| ObjectiveMicroglia are important immune cells in the central nervous system.Microglia remain in a resting state when they are normal.When microglia recognize injury stimuli through antennae,they are quickly activated and responded to and become macrophages.A large number of studies have shown that microglia play an important role in neuropathic pain.Calcitonin gene-related peptide(CGRP)is a major pro-inflammatory neuropeptide,which is widely expressed in the central and peripheral nervous systems and is considered to play an important role in pain sensory transmission.Therefore,the activation of microglia mediated by CGRP may play an important role in neuropathic pain,and the neuropeptide and its receptor may become an important target in the treatment of neuropathic pain.Although the biological role of CGRP has important clinical significance in the occurrence of some diseases and neuropathic pain,the clinical treatment targeting CGRP receptors has not progressed due to the lack of research on its functional receptors.This is a good entry point to clarify the pathophysiological mechanism of neuropathic pain and its clinical treatment.The research on CGRP and its receptors will be of great clinical value for neuropathic pain relief.Histone deacetylase is a kind of protease responsible for histone acetylation modification,which plays an important role in chromosome structure modification and gene expression regulation.In recent years,there is a lot of evidence that histone deacetylase is involved in chronic neuropathic pain,but the effect of CGRP on HDACs in neuropathic pain has not been clear progress.On the one hand,this study establishes a classical neuropathic pain model,the chronic constraining injury of the sciatic nerve(CCI),studies whether CCI increases the activation of microglia and the release of transmitters and the expression of CGRP by immunohistochemistry,studies the signal pathways of CGRP receptor protein RCP to RIG-I,IFN-1and NF-κB,and HDAC2,IFN-1 and NF-kappa B.The influence of RANTES and INOS.On the other hand,BV-2 microglia were incubated with CGRP and the expression of CGRP-related proteins was observed by Western bolt to explore the regulation and molecular mechanism of CGRP-activated microglia in neuropathic pain.Methord1.A rat CCI model was established.The paw withdrawal threshold(PWT)was measured by von Frey one day before operation and one day,three days,seven days,ten days and 14 days after operation.2.Immunohistochemical method was used to observe the difference of microglia activation and CGRP expression in spinal cord between CCI model group and blank control group.3.Immunohistochemical methods were used to observe whether CCI affected the expression of microglia in spinal cord,including receptor protein RCP,RIG-I,IFN-1,NF-κB,HDAC2,RANTES and so on.4.CGRP incubated microglia(BV-2)for 0 hours,1 hour,2 hours,4 hours and 6 hours respectively.Total protein was extracted.Western bolt method was used to observe the effects of CGRP on the expression of receptor proteins RCP,RIG-I,IFN-1,NF-κB,HDAC2,RANTES and INOS in microglia.5.HDACi(SAHA)incubated microglia(BV-2)for 0 hours,1 hour,2 hours,6 hours,12 hours and 24 hours respectively.Total protein was extracted.The effects of HDACi on INOS concentration in microglia were observed by Western bolt method.Result1.Establishment of rat CCI modelAfter the establishment of CCI model in rats,the threshold of contraction of right hind paw decreased slowly.It can be seen that the threshold began to decrease 1 day after operation,and then decreased 3,7 and 10 days after operation,and tended to be stable until14 days after operation.The data have statistical significance,indicating that the rat model of neuropathic pain has been established.2.CCI increases activation of spinal microglia and expression of CGRP(1)Immunohistochemistry showed that the activated microglia in the spinal cord of CCI group increased significantly,which was significantly different from that of sham-operated group(p < 0.05).(2)Immunohistochemistry showed that the expression of CGRP in spinal cord of CCI group was increased,and the difference was statistically significant compared with sham operation group(p < 0.05).(3)Ionized calcium binding adapter molecule 1(Iba1)and CGRP found that bead-like CGRP fibers surrounded the activated microglia were closely bound to each other,which indicated that CGRP played an important role in the activation of microglia in neuropathic pain.3.The double immunofluorescence staining of CRCP and Iba1 showed that CCI up-regulated the expression of CRCP in glial cells of spinal cord.Compared with sham-operated group,the difference was statistically significant(p < 0.05).4.Immunofluorescence double labeling of RIG-I,IFN-1,NF-κB,HDAC2,RANTES and Iba 1 showed that CCI up-regulated the expression of RIG-I,IFN-1,NF-kappa B and HDAC2 in spinal cord microglia,with significant difference compared with sham operation group(p < 0.05).5.CGRP incubated microglia(BV-2)for 0 hours,1 hour,2 hours,4 hours and 6 hours.Western bolt showed that CGRP increased the expression of CRCP and RIG-I,IFN-1,NF-κB,HDAC2,RANTES and INOS.6.CGRP and HDACi co-incubated microglia(BV-2).Western bolt showed that compared with the CGRP group,CGRP inhibited the down-regulation of INOS by HDACi.Conclusion1.CCI increased the activation of microglia and the expression of CGRP in spinal cord.CGRP fiber and the close contact of microglia around and activation,indicating that CGRP interaction for the activation of spinal microglia.2.CCI up-regulates the expression of CGRP receptor protein CRCP,HDAC2,RIG-I,IFN-1,NF-κB,RANTES and INOS in spinal microglia.3.CGRP plays an important role in regulating the synthesis and release of microglia activating mediators.It participates in the mechanism of neuropathic pain through receptor protein RCP and RIG-1 signaling pathway.4.CGRP up-regulates the expression of HDAC2 in microglia,and HDACi dow n-regulates the expression of INOS,suggesting that CGRP may regulate the express ion of INOS through HDAC2,thereby regulating neuropathic pain. |
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