Calycosin attenuates LPS-induced microglia inflammatory responses and microglia-mediated synaptic impairment via...

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.

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, ITDRF_CETSA 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.

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.

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.