and synthesis and cells were collected at 0,1,2,4,and 8 h after treatment and total protein was extracted for IκBα measurement,cells being divided into control group and fluoxetine group(10 μM).Results: the molecular docking data showed that there are multiple binding sites of fluoxetine in IκBα.DARTS experiment confirmed that in the absence of fluoxetine,the IκBα protein in the control group and vehicle group decreased after treatment by 1:52 and 1:26(mg/L)protease,and in the presence of fluoxetine,there was no detectable change in IκBα level.The ubiquitination assay showed that IκBα was increased over time in BV-2 cells of the control group and fluoxetine group,after the protein degradation pathway was inhibited.IκBα decreased in the control group and remained unchanged in the fluoxetine group over time after the protein synthesis was inhibited.3.Fluoxetine inhibits the inflammatory activation of the NF-κB pathway by suppressing the ubiquitination of IκBα.Methods: A siRNA sequence targeting mouse IκBα was designed,and corresponding vector and recombinant lentivirus were obtained,and IκBα gene was knocked down in BV-2 cells.BV-2 cells with or without IκBα knockdown were pretreated with fluoxetine and OGD/R treatment and supernatants were collected 24 hours after reoxygenation for examination of TNF α,IL-1β and IL-6 through ELISA.And total protein was extracted and the phosphorylation of p65 and p50,two subunits of NF-κB,and the content of IκBα were measured by western blotting.Results: The sequencing results suggested that si RNA was successfully designed and siRNA expression vector was constructed.72 hafter viral infection,observation of green fluorescent protein(GFP)confirmed that the gene delivery efficiency in BV-2 cells was close to 100%.Real-time PCR data suggest that compared with Lv-control and negative control,Lv-shRNA-IκBα effectively decreased IκBα mRNA in BV-2 cells(P < 0.05).ELISA results showed that 24 h after OGD/R treatment,the contents of TNF α,IL-1β and IL-6 in the supernatant of BV-2 cells were higher than that in the control group or the vehicle group(P < 0.05),and fluoxetine pretreatment reduced TNF α,IL-1β and IL-6 levels;the TNF α,IL-1β and IL-6 levels in the supernatant from the BV-2 cells infected with Lv-shRNA-IκBα were higher than that of the fluoxetine + OGD/R group,or the LV-NC + fluoxetine + OGD/R group(P < 0.05).Western blotting results showed that OGD/R treatment enhanced the phosphorylation of p65 and p50 in BV-2 cells and inhibited the expression of IκBα(P < 0.05),and fluoxetine pretreatment reduced the phosphorylation of p65 and p50 induced by OGD/R and increased IκBα expression as well.Lv-shRNA-IκBα reversed the effect of fluoxetine pretreatment.Conclusion:This paper mainly explores the role of fluoxetine in inhibiting the inflammatory activation caused by OGD/R and the mechanism.In the study,we first confirmed the dose-dependent inhibition of OGD/R induced inflammatory activation in BV-2 cells by fluoxetine.The mechanism study showed that the anti-inflammatory role of fluoxetine is associated with the activation of NF-κB: fluoxetine inhibits the ubiquitination of IκBα.The molecular docking suggested that IκBα has multiple theoretical binding sites of fluoxetine,and DARTS validated that fluoxetine can bind to IκBα directly,and the ubiquitination experiment showed that the binding can inhibit the ubiquitination of IκBα,so IκBα remained at a high level,and combined with p65 and p50 to form trimer and inhibited the activation of NF-κB,blocking the inflammatory pathway.The direct binding of fluoxetine to IκBα and inhibition of ubiquitination explains fluoxetine’s anti-inflammatory role,which is of great importance for using fluoxetine to prevent the inflammatory activation of MG and injury of neurons in ischemic brain injury.