Calycosin attenuates LPS-induced microglia inflammatory responses and microglia-mediated synaptic impairment via modulation of TLR4/MyD88/NF-κB signaling pathway

Abstract

Objective Neuroinflammation plays an important role in the initiation and progression of central nervous system (CNS) disorders. Calycosin (Caly) has been reported to exert anti-inflammatory and neuroprotective effects. However, little is known about its potential role in microglia-mediated neurotoxicity and the underlying mechanism. The aim of the present study is to investigate whether Caly attenuates LPS-induced inflammatory response in BV2 microglial cells and microglia-mediated neuronal injuries in HT-22 cells. Methods BV2 cells were stimulated with LPS (1 μg/mL, 24 h) and treated with Caly (5, 10, or 20 μM) or Dex (2 μM). Molecular docking, CETSA, ITDRFCETSA and DARTS assays were used to evaluate the potential target engagement between Caly and TLR4. Cell viability, microglial activation, inflammatory mediator expression, secreted cytokine levels, and TLR4/MyD88/NF-κB signaling pathway were examined. HT-22 cells were cultured with conditioned medium (CM) derived from LPS-stimulated or Caly-treated BV2 cells, cell viability and expression of synaptic plasticity-related proteins were evaluated. Supplementary validation was also performed in HMC3 human microglial cells and SH-SY5Y human neuronal cells. Results Stimulation with LPS could induce BV2 microglial activation, accompanied by the increased expression and secretion of pro-inflammatory mediators, and dysregulation of TLR4/MyD88/NF-κB signaling pathway. CM derived from LPS-stimulated BV2 cells decreased HT-22 cell viability and reduced synaptic plasticity-related protein expression levels. Caly treatment attenuated LPS-induced cytotoxicity and inflammatory responses, and reversed imbalanced expression of TLR4/MyD88/NF-κB signaling pathway in LPS-challenged BV2 cells, as indicated by the reduced Iba-1 fluorescence intensity, decreased mRNA expression levels and secretion of IL-1β, IL-6, and TNF-α, and decreased proteins expression of IL-17, TLR4, MyD88, p-NF-κB/NF-κB and p-IKB/IKB. Caly also increased the expression of the anti-inflammatory phenotype-associated markers CD206 and Arg1, while reducing the pro-inflammatory markers CD86 and iNOS. Moreover, CM derived from Caly-treated BV2 cells partially restored HT-22 cell viability and protein expression of β-Tubulin, PSD95, and Synapsin I. Supplementary experiments in HMC3 and SH-SY5Y cells showed similar anti-inflammatory and synaptic protein-preserving effects. Conclusion Caly could exert potential effects on suppressing LPS-induced microglia activation and inflammatory responses in immortalized BV2 cells by regulating TLR4/MyD88/NF-κB signaling pathway, and attenuating microglia-mediated neuronal injury and synaptic impairment in immortalized HT-22 cells.