Upregulation Of CX3CL1 Via STAT3 Contributed To SMIR-induced Chronic Postsurgical Pain

ObjectivesChronic postsurgical pain often occurs following surgeries,which affects life quality of patients seriously.Recent studies indicate that chemokine CX3CL1 plays an important role in neuropathic pain induced by nerve injury.However,whether CX3CL1 participates in chronic postsurgical pain is still unknown.In this study,we use a model of chronic postsurgical pain evoked by skin/muscle incision and retraction(SMIR),exploring whether CX3CL1 participates in mechanical allodynia induced by SMIR and its underlying mechanism.Methods1.Measuring the expression of CX3CL1 in spinal cord after SMIR and whether CX3CL1 participates in mechanical allodynia induced by SMIRPerforming SMIR surgery and measuring paw withdrawal threshold(PWT)of the ipsilateral and contralateral side to the operation before and 1,5,10 and 20 days after the operation.Intrathecal injection of CX3CL1 neuralizing antibody was performed once a day from 30 minutes before SMIR to the 10th day after SMIR or from the 11th day after SMIR for 5 consecutive days.PWT of the ipsilateral side was measured.Getting ipsilateral spinal cord before and 1,5,10 and 20 days after SMIR and detecting the level of CX3CL1 through western blot and qPCR.Getting spinal cord from blank control group,only SMIR group and SMIR+CX3CL1 antibody group and using immunofluorescence staining to detect the fluorescence intensity of microglia marker Ibal.Getting spinal cord 10 days after SMIR and using immunofluorescence staining to find the expression area of CX3CL1 in spinal cord.2.Detecting whether SMIR activate transcription factor STAT3 in spinal dorsal horn and whether STAT3 mediate the upregulation of CX3CL1 and mechanical allodyniaGetting spinal cord 10 days after SMIR and using immunofluorescence staining to find the expression area of p-STAT3 in spinal cord.Also getting spinal cord 10 days afterSMIR and using immunofluorescence staining to verify whether CX3CL1 and p-STAT3 were expressed in the same area.Getting ipsilateral spinal cord before and 1,5,10 and 20 days after SMIR and detecting the level of total STAT3 and p-STAT3 through western blot.Intrathecal injection of STAT3 inhibitor S3I-201 was performed once a day from 30 minutes before SMIR to the 10th day after SMIR.PWT of the ipsilateral side was measured.Rats were divided into three groups,one blank control group,one only SMIR group and one group performing intrathecal injection of STAT inhibitor S3I-201 once a day from 30 minutes before SMIR to the 10th day after SMIR.Detecting the level of p-STAT3 and CX3CL1 using western blot.Also detecting the level of CX3CL1 using qPCR.3.Detecting whether SMIR was able to enhance recruitment of STAT3 to the cx3cll gene promoterRats were divided into SMIR group and blank control group.Getting ipsilateral spinal dorsal horn and performing chromatin immunoprecipitation.DNA fragments which might combined with STAT3 were pulled down using STAT3 antibody.Then perform quantitative real-time and semiquantitative PCR analysis.Results1.The level of CX3CL1 in spinal cord was upregulated after SMIR and CX3CL1 participated in mechanical allodynia induced by SMIRObvious mechanical allodynia occurred 5 days after SMIR and maintained until at least 20 days after SMIR.Intrathecal injection of CX3CL1 neutralizing antibody in advance could attenuate mechanical allodynia induced by SMIR significantly.However,once mechanical allodynia has been developed,intrathecal injection of CX3CL1 neutralizing antibody could not reverse pain.Compared with control group,SMIR significantly increase the protein and mRNA levels of CX3CL1 in ipsilateral spinal cord.SMIR significantly activate microglia in spinal cord while intrathecal injection of CX3CL1 neutralizing antibody inhibited the increased Ibal immunostaining induced by SMIR.After SMIR,The immunofluorescence staining of increased CX3CL1 was colocalized with NeuN in spinal cord but not GFAP or Iba1.2.STAT3 mediate the upregulation of CX3CL1 in spinal cord and mechanical allodynia induced by SMIRAfter SMIR,The immunofluorescence staining of p-STAT3 was also colocalized with NeuN in spinal cord but not GFAP or OX42.Double immunofluorescence staining indicated the colocalization of p-STAT3 and CX3CL1 after SMIR.Compared with control group,SMIR significantly increase the the ratio of phosphorylated STAT3.Intrathecal administration of STAT3 inhibitor S3I-201 in advance could attenuate mechanical allodynia significantly.Intrathecal injection of S3I-201 in advance inhibit the upregulated protein level of p-STAT3 and CX3CL1 induced by SMIR.The upregulation of mRNA level of CX3CL1 was also inhibited.3.SMIR was able to enhance recruitment of STAT3 to the cx3cll gene promoterCompared with control group,data from ChIP demonstrated that SMIR was able to enhance recruitment of STAT3 to the cx3cll gene promoter.ConclusionsCX3CL1 was upregulated in spinal cord after SMIR and it participated in mechanical allodynia induced by SMIR.The activation of STAT3 after SMIR mediate the upregulation of CX3CL1 and mechanical allodynia,which might owing to the enhanced recruitment of STAT3 to the cx3cl1 gene promoter after SMIR.