Antinociceptive Effects And Underlying Mechanisms Of The GPR40 Agonist GW9508 And Opioid Analgesic Dezocine In Chronic Pain

PartⅠBackgroundThe G-protein-coupled receptor 40（GPR40）,broadly expressed in various tissues such as the spinal cord,exerts multiple physiological functions including pain regulation.This study aimed to elucidate the mechanism underlying GPR40activation-induced antinociception in neuropathic pain,particularly related to spinal glial expression of IL-10 and subsequentβ-endorphin.MethodsSpinal nerve ligation-induced neuropathic pain model was used in this study.β-Endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia,astrocytes,and neurons.Double/Trible immunofluorescence staining ofβ-endorphin or IL-10 with glial and neuronal cellular biomarkers was also detected in the spinal cord and cultured primary microglia,astrocytes,and neurons.Furthermore,the underlying mechanism of GPR40 was explored in vivo and in vitro by pain behavior study,PCR and ELISA.ResultsGPR40 was expressed on microglia,astrocytes and neurons in the spinal cords and upregulated by spinal nerve ligation-induced neuropathic pain.Intrathecal injection of the GPR40 agonist GW9508 dose dependently attenuated mechanical allodynia and thermal hyperalgesia in neuropathic rats,with E_max values of 80%MPE and 100%MPE and ED₅₀ values of 6.7 and 5.4μg respectively.Its mechanical antiallodynia was blocked by the selective GPR40 antagonist GW1100 but not GPR120 antagonist AH7614.Intrathecal GW9508 significantly enhanced IL-10-andβ-endorphin-immunostaining in spinal microglia and astrocytes but not neurons.GW9508 also markedly stimulated gene and protein expression of IL-10 andβ-endorphin in cultured primary microglia and astrocytes but not neurons,originated from 1-day-old neonatal rats.The IL-10 antibody inhibited GW9508-stimulated gene expression of theβ-endorphin precursor proopiomelanocortin（POMC）but not IL-10,whereas theβ-endorphin antibody did not affect GW9508-stimulated IL-10 or POMC gene expression.GW9508increased phosphorylation of mitogen-activated protein kinases（MAPKs）including p38,extracellular signal regulated kinase（ERK）and c-Jun N-terminal kinase（JNK）,and its stimulatory effects on IL-10 and POMC expression were blocked by each MAPK isoform inhibitor.Spinal GW9508 induced mechanical antiallodynia was completely blocked by intrathecal minocycline,IL-10neutralizing antibody,β-endorphin antiserum,andμ-opioid receptor-preferred antagonist naloxone.ConclusionsOur results illustrated that GPR40 activation produced antinociception was via the spinal glial IL-10/β-endorphin antinociceptive pathway.Part ⅡBackgroudDezocine is an opioid analgesic which is widely used in China.Its clinical use is far beyond the gold-standard painkiller morphine.It has high analgesic efficacy,wide analgesic activity and low tolerance.The previous study in our laboratory have demonstrated that dezocine produced antihypersensitivity activity was completely through spinal μ-opioid receptor（MOR）activation and norepinephrine reuptake inhibition（NRI）,without involving other mechanisms in neuropathic pain.Tapentadol,another painkiller with the dual mechanism of μ-opioid receptor activation and norepinephrine uptake inhibition,is the first recognized MOR-NRI dual-target analgesic.In this study,we aimed to make a head-to-head comparison regarding the analgesic effects and the underlying mechanisms between dezocine and tapentadol in bone cancer pain model.MethodsInoculating breast cancer cells Walker 256 into the tibia induced bone cancer pain rat model was utilized in the current study.Changes in pain reflex were investigated after administration of dezocine or tapentadol and intervention drugs,for further exploration of underlying analgesic mechanisms of dizocine and tapentadol in bone cancer pain.ResultsIn the bone cancer pain rat model,subcutaneous injections of dezocine and tapentadol exerted profound mechanical antiallodynia in a dose-and timedependent manner,with Emax values of 68% MPE and 75% MPE,ED50 values of 0.2 mg/kg and 5.1 mg/kg,respectively.Their analgesic effects were partially blocked by intrathecal injections of the selective μ-opioid receptor antagonist CTAP,the non-selective α-adrenoceptor antagonist phentolamine and the selective α2-adrenoceptor antagonist yohimbine.Moreover,depletion spinal norepinephrine by intrathecal administration of the selective norepinephrine depleting agent 6-OHDA also partially blocked the mechanical antiallodynia effects of subcutaneous dezocine and tapentadol.In addition,the combined antagonism of the selective μ-opioid receptor antagonist CTAP and the selective α2-adrenoceptor antagonist yohimbine,completely blocked the analgesic effects of dezocine and tapentadol.In contrast,the selective κ-opioid receptor antagonist GNTI and nor-BNI,the selective δ-opioid receptor antagonist naltrindole,or the selective serotonin depletor PCPA did not affect the analgesic effects of subcutaneous dezocine or tapentadol.Therefore,our study illustrated that both dezocine and tapentadol exerted significant antihypersensitivity effects in bone cancer pain model,and their antihypersensitivity effects were both through spinal μ-opioid receptor activation and norepinephrine reuptake inhibition.Activation of spinal κ-opioid receptors and δ-opioid receptors,inhibition of serotonin reuptake or other mechanisms were not involved in the antinociceptive effects of dezocine or tapentadol.ConclusionsOur study further proved that dezocine,like tapentadol,was a MOR-NRI drug,and provided pharmacological basis for the expansion of clinical application of dezocine.