Andrographolide (Andro), a labdane diterpene, possesses anti-inflammatory properties and has been used to treat numerous inflammatory diseases. Novel findings revealed that Andro might be vital in regulating pain. However, the contribution of Andro to chronic inflammatory pain has yet to be determined, and its underlying mechanism of action remains unknown. In this study, we observed that Andro attenuated mechanical allodynia in inflammatory pain mice induced by injecting complete Freund’s adjuvant (CFA) into the right hind paws. This analgesic effect of Andro is mainly dependent on its inhibition of microglial overactivation and the release of proinflammatory cytokines (TNF and IL-1β) in lumbar spinal cords of inflammatory pain model mice. More importantly, our data in vivo and in vitro revealed a negative role for Andro in regulating the TLR4/NF-κB signaling pathway, which might contribute to the inhibition of spinal microglial activation and proinflammatory cytokines production, and the improvement of paw withdrawal thresholds in a mouse model of chronic inflammatory pain evoked by CFA. We further found the potential interaction of Andro with TLR4/myeloid differentiation factor 2 heterodimer using molecular modeling, implying that TLR4 might be a potential target for Andro to exert an analgesic effect. Taken together, our findings demonstrated that the modulation of spinal microglial activation by Andro might be substantially conducive to managing chronic pain triggered by neuroinflammation.

穿心莲内酯 (Andro) 是一种拉丹烷二萜，具有抗炎特性，已用于治疗多种炎症性疾病。新的研究结果表明，Andro 可能对于调节疼痛至关重要。然而，Andro 对慢性炎症疼痛的贡献尚未确定，其潜在作用机制仍不清楚。在这项研究中，我们观察到 Andro 减轻了通过向右后爪注射完全弗氏佐剂 (CFA) 引起的炎性疼痛小鼠的机械异常性疼痛。Andro的这种镇痛作用主要依赖于其对炎性疼痛模型小鼠腰脊髓中小胶质细胞过度激活和促炎细胞因子（TNF和IL-1β）释放的抑制。更重要的是，我们的体内和体外数据揭示了 Andro 在调节 TLR4/NF-κB 信号通路中的负面作用，这可能有助于抑制脊髓小胶质细胞活化和促炎细胞因子的产生，并提高小鼠的缩爪阈值。 CFA 诱发慢性炎症疼痛的小鼠模型。我们进一步利用分子模型发现了Andro与TLR4/骨髓分化因子2异二聚体的潜在相互作用，这意味着TLR4可能是Andro发挥镇痛作用的潜在靶点。综上所述，我们的研究结果表明，Andro 对脊髓小胶质细胞激活的调节可能大大有助于控制神经炎症引发的慢性疼痛。