The Mechanisms Of NR2B-CaMKⅡα-HDAC4 In The Pathogenesis Of Remifentanil-Induced Hyperalgesia In Rats

Opioids are the main drugs in the treatment of acute and chronic pain,cancer pain and clinical anesthesia.But opioids cause hyperalgesia,which limits the use of them.Remifentanil has a rapid onset and clearance,and is widely used in general anesthesia,but studies have confirmed that remifentanil induced hyperalgesia（RIH）is more frequent than the others.At present,the mechanism of RIH is not clear,and there is a lack of effective drugs,so it is necessary to further clarify the specific molecular mechanism of RIH.Ca²⁺/calmodulin-dependent protein kinase II（CaMKII）,is an important protein component of postsynaptic dense components.CaMKII can sense the changes of postsynaptic calcium levels and maintain long-term activity.It plays an important role in regulating glutamate synaptic plasticity,LTP,learning and memory.Histone deacetylation regulated by Histone deacetylase 4（HDAC4）is an important process for chromosome recombination to regulate transcription.In model of hypersensitivity induced by spinal nerve ligation,the expression of phosphorylated HDAC4 in spinal cord and cytoplasmic HDAC4 were significantly increased.Administration of NMDA receptor antagonist or CaMKIIαinhibitor causes HDAC4 to shuttle from cytoplasm to nucleus（shuttling）,which significantly affected the gene transcription process related to synaptic plasticity.Thus,we hypothesize that remifentanil may enhance NMDAR-mediated calcium current and activate CaMKIIαand HDAC4 by increasing the expression of NR2B subunit.Shuttling from the nucleus to the cytoplasm promotes the transcription and expression of the target genes regulating synaptic plasticity,and then induces hyperalgesia.In this study,the rat model of incision-RIH model was established to explore the regulatory mechanism of NR2B-CaMKIIα-HDAC4 shuttling in RIH,so as to provide a new idea for the clinical prevention and treatment of RIH.Objective To investigate changes of NR2B subunit expression and membrane transport,CaMKIIαactivity,HDAC4 activity and shuttling in spinal dorsal horn of RIH rats and explore the possible mechanism of RIH;To investigate effects of NR2B on behavioral hyperalgesia,CaMKIIαphosphorylation,NR2B/CaMKIIαcomplex expression and HDAC4 activity in spinal dorsal horn of remifentanil induced hyperalgesia rats after intrathecal injection of NR2B antagonist Ro25-6981;To investigate effects of CaMKIIαon behavioral hyperalgesia and HDAC4 activity in spinal dorsal horn of RIH rats after intrathecal injection of CaMKIIαinhibitor KN-93;To investigate effects of HDAC4 on behavioral hyperalgesia,HDAC4 activity in spinal dorsal horn of RIH rats after intrathecal injection of HDAC4 selective inhibitor LMK235;To investigate effects of intrathecal Ro25-6981,KN-93 and LMK235injection on dendritic spine morphology of spinal dorsal horn in RIH rats.MethodsⅠ60 male SD rats（8¹2 weeks）were randomly divided into 5 groups（n=12）:C group（sham operation+saline group）:underwent sham operation and infusion of saline0.1 ml·kg^-1·min^-1,1h;G Group（sham operation+glycine group）:underwent sham operation and infusion of glycine solution 0.1 ml·kg^-1·min^-1,1h;R group（remifentanil group）:remifentanil was administered at the dose of 1.0μg·kg^-1·min^-1 for 1h;I Group（incision group）:underwent the infusion of saline 0.1 ml·kg^-1·min^-1 and incision surgery 10 mins later;IR group（remifentanil+incision group）:remifentanil was administered at the dose of 1.0μg·kg^-1·min^-1 for 1h and incision surgery was administered 10 mins later.At 1d before and the time of 2h、6h、1d、2d、3d、5d and7d after infusion PWT and PWL were measured（n=6）.Two days later after remifentanil infusion,we collected the L₄-₆ segments of spinal cord dorsal horn（SCDH）for determining changes of NR2B subunit expression and membrane transport,CaMKIIαactivity and HDAC4 activity using Western blot.II 48 male SD rats（8¹2 weeks）were randomly divided into 4 groups（n=12）:C group（sham operation+saline group）:underwent sham operation and infusion of saline 0.1 ml·kg^-1·min^-1,1h;IR group:remifentanil was administered at the dose of1.0μg·kg^-1·min^-1 for 1h and 10 mins later incision surgery was administered;C+Ro group（sham operation+NR2B antagonist Ro25-6981 group）:underwent sham operation and intrathecal injection 100nmol Ro25-6981 10 mins before the infusion of saline;IR+Ro（remifentanil+incision+Ro25-6981 group）:underwent intrathecal injection 100nmol Ro25-6981 10 mins before the infusion of remifentanil 1.0μg·kg^-1·min^-1 and incision surgery 10 mins later.To investigate the effects of inhibition of NR2B expression and membrane transport on CaMKIIαactivity,NR2B/CaMKIIαcomplex expression and HDAC4 activity in spinal dorsal horn of RIH rats using Western blot.