TRPC Channels Contribute To Morphine-Induced Antinociceptive Tolerance And Hyperalgesia

Research purposes:Morphine is an opioid receptor agonist which widely used in clinical analgesia.But long-term repeated use of morphine can lead to tolerance phenomenon of patients,and often accompanied by hyperalgesia and allodynia.For these reasons,the clinical application of morphine is greatly limited.However,Studies on morphine-induced analgesic tolerance and sensory sensitization are currently focused on both changes in central plasticity and desensitization and susceptibility of opioid receptors.the molecular mechanism of tolerance and hyperalgesia induced by morphine has remained largely unknown.In this study,in order to explore the mechanism of TRPC channel in morphine tolerance and hyperalgesia,we established the model of morphine tolerance in rats,and blocked the expression of TRPC channel,then detected the expression of TRPC channel in spinal cord.Research methods:(1)Male SD adult rats were randomly divided into control group and morphine group.Morphine group was injected with morphine hydrochloride(10 mg/kg,twice a day)for 7 days.The control group was injected intraperitoneally with normal saline.The mechanical pain threshold and thermal pain threshold were measured before and after injection of morphine or saline at 1 d,3 d,5 d,7 d,respectively.The mechanical pain threshold and thermal pain threshold were measured at 16 h after the last injection.After 30 min of intrathecal injection of morphine(5 μg),the above behavioral tests were repeated.The expression of mRNA and protein in the spinal cord of the control group and the morphine group was detected by qRT-PCR and Western blot.(2)Male SD adult rats were randomly divided into 8 groups:control group(Con group),morphine group(Mor group),2-APB blank group,2-APB Ⅰ group,2-APBⅡgroup,SKF blank group,SKF Ⅰ group,SKF Ⅱ group.Rats were injected intraperitoneally with morphine hydrochloride(10 mg/kg,twice a day)for 7 consecutive days in group A,2-APB Ⅰgroup,2-APB Ⅱgroup,SKF Ⅰ group and SKF Ⅱgroup.The rats in con group,2-APB blank group and SKF blank group were injected intraperitoneally with equal volume of saline after all rats in each group were treated with internal catheterization.During this period,0.1 p,g and 0.5 μg 2-APB(once a day)were injected into the 2-APB Ⅰgroup and the 2-APB Ⅱgroup by the intrathecal catheter for 7 days.Rats in SKF Ⅰ group and SKF Ⅱ group were injected with 0.1 μg and 0.5 μg SKF96365(once a day)for 7 days.0.5μg 2-APB and SKF96365(once a day)were injected into 2-APB blank group and SKF blank group respectively for 7 days.Mor group and Con group were injected intramuscularly with artificial cerebrospinal fluid 20 μg(once a day)for 7 days.The pain threshold(g)and the thermal pain threshold(s)of each group were measured after the last saline injection or saline for 16 h.Intrathecal indwelling catheter was injected with morphine(5 μg)for 30 min,and repeated behavioral testing was performed.(3)48 male SD adult rats were randomly divided into 6 groups:S + V group,M +V group,S + Si group,S + Sc group,M + Si group,M + Sc group,6 per group.M + V group,M + Si group and M + sc group were injected intraperitoneally with morphine hydrochloride(10 mg/kg,twice/day)for 7 days;S + V group,S + Si group and S +Sc blank group were injected intraperitoneally with equal volume of normal saline.During this period,TRPC6 siRNA(20 μl,once a day)was injected into the S + Si group and the M + Si group for 4 days,and the rats in the S + Sc group and the M + Sc group were injected intrathecally(20μl,once a day)for 4 days.The rats in the S + V group and the M + V group were injected intrathecally with artificial cerebrospinal fluid(20μl,once a day)for 4 days.After the last administration of morphine hydrochloride or saline for 16 h,the mechanical pain threshold(g)and the thermal pain threshold(s)were measured in each group.Intrathecal indwelling catheter was injected with morphine(5 μg)for 30 mins,and repeated behavioral testing was performed.The expressions of p-mTOR,PKCγ,nNOS,CaMKIIα,NF-κB,IL-1β,IL-6,TNF-α and GFAP and Ibal in the spinal cord of each group were detected by qRT-PCR,ELISA,Western blot and immunofluorescence expression change.Research results:(1)Intraperitoneal injection of morphine for 7 days could induce obvious susceptibility to chronic morphine in rats.The expression of TRPC1,TRPC3 and TRPC6 channels are up-regulated in the spinal cord in rats after chronic morphine treatment.(2)Intrathecal administration of TRPC channel blocker 2-APB or SKF96365,can inhibit morphine analgesic tolerance and hyperalgesia;(3)Intrathecal administration of TRPC6 siRNA inhibited morphine tolerance and sensory sensitization,and inhibited morphine-induced expression of nNOS and CaMKIIa in the spinal cord.Spinal cord TRPC6 knockout inhibits morphine-induced spinal cord immune activation.Analysis conclusion:TRPC6 channels may be involved in morphine-induced analgesic tolerance and hyperalgesia by promoting spinal cord inflammatory response and neuroimmunial responses.