Cynandione A alleviates neuropathic pain through spinal microglial interleukin-10/β-endorphin expression following α7 nicotinic acetylcholine receptor activation

Abstract Background Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, exhibits antihypersensitivity effects in neuropathic pain. This study sought to explore the target molecule and mechanisms underlying cynandione A mechanical antiallodynia, particularly related to the spinal glial expression of IL-10/β-endorphin, cAMP/PKA/p38/CREB signaling and α7 nicotinic acetylcholine receptor (α7 nAChR) activation. Methods IL-10 and β-endorphin in the spinal cord of spinal nerve ligation-induced neuropathic pain rats and cultured primary microglia were assessed by qRT-PCR and ELISA assays. Double immunofluorescence staining of IL-10, β-endorphin with glial and neuronal cellular biomarkers was also conducted in the spinal cord and cultured primary microglia. Microglial phosphorylation of PKA, p38, CREB and STAT3 were detected using western blot. Results Cynandione A significantly attenuated mechanical allodynia in neuropathic rats and substantially increased IL-10 and β-endorphin (but not dynorphin A) expression in the spinal cords and cultured primary microglia. The IL-10 antibody attenuated cynandione A-induced spinal or microglial gene expression of β-endorphin and mechanical antiallodynia, whereas the β-endorphin antiserum blocked cynandione A-induced mechanical antiallodynia but not spinal or microglial IL-10 gene expression. The α7 nAChR antagonist methyllycaconitine significantly declined cynandione A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. Cynandione A stimulated microglial phosphorylation of PKA, p38 and CREB, which was inhibited by methyllycaconitine. Treatment with the adenylyl cyclase inhibitor DDA, PKA inhibitor H-89, p38 inhibitor SB203580 and CREB inhibitor KG501 attenuated cynandione A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. Cynandione A stimulated spinal phosphorylation of the transcription factor STAT3, which was inhibited by methyllycaconitine, H-89 and the IL-10 antibody. The STAT3 inhibitor NSC74859 weakened cynandione A-induced mechanical antiallodynia and spinal expression of β-endorphin. Conclusion Our results illustrate that cynandione A produces mechanical antiallodynia through spinal microglial expression of IL-10 via the cAMP/PKA/p38/CREB signaling and subsequent β-endorphin expression via the IL-10/STAT3 signaling, following α7 nAChR activation.