Mechanisms Underlying Bulleyaconitine A Antinociception In Chronic Pain

Bulleyaconitine A(BAA)is an aconitine-like alkaloid initially isolated from Aconitum bulleyanum plants,which distinctive grows at yunnan province of China.After extensive clinical use and investigation,the orally and injection available BAA was approved by the Food and Drug Administration of China in 1985,although there appear no reports of clinical investigations outside China.BAA has been widely prescribed for the management of chronic pain and rheumatoid arthritis that is listed in the 2015 edition Chinese Pharmacopoeia.The mechanism underlying BAA analgesia is unclear,although some studies have postulated that its blockage of neuronal voltage-dependent sodium channels was responsible.However,the relatively wide therapeutic window(LD₅₀/ED₅₀)of BAA suggested that its anti-nociception could be separated from its toxicity and was possibly due to the mechanisms other than interference with sodium channels on neurons in the central nervous system.In additon,previous publications indicated that the anti-nociceptive effects of the Aconitum extracts were found to be mediated by the stimulation of dynorphin A release and the activation of spinal?-opioid receptors.Accordingly,we hypothesized that BAA produces its anti-nociception by stimulating the release of dynorphin A from spinal microglia,which is separate from the blockage of neuronal sodium channels that presumably mediate toxicity.The present study aimed to evaluate the anti-nociceptive properties of BAA in the rat models of pain and morphine anti-nociceptive tolerance,and explore whether BAA exhibited anti-nociception through the stimulation of the release of dynorphin A from spinal microglia.A single intrathecal or subcutaneous(but not intraventricular or local)injection of BAA blocked spinal nerve ligation-induced painful neuropathy,bone cancer-induced pain and formalin-induced hyperalgesia by 60–100%with median effective dose(ED₅₀)values of 73–120 ng/rat(intrathecal)and 37–50?g/kg(subcutaneous).Following chronic treatment,BAA did not induce either auto-tolerance to anti-nociception or cross-tolerance to morphine anti-nociception,and completely inhibited morphine tolerance.Spinal BAA anti-nociception,but not neurotoxicity,was completely blocked by the specific microglial inhibitor,minocycline.In a minocycline-sensitive and lidocaine-or ropivacaine-insensitive manner,BAA stimulated the release of dynorphin A from the spinal cord and primary cultures of microglia,but not from neurons or astrocytes.BAA anti-nociception was completely blocked by the specific dynorphin A antiserum and?-opioid receptor antagonist.Though our results have demonstrated that BAA anti-nociception through the direct stimulation of dynorphin A release from spinal microglia,which was not dependent on sodium channels,the upstream mechanism underlying analgesic signaling pathway has not been clarified.BAA analog mesaconitine produced antinociception through elevated intracellular c AMP levels and other analog aconitine can activate phosphorylation of P38 MAPK.In additon,the transcription factor CREB has been shown to regulate dynorphin A expression.Considered together,the similarity in structure among of mesaconitine,aconitine and BAA prompted us to explore whether BAA induced dynorphin A release from microglia is dependent upon activation of spinal c AMP/PKA/p38 MAPK/CREB signaling pathway.In order to validate these assumptions,spinal nerve ligation-induced neuropathic pain models were emoloyed,the expression of prodynorphin was measured using the real-time quantitative PCR and the phosphorylation of PKA,p38 MAPK,ERK,JNK,CREB and NF-?B were monitored by western blot.BAA dramatically enhanced c AMP accumulation in primary microglial cells in a dose-dependent manner compared to vehicle treatment.BAA anti-nociception were completely prevented by the adenylate cyclase inhibitor DDA,the PKA inhibitor H89,the p38 MAPK inhibitor SB203580and the CREB inhibitor KG501 rather than the ERK1/2 inhibitor U0126 and the JNK inhibitor SP600125.Moreover,BAA-increased prodynorphin gene expression were completely blocked by SB203580 and KG501,neither the SP600125 nor the U0126significantly inhibited BAA-increased prodynorphin expression.Furthermore,BAA significantly increased phosphorylation of PKA,p38 MAPK and CREB rather than NF-?B.Meanwhile,BAA-induced p38 MAPK activation were completely blocked by treatment with H89 and DDA,and SB203580 suppress BAA induced CREB activity.Our resultes demonstrated that BAA eliminated pain hypersensitivity and morphine tolerance through the direct stimulation of dynorphin A release from spinal microglia,which was not dependent on the sodium channel,and suggested that the anti-nociception of BAA can be separated from its neurotoxicity.Furtherore,our resultes also indicate BAA significantly induced the production of c AMP and activation of c AMP-dependent PKA,subsequently,PKA can regulate the activity of p38 and its translocation to the nucleus,and then phosphorylates the transcription factor CREB,leading to increase prodynorphin expression and produce anti-nociception.As summarized these process,BAA exert its analgesia by activation of the spinal cyclic AMP/PKA/p38 MAPK/CREB signaling pathway.On the other hand,we also compare to aconitine and its hydrolytic analogs benzolyaconine and aconine on antinociception,dynorphin A release and neurotoxicity:implications of the Aconitium processing.As we all known,aconitines,probably the most bioactive and abundant in Aconitum plant,are a group of diester C19-diterpenoid alkaloids with one acetylester group attached to the C8 of the skeleton and one benzoylester group to the C14,both of which are involved in the Aconitum processing from the traditional Chinese medicinal approach.We have recently made conceptual advancement and demonstrated that bulleyaconitine produced anti-hypersensitivity,which was entirely mediated by the stimulation of the spinal microglial dynorphin expression.The present study aimed to elucidate whether the acetyl and benzoylester groups are involved in aconitine-induced dynorphin A expression,anti-nociception and neurotoxicity in a rat model of neuropathy.Intrathecal aconitine and benzoylaconine but not aconine blocked mechanical allodynia and heat hyperalgesia with the normalized ED₅₀ of 35 pmol and 3.6 nmol,which were totally inhibited by the specific microglial inhibitor minocycline,dynorphin A antiserum and the selective?-opioid receptor antagonist Nor-BNI.Aconitine and benzoylaconine but not aconine stimulated the dynorphin A expression in primary cultures of spinal microglia,with the EC₅₀ of 32 n M and 3?M.Intrathecal aconitine,benzoylaconine and aconine induced acute neurotoxicity characterized by paralysis and mortality,with the normalized TD₅₀ of 0.5 nmol and 0.2 and 1.6 mol.Our results suggest that the C8-acetyl and C14-benzoyl groups are essential for aconitines to stimulate spinal microglial dynorphin A expression and subsequent anti-hypersensitivity,which can be separated from the neurotoxicity as benzoylaconine has a broader normalized therapeutic index(TD₅₀/ED₅₀)of 56:1 than that of aconitine(14:1),in addition to its greater stimulatory effect on the dynorphin expression.Our results support the scientific rational for the Aconitum processing,but caution should be taken to minimize the aconine content.