Ⅲ48 male SD rats（8¹2 weeks）were randomly divided into 4 groups（n=12）:C group（sham operation+saline group）:underwent sham operation and infusion of saline 0.1 ml·kg^-1·min^-1,1h;IR group:remifentanil was administered at the dose of1.0μg·kg^-1·min^-1 for 1h and 10 mins later incision surgery was administered;C+K group（sham operation+CaMKIIαinhibitor KN-93 group）:underwent sham operation and intrathecal injection 100nmol KN-93 10 mins before the infusion of saline;IR+Ro（remifentanil+incision+KN-93 group）:underwent intrathecal injection 100nmol KN-93 10 mins before the infusion of remifentanil 1.0μg·kg^-1·min^-1 and incision surgery10 mins later.To investigate the effects of inhibition of CaMKIIαon behavioral hyperalgesia,NR2B/CaMKIIαcomplex expression and HDAC4 activity using Western blot and immunofluorescence staining.Ⅳ72 male SD rats（8¹2 weeks）were randomly divided into 6 groups（n=12）:C group（sham operation+saline group）:underwent sham operation and infusion of saline 0.1 ml·kg^-1·min^-1,1h;IR group:remifentanil was administered at the dose of1.0μg·kg^-1·min^-1 for 1h and 10 mins later incision surgery was administered;C+L3group（sham operation+high dose HDAC4 selective inhibitor LMK235 group）:underwent sham operation and intrathecal injection 100nmol LMK235 10 mins before the infusion of saline;IR+L1 group,IR+L2 group and IR+L3 group:underwent intrathecal injection 10nmol,30nmol and 100nmol LMK235 10 mins before the infusion of remifentanil 1.0μg·kg^-1·min^-1 and incision surgery 10 mins later.To investigate the effects of inhibition of HDAC4 on behavioral hyperalgesia and HDAC4phosphorylation using Western blot.Ⅴ30 male SD rats（8¹2 weeks）were randomly divided into 5 groups（n=6）:C group:underwent sham operation and infusion of saline 0.1 ml·kg^-1·min^-1,1h;IR group:remifentanil was administered at the dose of 1.0μg·kg^-1·min^-1 for 1h and 10 mins later incision surgery was administered;IR+Ro group:underwent intrathecal injection100nmol Ro25-6981 10 mins before the infusion of remifentanil 1.0μg·kg^-1·min^-1 and incision surgery 10 mins later;IR+K group:underwent intrathecal injection 100nmol KN93 10 mins before the infusion of remifentanil 1.0μg·kg^-1·min^-1 and incision surgery10 mins later;IR+L3 group:underwent intrathecal injection 100nmol LMK235 10mins before the infusion of remifentanil 1.0μg·kg^-1·min^-1 and incision surgery 10 mins later.Two days later after remifentanil infusion,we collected the L₄-₆ segments of spinal cord dorsal horn for detecting the changes of dendritic spines’length and number using Golgi staining method.ResultsⅠCompared with C group,PWT and PWL were significantly decreased in rats of I,R and IR group.And increasement in NR2B expression and membrane transport,CaMKIIαactivity,NR2B/CaMKIIαcomplex expression and HDAC4 activity were seen in I,R and IR group（P<0.01）;Compared with I group,PWT and PWL were significantly decreased in rats of IR group and increasement in NR2B expression and membrane transport,CaMKIIαactivity,NR2B/CaMKIIαcomplex expression and HDAC4 activity were seen in IR group（P<0.01）.II Compared with C group,PWT and PWL were significantly decreased in rats of IR and IR+Ro group and increasement in NR2B expression and membrane transport,CaMKIIαactivity,NR2B/CaMKIIαcomplex expression and HDAC4 activity were seen in IR and IR+Ro group（P<0.01）;Compared with IR group,IR+Ro group showed a significant increase in PWT and PWL and decrease in NR2B expression and membrane transport,CaMKIIαactivity,NR2B/CaMKIIαcomplex expression and HDAC4 activity（P<0.01）.ⅢCompared with C group,PWT and PWL were significantly decreased in rats of IR and IR+K group and increasement in CaMKIIαactivity,NR2B/CaMKIIαcomplex expression and HDAC4 activity were seen in IR and IR+K group（P<0.01）;Compared with IR group,IR+K group showed a significant increase in PWT and PWL and decrease in CaMKIIαactivity,NR2B/CaMKIIαcomplex expression and HDAC4activity（P<0.01）.ⅣCompared with C group,PWT and PWL were significantly decreased and HDAC4 activity was increased in rats IR,IR+L1,IR+L2 and IR+L3 group（P<0.01）;Compared with IR group,a significant increase in PWT and PWL and decrease in HDAC4 phosphorylation in a dose-dependent manner were seen in IR+L2 and IR+L3group（P<0.05）.ⅤCompared with C group,the number and length of dendritic spines in IR,IR+Ro,IR+K and IR+L3 groups were significantly increased（P<0.05）;compared with IR group,the number and length of dendritic spines in IR+Ro,IR+K and IR+L3 groups were significantly decreased（P<0.05）.Conclusion Intraoperative infusion of remifentanil may aggravate postoperative mechanical and thermal hyperalgesia caused by incision.Upregulation of NR2B expression,CaMKIIαactivity,the expression of NR2B/CaMK IIαcomplex and the HDAC4 activity in spinal dorsal horn of rats with incision may be involved in the development of RIH.Inhibition of spinal cord level NR2B could alleviate RIH in rats and the mechanism may be related to the decrease of CaMKIIαand HDAC4 activity in spinal dorsal horn;the inhibition of CaMKIIαcan alleviate the RIH and may be related to the inhibition of HDAC4 activity.Selective inhibition of HDAC4 may alleviate RIH in rats.Remifentanil anesthesia may increase the expression of NR2B in spinal dorsal horn and increase the activity of CaMKIIαand HDAC4.Furthermore,the regulation of the number and structural remodeling of dendritic spines may be the key for the development of RIH.