The Role And Mechanism Of Rheb-mediated Activation Of MTORC1 In Morphine Tolerance And Neuropathic Pain

Objective:To investigate the role of Rheb-mediated mTORC1 activation in central sensitization of chronic morphine tolerance and neuropathic pain model in mice.Methods:Firstly,chronic morphine tolerance model and neuropathic pain model in mice were established as follows:intrathecal injection of 10?g morphine twice a day for five consecutive days to induce chronic morphine tolerance;chronic sciatic nerve constriction injury was used to simulate neuropathic pain.(1)The expression of Rheb and the activation of mTORC1 pathway(mTOR,S6K,S6 and 4E-BP1)in the spinal cord in morphine tolerance model and neuropathic pain model mice were compared by western blot;(2)Immunofluorescence double-labeled technique was used to detect the cell types in the spinal cord where Rheb-mTORC1 signaling pathway was activated in morphine tolerance model and neuropathic pain model;(3)Intrathecal injection of mTOR inhibitor rapamycin(10?g)30 min before morphine injection during tolerance inducing,and intrathecal injection of mTOR inhibitor rapamycin(10?g)3 days before CCI surgery until 7 days after surgery were used to inhibit the activation of mTORC1.Tail-flick test(52±0.5?water bath,60min after morphine injection,tail flick latency was recorded)and plantar heat radiation test(plantar radiometer,paw withdrawal latency was recorded)were used to observe the formation of morphine tolerance and neuropathology Pain;(4)After intrathecal injection of 10?g rapamycin in the established morphine tolerance and neuropathic pain model,TFL and PWL were recorded 4h/24h after injection to observe the maintenance of morphine tolerance and neuropathic pain;(5)After intrathecal co-injection of 10?g rapamycin and 10?g morphine in neuropathic pain model,PWL was record to test the acute morphine analgesic effect and tolerance formation.Then,using Rosa and loxp/cre technology to make Rheb S16H nestin Cre Ms(KI)and Rheb conditional knockout transgenic mice(Rheb^F/F;CAMK II^cre+Ms,CKO).(1)The expression of Rheb and the activation of mTORC1 pathway(mTOR,S6K,S6 and 4E-BP1)in the spinal cord of these mice were detected by western blot;(2)30-60 minutes after single injection of morphine in the KI(10?g)and CKO(1?g)Ms,tail-flick latency was recorded to observe the acute analgesic effect of morphine;(3)Chronic morphine tolerance was induced in KI and CKO transgenic mice,and the formation of tolerance was observed.Results:(1)Rheb expression in the spinal cord of chronic morphine tolerance and neuropathic pain mice was significantly higher than that of the control group(p<0.05,compared with the saline group or sham group),and so was the activity of Rheb.The phosphorylation of mTOR,phosphorylated mTOR(p<0.05,compared with saline group or sham operation group),as well as mTORC1 downstream effector proteins S6K,S6 and 4E-BP1 also increased(p<0.05,compared with the saline group or sham group);(2)Co-localization of p S6 and Neu N suggested that chronic morphine tolerance and neuropathic pain-induced activation of Rheb-mTORC1 mainly occur in the spinal cord neurons;(3)Continuous application of mTOR inhibitor rapamycin can significantly inhibited the formation of chronic morphine tolerance and neuropathic pain(p<0.05,compared with the vehicle group);(4)Rapamycin can reverse the chronic morphine tolerance and thermal hyperalgesia induced by neuropathic pain f(p<0.05,compared with the vehicle group);(5)Blocking mTORC1 activation by rapamycin can potentiate the analgesic effect of morphine in neuropathic pain model and prevent the onset of tolerance(p<0.05,compared with the vehicle group);(6)acute analgesic effect of morphine in Rheb KI mice was impaired compared to wild-type mice(p<0.05)(7)Meanwhile,acute analgesic effect of morphine in Rheb CKO mice was potentiated compared to wild-type mice(p<0.05),and the onset of morphine tolerance was delayed to some extent(p<0.05,compared with wild-type mice).Conclusions:The Rheb-mediated activation of mTORC1 is involved in the formation and maintenance of chronic morphine tolerance and neuropathic pain,which is their the common pathological basis.Inhibition of Rheb-mediated activation of mTORC1 is useful for the treatment of neuropathic pain and inhibition of morphine tolerance,which provide a new therapeutic strategy.mTOR inhibitor,rapamycin,may be a powerful adjunct to morphine in the treatment of neuropathic pain.