| Objective:Postsurgical pain is defined as the pain caused by surgical trauma during routine surgery.More than 80%of patients who undergo surgical procedures experience acute postsurgical pain,and an estimated 10-50%of them develop persistent postsurgical pain.Evidence suggests that less than half of patients who undergo surgery report adequate postsurgical pain relief.Inadequately controlled pain negatively affects quality of life,function,and functional recovery,the risk of post-surgical complications.It has brought enormous socioeconomic and humanitarian burdens to individuals and societies.postsurgical pain represents a major,largely unrecognized clinical problem.The purpose of this study is to investigate the possibility of alleviating postsurgical pain by opening the KATP Channel and to discuss the underlying analgesic mechanisms.In this study,we try to propose new targets and strategies for the treatment of postsurgical pain.Methods:?1?The transcription and expression levels of SOCS3 after Cromakalim administration were detected by western blot and real-time fluorescence quantitative PCR both in microglia and in spinal cord.?2?The cell specificity of SOCS3 increasing after Cromakalim administration was investigated by immunofluorescence co-localization.?3?It was demonstrated that up-regulating of SOCS3 expression is associated with the opening of KATPTP channel but not the unique pharmacological effect of Cromakalim by trying more KATP channel openers beside Cromakalim and Kir6.1siRNA knocking down.?4?It was demonstrated that the up-regulation of SOCS3expression after Cromakalim administration was dependent on TLR4-NF-?B activating by using several specific inhibitors of TLR4-NF-?B signal pathway.?5?The level of HSP70 in cell culture supernate was detected by western blot.It was demonstrated that the up-regulation of SOCS3 expression after Cromakalim administration was dependent on HSP70 releasing by the reverse experiment using siRNA or neutralizing antibody and the mimic experiment using exogenous recombinant HSP70.?6?The specific inhibitors of classic endoplasmic reticulum/Golgi apparatus exocytosis pathway and lipid raft were used to study mechanism of HSP70release.?7?The levels of ERK and AKT phosphorylation after Cromakalim administration were detected by western blot.The specific inhibitor of MEK1/2,the up-stream kinase of ERK,was used to demonstrate whether HSP70 releasing was dependent on ERK phosphorylation.?8?The cerebrospinal fluid?CSF?of rats was collected and the HSP70 releasing in it was detected.?9?The effect of endotoxin tolerance induced by opening KATP channel was investigated by stimulating BV2 cells with LPS,IL-12?or IL-6.?10?Plantar incision model was used to investigate the analgesic effect of KATP channel opener,such as Cromakalim.?11?In vivo knocking down or overexpression of SOCS3 by lentivirus were performed to investigate whether the analgesic effect of Cromakalim was dependent on SOCS3.?12?Specific inhibitors of TLR4-NF-?B signal pathway were also administrated to demonstrate whether this pathway was involved in the analgesic effect of Cromakalim.Results:?1?Cromakalim significantly increased the SOCS3 transcription and expression both in microglia and in mice spinal cord.?2?Cromakalim mainly up-regulated SOCS3 expression level in microglia.?3?All of the KATP channel openers used in this study could promote SOCS3 expression.Moreover,specific knocking down Kir6.1 could abolish the effect of Cromakalim administration.?4?Specific inhibitors of TLR4 or NF-?B could block the effect of Cromakalim both in vitro and in vivo.?5?Knocking down HSP70 by siRNA could abolish the effect of Cromakalim.?6?The HSP70 neutralizing antibody blocked the effect of Cromakalim administration.?7?Exogenous recombinant HSP70 could mimic the effect of Cromakalim administration.?8?HSP70 was detected both in cell culture supernatant and CSF after Cromakalim administration.?9?Inhibitor of lipid raft,but not endoplasmic reticulum/Golgi apparatus exocytosis pathway,could block the effect of Cromakalim administration.?10?Cromakalim could increase ERK phosphorylation in BV2 cells.?11?Specific inhibitor of MEK1/2,reduced ERK phosphorylation,and abolished HSP70 release and up-regulation of SOCS3 after Cromakalim administration.?12?Pretreating BV2 cells with Cromakalim induced neuroinflammation tolerance,and suppressed inflammation induced by LPS,IL-12?and IL-6.?13?Cromakalim could ameliorate postsurgical pain in plantar incision model in a dose-dependent manner.?14?Knocking down SOCS3 abolish the analgesic effect of Cromakalim in vivo.?15?Overexpression of SOCS3 could mimic the effect of Cromakalim in vivo.?16?Intrathecal injection of TLR4-NF-?B signal pathway inhibitors could block the analgesic effect of Cromakalim in vivo.Conclusions:These results indicat that opening KATPTP channel increase the expression of SOCS3 in microglia and spinal cord,and this effect depends on the activation of TLR4-NF-?B signal pathway by released HSP70,one of the endogenous ligands of TLR4.HSP70 releases in a ERK phosphorylation mediated lipid raft form.Opening KATP channel induces neuroinflammation tolerance in microglia,and preemptive analgesia effect in vivo.Opening KATP channel can ameliorate both acute and chronic postsurgical pain in plantar incision model.The preemptive analgesia effect is dependent on activating TLR4-NF-?B signal pathway by HSP70,and SOCS3mediating neuroinflammation tolerance.In conclusion,our present study indicates that opening KATP channel increases SOCS3 expression in microglia and induces neuroinflammation tolerance.It may be a potential therapeutic strategy to postsurgical pain.Our study focuses on neuroinflammation in postsurgical pain.We try to induce neuroimmune tolerance and look for new targets and new treatment strategy to postsurgical pain based on the characteristics of innate immune system. |
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