Mechanisms Underlying Bullatine A And Cynandione A Antinociception In Chronic Pain

Aconiti brachypodi Radix（Xue-shang-yi-zhi-hao）has been prescribed to manage chronic pain,arthritis and traumatic injuries.Bullatine A,a C₂₀-diterpenoid alkaloid,is one of its principle effective compounds.The first part of the study aimed to investigate the anti-hypersensitivity of bullatine A in a variety of rat pain models and explore its mechanisms of action.Rat neuropathic pain,inflammatory pain,diabetic neuropathic pain,and bone cancer pain models were used.Dynorphin A and pro-inflammatory cytokines were measured in the spinal cord and cultured primary microglia.Double immunofluorescence staining of dynorphin A and glial and neuronal cellular markers was also measured in the spinal cord.Our results showed subcutaneous and intrathecal injection of bullatine A dose-dependently attenuated spinal nerve ligation-,complete Freud’s adjuvant-,diabetes-,and bone cancer-induced mechanical allodynia and thermal hyperalgesia,with the efficacies of 45-70%inhibition,and ED₅₀s of 0.9-1.9 mg/kg for subcutaneous injection.However,bullatine A was not effective in blocking acute nociceptive response in the normal condition.Bullatine A specifically stimulated dynorphin A expression in microglia in the spinal cord in vivo and cultured primary microglia in vitro;the stimulatory effects were completely inhibited by the microglial inhibitor minocycline.In contrast,bullatine A did not have an inhibitory effect on peripheral nerve injury-or lipopolysaccharides-induced pro-inflammatory cytokine expression.The spinal anti-allodynic effects of bullatine A were entirely blocked by intrathecal injection of minocycline,the specific dynorphin A antiserum,and the selectiveκ-opioid receptor antagonist.We,for the first time,demonstrate that bullatine A specifically attenuates pain hypersensitivity,regardless of the pain models employed.The results also suggest that stimulation of spinal microglial dynorphin A expression mediates bullatine A anti-nociception in pain hypersensitivity conditions.Next,We assessed the interaction between bullatine A and morphine on antinociception in acute nociception and pain hypersensitivity states,with the exogenous synthetic dynorphin A as a comparison.Single subcutaneous injection of bullatine or dynorphin A（1-17）did not alter formalin-or thermally（hot-plate and water immersion tests）-induced acute nociception in na?ve mice.In contrast,bullatine A dose-dependently inhibited formalin-induced tonic pain with the efficacy of 54%inhibition and the half-effective dose of 0.9 mg/kg.Concurrent bullatine A additively enhanced morphine antinociception.In neuropathic rats,the antinociceptive effects of multiple bidaily intrathecal injections of bullatine A and dynorphin A remained consistent over 13 days,whereas morphine produced progressive and complete tolerance to antinociception,which was completely inhibited by concurrent bullatine A and dynorphin.A single injection of bullatine A and dynorphin A immediately reversed established morphine tolerance and also blocked morphine tolerance,although there was a less degree in the thermally-induced mouse acute nociceptive tests.The inhibitory effects of bullatine A and dynorphin A on morphine tolerance were immediately and completely attenuated by intrathecal dynorphin A antiserum and/or selectiveκ-opioid receptor antagonist GNTI.Our results suggests that bullatine A produce antinociception without induction of tolerance and inhibit morphine antinociceptive tolerance,and provided pharmacological basis for concurrent bullatine A and morphine treatment of chronic pain and morphine analgesic tolerance.Cynanchi Wilfordii Radix（baishouwu）,a medicinal herb,has been widely used in Asia to treat a variety of diseases or illnesses.Cynandione A isolated from C.Wilfordii is the principle acetophenone and exhibits neuroprotective and anti-inflammatory activities.This study aims to evaluate the antihypersensitivity activities of cynandione A in neuropathy and explored its mechanisms of action.Intrathecal injection of cynandione A dose-dependently attenuated spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia,with maximal possible effects of 57%and 59%,ED₅₀s of 14.9μg and 6.5μg,respectively.Intrathecal injection of cynandione A significantly increasedβ-endorphin levels in spinal cords of neuropathic rats and its treatment concentration-dependently inducedβ-endorphin expression in cultured primary microglia（but not in neurons or astrocytes）,with EC₅₀s of 38.8 and 20.0μM,respectively.Cynandione A also non-selectively upregulated phosphorylation of mitogen-activated protein kinases（MAPKs）,including p38,extracellular signal regulated kinase（ERK1/2）,and extracellular signal regulated kinase（JNK）in primary microglial culture;however,cynandione A-stimulatedβ-endorphin expression was completely inhibited by the specific p38 activation inhibitor SB203580,but not by the ERK1/2 or JNK activation inhibitors.Knockdown of spinal p38βbut not p38αusing siRNA also completely blocked cynandione A-inducedβ-endorphin expression in cultured microglial cells.Furthermore,cynandione A-induced antiallodynia in neuropathy was totally inhibited by the microglial inhibitor minocycline,SB203580,anti-β-endorphin antibody,andμ-opioid receptor antagonist CTAP（but not theκ-orδ-opioid receptor antagonist）.These results suggest that cynandione A attenuates neuropathic pain through upregulation of spinal microglial expressionβ-endorphin via p38βMAPK activation.Our study,for the first time,charaterized the antinociceptive effects of bullatine A and cynandione A,two priniciple effective compounds from herbal medicine in chronic pain.Also,for the first time,we revealed that bullatine A and cynandione A exhibited antinociception in chronic pain through stimulating spinal microglial expression of opioid peptides dynorphin A andβ-endorphin,respectively.Furthermore,Our study provided new evidence for the important role of expression of microglial opioid peptides in chronic pain.