Glycogen Synthase Kinase-3β Modulate NMDA And AMPA Receptor In The Mechanism Of Remifentanil-induced Hyperalgesia

Opioids have been regarded as the most effective analgesics for management of acute or chronic pain and cancer pain. However, they also produce a lot of adverse effects, including hyperalgesia, which limits their clinical use. Remifentanil is a potent short-acting μ-opioid receptor agonist, as it is a rapid-onset opioid and has short duration, remifentanil has been widely used as analgesic in general anesthesia during operation. Many researches show that the incidence rate of remifentanil-induced hyperalgesia is significantly higher than other opioids.There is increasing evidence that N-methyl-d-aspartate(NMDA) receptor plays a critical role in the development of opioid-induced hyperalgesia and NMDA receptor antagonistketamine infusion can significantly decrease remifentanil-induced hyperalgesia. However, ketamine infusion could increase some side effects, such as hallucination, sedation, dizziness and drowsiness. So, the significance obviously is there for make sure the mechanism of remifentanil-induced hyperalgesia and finds a new drug target to treat and prevent opioid-induced hyperalgesia.Glycogen synthase kinase-3(GSK-3) is amultifunctional serine/threonine protein kinase, ubiquitous in eukaryotes. GSK-3contains two subtypes, GSK-3a and GSK-3β, in mammals. The main function of this enzyme includes regulating the synthesis of glycogen metabolism, differentiation and proliferation. Recentstudieshavefoundthat, GSK-3βaffects synaptic plasticity, and has an important role in mediating NMDA receptor and AMPA receptor trafficking. However, whether GSK-3mediated NMDA receptor and AMPA receptor trafficking in incisional pain-remifentanil-induced hyperalgesia model has not been studied systematically.In this study, we evaluated the role of GSK-3β in mediating NMDA receptor and AMPA receptor trafficking and function in incisional pain-remifentanil-induced hyperalgesia model in vivo and remifentanil infusion model in vitro. We had conducted a thorough research on the mechanism of remifentanil-induced hyperalgesia and hope to find a new target for the treatment of opioid-induced hyperalgesia. Part1Changes of GSK-3β protein activity in spinal cord in rats with incisional pain-remifentanil-induced hyperalgesiaObjective To evaluate the protein activity of GSK-3β in spinal cord in rats with incisional pain-remifentanil-induced hyperalgesia and discuss the possible mechanism of GSK-3β in remifentanil-induced hyperalgesia.Methods Thirty-two health male SD rats (240～260g) with caudal vein catheter were randomly divided into four groups (n=8):Control group (C):underwent saline infusion0.1ml·kg-1·min-1; Remifentanil group (R):underwent remifentanil infusion1.0μg·kg-1·min-1; Incisional pain group (I):underwent a surgical incision with saline infusion0.1ml·kg-1·min-1; Remifentanil+incisional pain group (RI):underwent a surgical incision with remifentanil infusion1.0μg·kg-1·min-1; all rats were continuously exposed to saline or remifentanil for60min. The paw withdraw threshold (PWT) and paw withdraw latency (PWL) were measured at1d before and2h,6h,1d,2d,3d,5d,7d after infusion by Von Frey filaments test and Hot Plate test. The rats were sacrificed at2d after infusion, and then the lumbar segment (L4-6) was immediately separated for the evaluation of the expression of pTyr216-GSK-3β, pSer9-GSK-3β and total GSK-3β in spinal cord.Results Compared with group C, the PWT and PWL were significantly decreased in group R, I and RI (P<0.05), and the decreased level of PWT and PWL in group RI was higher than group R and I (P<0.05). Compared with group C, the expression level of pTyr216-GSK-3β was significantly higher and pSer9-GSK-3β was significantly lowered in group R, I and RI (P<0.05). However, there is no significant difference in the