The Mechanism Of Glucocorticoid Receptor In Microglia On Early-phase Neuropathy Pain

Background The International Association for the Study of Pain defined neuropathic pain as the abnormal pain caused by central or peripheral nervous system injury.Neuropathic pain belongs to a kind of chronic pain,the major clinical manifestations were hyperalgesia,paresthesia,abnormal spontaneous pain,etc.Because of its high incidence,the poor overall effect of the pain management,it is essential to explore the pathogenesis of neuropathic pain thus to provide reliable basis for pain management.Glucocorticoid receptor(GR)is not only a type of hormone-dependent nuclear receptor,but an important transcription factor.When it is activated,it enters into the nuclear thus to negative or positive its target genes,and participate in different physiological and pathological process.In recent years,more and more evidences showed that the central glucocorticoid receptor involved in the central pain sensitivity formation and the transmission of abnormal information,but the specific mechanism is not clear.As is known that,central activated microglia could synthesize and release the pain and inflammatory medium(such as COX 2,IL-6,TNF alpha,etc.),thus to active the neurons or surounding astrocytes,then enhanced the sensitivity and reactivity of neurons,causing mental derangement of rational persistent neuropathic pain.Then,how the central GR in the microglia involve in the prosess of neuropathic pain induced by nerve injury?Rensent study found that the activation of the protein kinase p38 MAPK participate in the process of the central sensitization,and the activated p38 MAPK specifically express in microglial.We used spared nerve injury(SNI)rats as pain models and intrathecal injected p38 MAPK inhibitor thus to explore the regulation of microglia p38 MAPK on GR.Than intrathecal injected GR inhibitor to test the regulation of GR onto the downstream transcription factor nuclear factor kappa B(NF-?B)and proinflammatory factor.We aim at exploring the mechanism of GR in the development of neuropathic pain,providing new experimental basis for pain treatment.Objective(1)Established SNI neuropathy pain model in rats,observed the changes of mechanical pain behavior and spinal cord GR protein expression levels after SNI operation.(2)Blocked the p38 MAPK pathway by intrathecally injecting SB203580 and used RU38486 to inhibit the function of GR,than test the changes of the mechanical behavior and spinal cord GR ? phosphorylation NF-kappa B(p NF-?B).Thus to explore the regulation of p38 MAPK signaling pathway on GR.(3)Blocked the GR with dexamethasone(DEX)treatment,than test the changes of the mechanical behavior and spinal cord p NF-?B as to check the regulation of GR on the downstream transcription factor p NF-?B.Methods(1)Established SNI model,tested the reliability of the model and the changes of GR protein of spinal cord Divide SD male rats into nine groups: normal group(n = 12),spared nerve injury SNI group(n = 12),sham group(n = 12),Sham+Vehicle(n = 12),SNI +SB203580 group(n = 12),SNI+Vehicle(n = 10),SNI+SB203580+RU38486 group(n= 10),SNI + DEX group(n = 12).For SNI group,cut open the skin,subcutaneous tissue,muscle and fascia,make the the sciatic nerve and three branch fully showed,cut off the nerve stems in the middle of two ligation points and removed a part of nerve stems.Sham group only exposed the sciatic nerve,and do nothing with ligation processing.Von Frey to test the mechanical pain behavior as to ensure the successful establishment of pain model;Enzyme-linked immunosorbent(ELISA)to test the change of plasma corticosterone;Western blot to test the expression of spinal cord GR?phosphorylated p38(p-p38)and p-NF-?B;Immunofluorescence chemical to test the colocalization of GR with microglia?astrocytes and neurons.(2)Regulation of p38 on GR Blocked the p38 MAPK pathway by intrathecal injecting SB203580 in early-phase of neuropathic pain,than tested the changes of mechanical pain behavior of rat.Western blot and immunofluorescence techniques to detect the changes of the downstream transcription factor p-NF-?B;ELISA to detect the changes of serum pro-inflammatory factor IL-6 and TNF-alpha(TNF-?).Using RU38486 to block down the function of GR,than observed whether the phenomenon was reversed by RU38486.(3)Regulation of GR on phosphorylated NF-?B Blocked the GR by intrathecal injecting DEX in early-phase of neuropathic pain,Than used Western-blot and ELISA assay to test the changes of mechanical pain behavior and the phosphorylated NF-?B and the changes of serum pro-inflammatory factor IL-6 and TNF-?.Thus to test whether the RU38486 reversed the reduction trend of phosphorylated NF-?B and pro-inflammatory factor induced by SNI.Results1.The establishment of SNI model was successful.Compared with the sham group,the mechanical withdrawal threshold of SNI group was lower at 3 d,7 d and 14 d and21 d and 28 d after SNI operation.Western-blot results showed that,the GR expression in the SNI group was gradually reduced compared with the sham group and the phosphorylated p38 and TNF-? ? IL-6 expression was increased.Immunofluorescence double staining showed the GR was expressed in microglia?neurons and astrocytes.2.Western blot and immunofluorescence results showed that,following intrathecal injection SB203580 for consecutive 7 days in SNI rat,the GR was increased and the the phosphorylated NF-?B expression was decreased,and ELISA showed the serum pro-inflammatory factor IL-6 and TNF-? was decreased.Intrathecal injection of RU38486 reversed the phenomenon induced by SB203580 in SNI rats.3.Intrathecal injection of DEX increased the expression of GR in the early-phase neuropathic pain,accompanying with reduction of phosphorylated NF-?B and the pro-inflammatory factors.Western blot and immunofluorescence assay showed that the GR negatively regulated the expression of phosphorylated NF-?B.Conclusions1.The activation of spinal microglia p38 MAPK signaling pathways promoted the occurrence and development of pain by down-regulating the expression of GR on the early-phase of neuropathic pain.2.The activation of GR eased the pain by inhibiting the phosphorylation of spinal cord NF-?B and the expression of IL-6 and TNF-?.