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Wu-tou Decoction (WTD) is a classic herbal formula in traditional Chinese medicine for the treatment of joint diseases, neuropathic pain (NP) and inflammatory pain. In this study we investigated whether WTD produced analgesic action in a mouse spinal nerve ligation (SNL) model and elucidated the underlying molecular mechanisms. Mice were subjected to SNL and orally treated with WTD (3.15, 6.30 or 12.60 g.kg11d-1) for 21 d. SNL induced mechanical hyperalgesia and heat hyperalgesia characterized by rapid and persistent pain hypersensitivity. In addition, the expression levels of IL-1β, TNF-α, CCL2 and CXCL1 in the spinal cord dorsal ho were dramatically increased on the 10th d post-surgery. Oral administration of WTD dose-dependently suppressed both mechanical and heat hyperalgesia as well as the expression levels of inflammatory cytokines in the spinal cord dorsal ho on the 21st d post-surgery. Then whole-genome microarray analyses were conducted to detect the gene expression profiles of spinal cord dorsal ho in SNL mice with or without WTD treatment. After construction of the WTD-SNL-network and topological analysis, a list of candidate target genes of WTD acting on SNL-induced NP was identified and found to be functionally enriched in several glial cell activation-related pathways and neuroinflammatory pathways. Our data have clarified the gene expression pattes in the mouse spinal cord under the NP condition. We also demonstrate the analgesic action of WTD through suppression of glial cell activation and neuroinflammation,which suggest the potential of WTD as a promising candidate for the treatment of NP.