| BackgroundDexamethasone(Dex)is a synthetic glucocorticoid, which is both a important physiological substance and widely used in clinical immunosuppression. Glucocorticoid receptor(GR) is a hormone-dependent transcription factor, which is one of the members of nuclear receptor superfamily. Studies have found that GR is widely expressed in peripheral and central nervous system. It was found that the generation and development of central pain sensitization was closely related to GR in the transmission of nociceptive information and the development of pathological pain.Neuropathic pain is a chronic pain condition that occurs after nerve damage, such as that induced by bone compression in cancer, infection, autoimmune disease, trauma and diabetes. In addition to spontaneous pain and hyperalgesia(the increased pain perception of noxious stimuli), a troublesome symptom of neuropathic pain is pain hypersensitivity to normally innocuous stimuli, called tactile allodynia. We are now beginning to understand that neuropathic pain is not just a symptom of disease, but is a consequence of disordered functioning of the nervous system. Unraveling the mechanisms of pain hypersensitivity caused by nerve damage is therefore essential for the development of new therapeutic drugs for neuropathic pain.Recent studies showed that the activation of glial cells in spinal cord dorsal horn could be observed on peripheral nerve injury, cancer pain and inflammatory pain model at different degrees. And glial cells play an important role in neuropathic pain, are key factors in the occurrence and maintenance of central hyperalgesia.As a member of mitogen-activated protein kinase signaling pathway, p38 is activated in spinal cells labeled with microglial marker CD11b(recognized by OX-42 antibody),and its activation in spinal microglia was reported in the SNL model, spared nervous injury model, after ventral root lesion and spinal cord injury(SCI) model. A lot of evidence showed that the development of neuropathic pain and activation of p38 MAPK in spinal microglia were closely related.Studies showed that glucocorticoid could regulate peripheral immune response and had anti-inflammatory effects on the brain, whie microglial cells played an important role in immune and inflammatory reaction in the brain tissues;The hyperplasia of microglia in the spinal dorsal horn had effect on the formation of neural sensitization during the process of neuropathic pain; Glucocorticoids could affect the BV-2 microglial proliferation and activation in vitro. However, whether glucocorticoids could affect the development of central hyperalgesia through activation of p38 MAPK signaling pathway in microglia during neuropathic pain in vivo is needed to verify. ObjectiveTo evaluate the effect of Dex and p38 MAPK inhibitor sb203580 on mechanical pain threshold, we established SNI model in rats and observed the change of 50% mechanical withdrawal threshold(MWT), GR and phosphorylated p38 expression in the spinal cord, and the expression of inflammatory cytokines IL-6 protein though injecting Dex and sb203580.Then primary microglial cells were cultured in vitro, and we observed the effects of Dex and sb203580 on expression of microglia activation marker CD11b/c in order to explore the analgesic effect of dexamethasone on rat SNI model and possible mechanism. Method1.Animal grouping: 90 SD male rats were randomly divided into nine groups: Naive group(n = 10), SNI group(n = 10), Sham group(n = 10),SNI + Dex group(n = 10), Dex group(n = 10), SNI + NS group(n = 10), SNI + sb203580 group(n = 100, sb203580 group(n = 10), SNI + DMSO group(n = 10); Cell grouping: cells were divided into 5 groups: DPBS group(n = 4), GLU group(n = 4), GLU + Dex group(n = 4), GLU + DMSO(n = 4), GLU + sb203580 group(n = 4).2.The establishment of SNI model: cut the skin and muscle,explored the sciatic nerve and the branch of sciatic nerve. Ligated the tibial nerve and peroneal nerve, sheared and removed a part of nerve stem. The Sham group exposed to sciatic nerve without surgery.3.Subarachnoid catheterization: PE10 was put into the endorhachis at L5-L6. The outflow of clear CSF from the catheter or the tail flick reflex showed the success of subarachnoid catheterization.4.50%MWT: 50% paw withdrawal threshold of Rats was measured by the Von frey wire.5.Western-blot: detected the expression of GR protein and p-p38 in the spinal cord on postoperative day of 3,7,14,21 in the Sham group and SNI group, and on the seventh postoperative day in the others.6.Enzyme linked immunosorbent assay(ELisa) detection: detected IL-6 protein levels in serum on the seventh day after operative.7.Primary microglia cell culture: the primary cultured spinal cord glial cells were purified and identified.8.Immunofluorescence: detected the fluorescence intensity of GR and CD11b/c in each group of cells. Result1.MWT results showed that compared with the Sham group, 50% paw withdrawal threshold(PWT) of rats in SNI group decreased significantly on postoperative day of 7,14,21,28(P<0.05); Compared with SNI group, 50%PWT of rats in SNI+Dex group injecting Dex earlier increased significantly on the postoperative day of 7(P<0.05),50%PWT of rats in SNI+Dex 10 group injecting Dex later increased on the postoperative day of 10,11,12(P<0.05), and 50%PWT of rats in SNI+sb203580 group was elevated on the postoperative day of 7,9,11(P<0.05).2.Western-blot results showed that the level of GR protein in SNI group was significantly decreased on the postoperative day of 7,14,21( P<0.05),and phosphorylated p38 expression level was increased on the postoperative day of 3,7(P<0.05); The level of GR expression in SNI+Dex group was significantly increased(P<0.05), and the phosphorylation of p38 was decreased(P<0.05); Compared with the SNI+DMSO group, phosphorylated p38 expression level of SNI+sb203580 group was decreased significantly(P<0.05) but changes of GR expression had no statistical significance(P>0.05).3.Elisa results showed that compared with the Sham group, IL-6 protein expresion in serum of SNI rats was significantly increased(P<0.05); Compared with SNI group, the IL-6 levels of SNI + Dex group and SNI + sb203580 group were significantly lower(P<0.05).4. Immunofluorescence results in GLU group showed that compared with the DPBS group, morphological features of microglial activation were a cell body hypertrophy and retracted processes, but in GLU+Dex group cells had slender and long branches; The immunofluorescence intensity of GR in GLU+Dex group was significantly increased,but the intensity of CD11b/c was significantly lower as the same as the intensity of CD11b/c in GLU+sb203580 group(P<0.05). ConclusionsThe analgesic effect of dexamethasone on early mechanical pain sensitivity of SNI model rats can be mediated by p38 MAPK in activated microglia. |
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