Trifluoro-icaritin alleviates chronic inflammatory pain through α7nAChR-mediated suppression of HMGB1/NF-κB signaling in the spinal cord of rats

Inflammatory pain is a chronic, persistent and serious disease that greatly impacts public health, which is often accompanied by allodynia, hyperalgesia, and spontaneous pain. It is evident that α7 nicotinic acetylcholine receptor (α7nAChR) plays a key role in cholinergic anti-inflammatory pathway and exhibits the inhibition of neuroinflammation in chronic pain. Trifluoro-icaritin (ICTF), a derivative of icaritin from the extract of a genus of Epimedium plant, is identified to possess profound anti-inflammatory activity. However, whether ICTF has anti-nociceptive effect on inflammatory pain and its potential mechanisms remain poorly elucidated. Intraperitoneal injection (i.p.) of ICTF to complete Freund's adjuvant (CFA)-induced inflammatory pain rats once daily for 21 consecutive days. Pain-related behaviors were evaluated with paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and CatWalk gait analysis. Expression of pain-related signaling molecules in the spinal cord were detected using qRT-PCR, western blot assay, and immunofluorescence staining. This results showed that ICTF (3.0 mg/kg, i.p.) effectively alleviated mechanical allodynia and thermal hyperalgesia not 0.3 and 1.0 mg/kg in CFA rats. Subsequently, we further observed that ICTF (3.0 mg/kg) dramatically decreased the mRNA and protein levels of HMGB1, NF-κB p65, and IL-1β but markedly enhanced α7nAChR and IL-10 expression in the spinal cord of CFA rats, and Immunofluorescence staining also showed that ICTF (3.0 mg/kg) significantly increased the expression of α7nAChR and reduced IBA1 in the spinal cord of CFA rats, along with suppressing the alterations of gait parameters induced by CFA. Moreover, Intrathecal injection (i.t.) of α7nAChR antagonist alpha-bungarotoxin (α-Bgtx, 1.0 μg/kg) not only reversed the anti-nociceptive effect of ICTF on pain hypersensitivity, but also inhibited the down-regulation of HMGB1, NF-κB p65, and IL-1β as well as the up-regulation of α7nAChR and IL-10 protein expression induced by ICTF treatment. Altogether, our results illustrate that ICTF enables to ameliorate CFA-induced inflammatory pain through α7nAChR-mediated inhibition of HMGB1/NF-κB signaling pathway in the spinal cord of rats, suggesting that ICTF may be exploited as a potential painkiller against chronic inflammatory pain.