expression level of total GSK-3β in all groups (P>0.05).Conclusion Incision can induce incisional hyperalgesia and remifentanil infusion aggravated incisional pain-induced hyperalgesia; the potential mechanism of incisional pain-remifentanil-induced hyperalgesia may be related to the up-regulation of pTyr216-GSK-3β and down-regulation of pSer9-GSK-3β in spinal cord. Part2The effect of GSK-3β on the trafficking and function of NMDA receptorin remifentanil-induced hyperalgesia rats.Experiment1The effect of GSK-3β on the trafficking of NMDA receptor NR1, NR2A and NR2B subunit in spinal cord in remifentanil-induced hyperalgesia.Objective To evaluate the effect of GSK-3β inhibitor on the trafficking of NMDA receptor NR1, NR2A and NR2B subunit in spinal cord in remifentanil-induced hyperalgesiaand discuss the possible mechanism of GSK-3β modulate NMDA receptor trafficking in remifentanil-induced hyperalgesia model.Methods Forty health male SD rats (240～260g) with caudal vein catheter were randomly divided into four groups (n=8):Control group (C):underwent saline infusion0.1ml·kg-1·min-1; Glycine group(G):underwent glycine infusion15μg·kg-1·min-1; Remifentanil group (R):underwent remifentanil infusion1.0μg·kg-1·min-1; Remifentanil+GSK-3β inhibitor TDZD group (RT):underwent remifentanil infusion1.0μg·kg-1·min-1and TDZD infusion1ug·kg-1; TDZD group (T): underwent saline infusion0.1ml·kg-1·min-1and TDZD infusion1μg·kg-1; all rats were continuously exposed to saline or remifentanil for60min. The paw withdraw threshold (PWT) and paw withdraw latency (PWL) were measured at24h before and2h,6h,24h,48h after infusion by Von Frey filaments test and Hot Plate test. The rats were sacrificed at48h after infusion, and then the lumbar segment (L4-6) was immediately separated for the evaluation of the expression of membrane and total protein of NMDA receptor NR1, NR2A and NR2B in spinal cord.Results Compared with group C, the PWT and PWL were significantly decreased in group R, the membrane NR1and NR2B were significantly increased and the total NR1and NR2B were significantly increased in group R(P<0.05).Compared with group R, the PWT and PWL were significantly increased in group RT, the membrane NR1and NR2B were significantly decreased and the total NR1and NR2B were significantly decreased in group RT(P<0.05). However, there is no significant difference in the expression level of membrane and total NR2A in all groups (P>0.05).There is no significant difference in the expression level of membrane and total NMDA receptor subunits in C, G and T group (P>0.05). Conclusion Remifentanil-induced hyperalgesia may be related to the up-regulation of membrane and total protein level of NR1and NR2B subunits in spinal cord. And TDZD could decrease up-regulated membrane and total protein level of NR1and NR2B subunits in spinal cord in remifentanil-induced hyperalgesia rats. The thermal hyperalgesia and mechanical hyperalgesia was decreased after the application of GSK-3β inhibitor TDZD. These results indicate that GSK-3β mediated NMDA receptor NR1and NR2B subunits trafficking and expression in spinal cord may releated to remifenanil-induced hyperalgesia.Experiment2The effect of GSK-3β on the function of NMDA receptor in spinal cord in remifentanil-induced hyperalgesia.Objective To investigate the role of glycogen synthesis kinase-3β (GSK-3β) in remifentanil induced NMDA receptor miniature excitatory postsynaptic currents (mEPSCs) in rat spinal dorsal horn neurons.Methods Thirty-two14-18day old Wistar rats weighing50-60g were randomly divided into4groups (n=8each):Control group (group C), Glycine group (group G), Remifentanil group (group R); Remifentanil+GSK-3β inhibitor TDZD-8group (group RT). L1-S1spinal tissues were removed for preparation of spinal cord slices. Spinal cord slices were incubated in artificial cerebrospinal fluid (ACSF)(group C), ACSF containing