Ruscogenin Exerts Anxiolytic‐Like Effect via Microglial NF‐κB/MAPKs/NLRP3 Signaling Pathways in Mouse Model of Chronic Inflammatory Pain

Long‐term inflammation can cause chronic pain and trigger patients' anxiety by sensitizing the central nervous system. However, effective drugs with few side effects for treating chronic pain‐induced anxiety are still lacking. The anxiolytic and anti‐inflammatory effects of ruscogenin (RUS), an important active compound in Ophiopogon japonicus, were evaluated in a mouse model of chronic inflammatory pain and N9 cells. RUS (5, 10, or 20 mg/kg/day, i.g.) was administered once daily for 7 days after CFA injection; pain‐ and anxiety‐like behaviors were assessed in mice. Anti‐inflammatory effect of RUS (0.1, 1, 10 μM) on N9 microglia after LPS treatment was evaluated. Inflammatory markers (TNF‐α, IL‐1β, IL‐6, CD86, IL‐4, ARG‐1, and CD206) were measured using qPCR. The levels of IBA1, ROS, NF‐κB, TLR4, P‐IKK, P‐IκBα, and P65, MAPKs (ERK, JNK, and P38), NLRP3 (caspase‐1, ASC, and NLRP3) were detected by Western blotting or immunofluorescence staining. The potential target of RUS was validated by molecular docking and adeno‐associated virus injection. Mice in CFA group exhibited allodynia and anxiety‐like behaviors. LPS induced neuroinflammation in N9 cells. Both CFA and LPS increased the levels of IBA1, ROS, and inflammatory markers. RUS (10 mg/kg in vivo and 1 μM in vitro) alleviated these alterations through NF‐κB/MAPKs/NLRP3 signaling pathways but had no effect on pain hypersensitivity. TLR4 strongly interacted with RUS, and TLR4 overexpression abolished the effects of RUS on anxiety and neuroinflammation. RUS exerts anti‐inflammatory and anxiolytic effects via TLR4‐mediated NF‐κB/MAPKs/NLRP3 signaling pathways, which provides a basis for the treatment of chronic pain‐induced anxiety.