Abstract
We aimed to investigate the anti-inflammatory role of fluoxetine, a selective serotonin reuptake inhibitor, in microglia (MG) and the mechanisms under oxygen glucose deprivation/reoxygenation (OGD/R). An OGD/R model on BV-2 cells was used for the study of microglia under ischemia/reperfusion injury in ischemic stroke. Lentiviral transfection was applied to knock down IκB-α. Enzyme-linked immunosorbent assay (ELISA) was used for detecting levels of TNF-α, IL-1β, and IL-6, and real-time PCR was used to assess the expression of IκB-α protein. Western blotting was applied to analyze NF-κB-signaling related proteins and Cell Counting Kit-8 (CCK-8) was used for assessing cell viability. Molecular docking and drug affinity responsive target stability (DARTS) assay were used for the detection of the interaction between IκB-α and fluoxetine. We found that fluoxetine decreased the levels of TNF-α, IL-1β, and IL-6 in supernatant as well as NF-κB subunits p65 and p50 in BV-2 cells under OGD/R. Fluoxetine significantly increased the level of IκB-α through the inhibition of IκB-α ubiquitylation and promoted the bonding of IκB-α and fluoxetine in BV-2 cells under OGD/R. Knocking down IκB-α attenuated the decreasing effect of TNF-α, IL-1β, and IL-6 as well as p65 and p50 in BV-2 cells under OGD/R led to by fluoxetine. In conclusion, our present study demonstrated the anti-inflammatory role of fluoxetine and its mechanisms related to the modulation of NF-κB-related signaling in MG under ischemia/reperfusion challenge.
Highlights
Ischemic stroke is one of the common age-related diseases worldwide [1,2,3]
Cells subjected to oxygen glucose deprivation/reoxygenation (OGD/R) challenge were kept in glucose-free Dulbecco’s modification of Eagle’s medium (DMEM) (Gibco) under 5% CO2 and 95% N2 for 2 h followed by 48-h culture under normal condition. 293TN cells, obtained from ATCC, were cultured in DMEM supplemented with 10% fetal calf serum (FCS) at 37◦C under 5% CO2
The levels of TNF-α, IL-1β, and IL-6 in supernatant obtained from the OGD/R group were increased compared with the control group, while the administration of fluoxetine significantly decreased the levels of TNF-α, IL-1β, and IL-6 in supernatant compared with the OGD/R group in a dose-dependent manner (Figure 1B)
Summary
Ischemic stroke is one of the common age-related diseases worldwide [1,2,3]. On the occurrence of ischemic stroke, rapture or blockage of blood vessels causes obstructed circulation, resulting in hypoxia injury of cerebral cells and tissues [4]. The main therapeutic approach is to recover blood and nutrient supply, which, if applied in time, will effectively alleviate the severity of ischemic stroke. This traditional therapy has been increasingly questioned by modern clinical studies, which pointed out several related adverse effects [5,6]. Circulation recovery, commonly referred to as reperfusion, has been considered to lead to cerebral inflammatory responses via the activation of microglia (MG) after ischemia. Suppressing the inflammatory responses triggered by the activation of MG under ischemia/reperfusion challenge might provide a potential therapeutic pathway in the alleviation of damage of neuronal function under cerebral ischemia [9,10,11]
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