glycine0.24umol·L-1(group G), ACSF containing remifentanil4nmol·L-1(group R) and ACSF containing remifentanil4nmol·L-1+TDZD-810μmol·L-1(group RT) for60min. The whole cell patch clamp technique was used to measure NMDA receptor induced mEPSCs.Results Compared with group C, the amplitude and frequency of mEPSCs were significantly increased in group R (P<0.01). Compared with group R, the amplitude and frequency of mEPSCs were significantly decreased in group RT(P<0.01). There were no significantly differences in amplitude and frequency of mEPSCs among gorap C, G and RT (P>0.05).ConclusionGSK-3β activity increase can enhance NMDA receptor function in spinal dorsal horn neurons in rats which may be related to remifentanil-induced hyperalgesia. Part3The effect of GSK-3β on the trafficking and function of AMPA receptorin remifentanil-induced hyperalgesia rats. Experiment1The effect of GSK-3β inhibitor on the trafficking of NMDA receptor GluR1and GluR2subunit in spinal cord in remifentanil-induced hyperalgesia.Objective To evaluate the effect of GSK-3β inhibitor on the trafficking of AMPA receptor GluR1and GluR2subunit in spinal cord in remifentanil-induced hyperalgesiaand discuss the possible mechanism of GSK-3β modulate AMPA receptor trafficking in remifentanil-induced hyperalgesia.Mehthods Health male SD rats (240～260g) with caudal vein catheter were randomly divided into six groups (n=8):Control group (C):underwent saline infusion0.1ml·kg-1·min-1; remifentanil group (R):underwent remifentanil infusion1.0μg·kg-1·min-1; incisional pain group(I):underwent a surgical incision with saline infusion0.1ml·kg-1·min-1; remifentanil+incisional pain group(RI):underwent a surgical incision with remifentanil infusion1.0μg·kg-1·min-1;RI+GSK-3β inhibitor LiCl group (LiC3):underwent a surgical incision with remifentanil infusion1.0μg·kg-1·min-1and LiCl infusion100mg·kg-1; RI+GSK-3β inhibitor TDZD group (TDZD):underwent a surgical incision with remifentanil infusion1.0μg·kg-1·min-1and TDZD infusion1μg·kg-1; all rats were continuously exposed to saline or remifentanil for60min. The paw withdraw threshold (PWT) and paw withdraw latency (PWL) were measured at1d before and2h,6h,1d,2d,3d,5d,7d after infusion by Von Frey filaments test and Hot Plate test. The rats were sacrificed at48h after infusion in group C, R, I, LiCl, TDZD and at1d before and2h,6h,1d,2d,3d,5d,7d after infusion in group RI, then the lumbar segment (L4-6) was immediately for the evaluation of the expression of membrane and cytoplasm protein of AMPA receptor GluR1and GluR2in spinal cord.Results The expression level of membrane GluRl was significant increased at2h after RI treatment, reached the peak level at2d after RI treatment and get back to normal level at5d after RI treatment (P<0.05). However, there is no significant difference in the expression of cyto GluR1at any time point in group RI(P>0.05). Compared with group C (2d), the PWT and PWL were significantly decreased and the membrane GluRl were significantly increased in group R, I and RI (P<0.05). Compared with group RI, the PWT and PWL were significantly increased and the membrane GluRl were significantly decreasedin group LiCl and TDZD (P<0.05). However, there is no significant difference in the expression level of cyto GluRl in all groups (P>0.05).There is no significant difference in the expression level of membrane and total AMPA receptor GluR2subunit in all groups (P>0.05).Conclusion Remifentanil-induced hyperalgesia may be related to the up-regulation of membrane protein level of GluRl subunit in spinal cord, which reached a peak level at2d after RI treatment. GSK-30inhibitors LiCl and TDZD could decrease up-regulated membrane GluRl in spinal cord in incision pain-remifentanil-induced hyperalgesia rats. These results indicate that the potential mechanism of remifentanil-induced hyperalgesia may be related to GSK-3β mediated