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Assessing the Anti-Inflammatory Activity of the Anxiolytic Drug Buspirone Using CRISPR-Cas9 Gene Editing in LPS-Stimulated BV-2 Microglial Cells.

ABSTRACT: Buspirone is an anxiolytic drug with robust serotonin receptor 1A (Htr1a) agonist activities. However, evidence has demonstrated that this drug also targets the dopamine D3 receptor (Drd3), where it acts as a potent antagonist. In vivo, Drd3 blockade is neuroprotective and reduces inflammation in models of Parkinson's disease. To test if buspirone also elicited anti-inflammatory activities in vitro, we generated stable Drd3^-/- and Htr1a^-/- BV2 microglial cell lines using CRISPR-Cas9 technology and then tested the effects of buspirone after lipopolysaccharide (LPS) challenge. We found that LPS exposure had no effect on cell viability, except in Htr1a^-/- cells, where viability was reduced (p < 0.001). Drug treatment reduced viability in Drd3^-/- cells, but not in WT or Htr1a^-/- cells. Buspirone counteracted LPS-induced NO release, NOS2, IL-1β and TNF-α gene expression in WT cells, whereas it exerted limited effects in Drd3^-/- or Htr1a^-/- microglia. In summary, our findings indicate that buspirone attenuates microglial polarization after LPS challenge. These results also highlight some major effects of Drd3 or Htr1a genetic ablation on microglial biology, raising important questions on the complex role of neurotransmitters in regulating microglia functions.

SUBMITTER: Thomas Broome S

PROVIDER: S-EPMC8229595 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.