| Research background: Neuropathic pain is mostly chronic and paroxysmal,which can cause insomnia,lack of energy,anorexia,nervousness and other symptoms,which can cause great distress to patients.Previous drug treatments have mostly targeted neurons.In recent years,more and more evidences show that glial cells are involved in the occurrence and regulation of chronic pain,which suggests that the study on the mechanism of glial cells in chronic pain may become a new target for the treatment of chronic pain.P2X4 receptors is a subtype for purine P2 X receptors(ion channel receptors),in the Spinal dorsal horn(Spinal dorsal horn,SDH)microglia and dorsal root ganglion(dorsal root ganglion,DRG)satellite glial cells(SGCs)were expressed,and express more during chronic nerve pathological pain,P2X4 receptor blockers or knockout P2X4 receptors can reduce nerve pathological pain,this study suggests that glial cells P2X4 in chronic nerve pathological pain plays an important role in the process of development.Studies have shown that there is an interaction between neurons and glial cells.When P2X4 receptor is activated in neuropathic pain,glial cells can be activated at the same time,causing the release of inflammatory factors,thus improving the sensitivity of neurons and leading to chronic pain.Therefore,purine P2X4 receptor is involved in the interaction between neurons and glial cells.Neohesperidin(NH)can inhibit the activity of inflammatory factors,reduce the effect of immune response and nourish the nervous system.Studies have shown that nehesperidin can effectively alleviate chronic pain in neuropathologic pain model mice,suggesting that NH may be involved in the occurrence and regulation of chronic pain,but whether it plays a role by acting on P2X4 receptors is unclear.Purpose: To investigate the role of neohesperidin in chronic neuropathologic pain mediated by P2X4 receptors in spinal dorsal horn and dorsal root ganglion.Methods: A rat model of chronic neuropathic pain caused by chronic constriction injury of sciatic nerve(CCI)was established.The success of the model was determined by measurement of mechanical and thermal pain sensitivity.The experiment was divided into 6 groups: control group(Ctrl group),control group +neohesperidin group(Ctrl +NH group),Sham group(Sham group),CCI model group(CCI group),CCI+neohesperidin group(CCI+NH group),and model + drug solvent group(CCI+DMSO group).1)To measure the pain behavior of rats: The mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were used to detect the pain threshold of rats in each group.2)The dorsal root ganglia and corresponding spinal cord segments of rats from L4-L6 were removed,and the changes of P2X4 m RNA in DRG and SDH were detected by real-time quantitative PCR.3)The levels of P2X4 receptor and satellite glial cell activation marker(glial fibrillary acidic protein,GFAP)in DRG and the expression of P2X4 receptor and microglial marker OX-42 in SDH were detected by double-label immunofluorescence assay.4)The changes of P2X4,tumor necrosis factor alpha receptor 1(TNF-R1),GFAP,OX-42,phosphorylated extracellular signal-regulated kinase(p-ERK),total ERK and upstream regulator IRF5 in DRG and SDH of rats in each group were detected by Western blot,and the effects of neohesperidin on the expression of these proteins were observed.Results:(1)In this study,CCI neuropathic pain model was established in rats,and on this basis,neohesperidin was used for drug intervention in CCI rats.The results of mechanical pain threshold and thermal pain threshold measurement showed that the MWT and TWL in CCI model rats were significantly lower than those of control rats(p <0.001),indicating the successful establishment of neuropathic pain model.In CCI model rats after neohesperidin intervention,the above indexes were significantly increased(p <0.001),while there was no significant difference in DMSO intervention group rats(p >0.05).(2)Neohesperidin intervention can reduce the expression of P2X4 receptor in DRG and SDH tissues of CCI model rats.The level of P2X4 m RNA in DRG and SDH tissues of CCI model rats was significantly higher than that of normal control rats,the difference was statistically significant(p <0.001).In CCI rats treated with neohesperidin,P2X4 m RNA levels were significantly decreased compared with the model group(p<0.001),while there was no difference in P2X4 m RNA levels in CCI rats treated with DMSO compared with the model group(p >0.05).Western blot analysis of P2X4 receptor protein showed that the P2X4 protein in DRG and SDH tissue of CCI model rats was significantly higher than that of normal control rats,the difference was statistically significant(p <0.001).Compared with the model group,the expression level of P2X4 receptor protein was significantly decreased in CCI rats treated with neohesperidin,with statistical significance(p <0.001).However,DMSO intervention did not decrease the expression of P2X4 receptor protein in CCI rats(p >0.05).(3)Neohesperidin drug intervention reduced the activation of glial cells in CCI model rats.Immunofluorescence showed that microglia marker OX-42 was co-localized with P2X4 receptor in SDH.In DRG,satellite glial cell activation marker GFAP was co-localized with P2X4 receptor.The fluorescence intensity of CCI model rats was significantly higher than that of normal control rats(p <0.001).In the CCI group treated with neohesperidin,the fluorescence intensity was significantly lower than that in the CCI group(p <0.001).However,DMSO intervention could not reduce the fluorescence intensity of CCI rats(p >0.05).(4)Neohesperidin decreased the expression of TNF-R1 in CCI rats: The results showed that TNF-R1 in CCI model rats was significantly higher than that in normal control rats(p <0.001);However,TNF-R1 expression was significantly reduced in the CCI group treated with neohesperidin compared with the model group(p <0.001).After DMSO intervention in CCI rats,TNF-R1 expression showed no statistically significant difference compared with that in CCI group(p >0.05).(5)P2X4 receptor activates ERK signaling pathway to participate in chronic neuropathologic pain.The results showed that the p-ERK1/2 protein level of CCI model rats was significantly higher than that of normal control rats(p <0.001).After CCI rats were treated with neohesperidin,the expression of p-ERK1/2 protein was significantly decreased compared with CCI rats,and the difference was statistically significant(p <0.001).However,the expression of p-ERK1/2 protein in CCI rats was not decreased after DMSO intervention(p >0.05).(6)The effect of neohesperidin on IRF5,the upstream regulator of P2X4 receptor expression.IRF5 transcription factor can regulate the transcription of P2X4 receptor and increase the expression of P2X4 receptor.It was observed that the level of IRF5 in CCI rats was significantly higher than that in the control group,and the expression of IRF5 in CCI rats was significantly lower than that in the model group after treatment with neohesperidin(p <0.001).Conclusion: Neohesperidin can effectively inhibit the expression of P2X4 receptor in SDH and DRG,inhibit the the activation of SGCs in DRG and microglia cells in SDH,reduce the activity of ERK1/2 pathway,reduce the expression of TNF-R1,inhibit the transmission of pain signals,and finally alleviate the pain behavior of CCI rats.Therefore,neohesperidin can reduce the pain behavior of neuropathologic pain rats by reducing the expression of P2X4 receptor in glial cells and the activation of glial cells. |
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