AMPA receptor GluRl subunit trafficking and expression in spinal cord.Experiment2The effect of GSK-3β inhibitor on the function of AMPA receptorin spinal cord in remifentanil-induced hyperalgesia.Objective To investigate the role of glycogen synthesis kinase-3β (GSK-3β) in remifentanil induced AMPA receptor mEPSCs in rat spinal dorsal horn neurons.Methods Thrity-twol4-18day old Wistar rats weighing50～60g were randomly divided into4groups (n=8each):Control group (group C), Remifentanil group (group R); Remifentanil+LiClgroup (group LiCl); Remifentanil+TDZD-8group (group TDZD). L1-S1spinal tissues were removed for preparation of spinal cord slices. Spinal cord slices were incubated in artificial cerebrospinal fluid (ACSF)(group C), ACSF containing remifentanil4nmol·L-1(group R), ACSF containing remifentanil4nmol·L-1+LiC120mmol·L-1(group LiCl)and ACSF containing remifentanil4nmol·L-1+TDZD-810μmol·L-1(group TDZD) for60min. The whole cell patch clamp technique was used to measure AMPA receptor induced mEPSCs.Results Compared with group C, the amplitude and frequency of mEPSCs were significantly increased in group R (P<0.01). Compared with group R, the amplitude and frequency of mEPSCs were significantly decreased in group LiCl and TDZD (P<0.01). There were no significantly differences in amplitude and frequency of mEPSCs among group C, LiCl and TDZD(P>0.05).ConclusionIncreased GSK-3β activitycan enhance AMPA receptor function in spinal dorsal horn neurons in rats which may be related to remifentanil-induced hyperalgesia.Experiment3The related mechanism of GSK-3βom the trafficking and function of AMPA receptorin incisonal pain-remifentanil-induced hyperalgesia rats.Objective To find the possible mechanism of GSK-3β on the trafficking and function of AMPA receptor in spinal cord in remifentanil-induced hyperalgesia.Methods Thirty-two health male SD rats (240～260g) with caudal vein catheter were randomly divided into four groups (n=8):Control group (C):underwent saline infusion0.1ml·kg-1·min-1; Remifentanil+incisional pain group(RI):underwent a surgical incision with remifentanil infusion1.0μg·kg-1·min-1;RI+GSK-3β inhibitor LiCl group (LiCl):underwent a surgical incision with remifentanil infusion1.0μg·kg-1·min-1and LiCl infusion100mg·kg-1; RI+GSK-3β inhibitor TDZD group (TDZD):underwent a surgical incision with remifentanil infusion1.0μg·kg-1·min-1and TDZD infusion1μg·kg-1; all rats were continuously exposed to saline or remifentanil for60min. The rats were sacrificed at48h after infusion, then the lumbar segment (L4-6) was immediately separated for the evaluation of the expression of pSer845-GluRl, pSer880-GluR2, Rab5, Rab4and PSD-95in spinal cord.Results Compared with group C, pSer845-GluRl was significantly increased in group RI, LiCl and TDZD (P<0.05).Compared with group RI, pSer845-GluRl was significantly were significantly decreased in group LiCl and TDZD (P<0.05). However, there is no significant difference in the expression level of pSer880-GluR2in all groups (P>0.05).Compared with group C, Rab5was significantly decreased in group RI (P<0.05). Compared with group RI, Rab5was significantly were significantly increased in group LiCl and TDZD (P<0.05). However, there is no significant difference in the expression level of Rab4and PSD-95in all groups (P>0.05). Conclusion The increased membrane GluRl level in Remifentanil-induced hyperalgesia may be related to the up-regulation of pSer845-GluRl and down-regulation of Rab5. And LiCl and TDZD could reverse up-regulation of pSer845-GluRl and down-regulation of Rab5in spinal cord in remifentanil-induced hyperalgesia rats. These results indicate that the potential mechanism of remifentanil-induced hyperalgesia may be related to GSK-3β mediated up-regulation of pSer845-GluRl and down-regulation of Rab5and then increased the membrane AMPA receptor CluR1subunit trafficking in spinal cord.