Up-regulation Of P2X7 Receptor In Satellite Glial Cells Of Drg Contributes To Chronic Pain Transition

Background and Objective The International Pain Association(IASP)defines chronic post-surgical pain(CPSP)as occurring after surgery,with surgery-related,other causes and lasting at least 2 months of chronic pain.CPSP has a high incidence,a recent large sample of domestic studies showed that the incidence of CPSP was 29.6% in 3110 patients,30.3% of whom were in anxious state and 24.4% were in a depressed state.P2X7 receptor is adenosine triphosphate(ATP)gated ion channel,belonging to the purine can P2 receptor family,involved in cell signaling,cytokine secretion and other physiological functions.Recent experimental studies have shown that P2X7 receptor not only in immune cells can cause apoptosis,but also can affect the cell many complex physiological and biochemical functions.Studies have shown that in rats with neuropathic pain and inflammatory pain.P2X7 receptors in the dorsal horn of the spinal cord,by regulating inflammatory factors such as TNF-α and IL-1β,Such as the differentiation and release,participation in neurons and glial cell dialogue,in the development of central sensitization plays an important role.Our previous study has confirmed that P2X7 receptors can participate in the transformation of acute and chronic pain by regulating the activation of microglia and the release of inflammatory factors in the dorsal horn of the spinal cord.Inhibition of P2X7 receptor activation in the spinal dorsal horn can down-regulate the release of tumor necrosis factor-α and significantly improve chronic pain after SMIR.Dorsal root ganglion(DRG)is located in the posterior root of the spinal nerve,the peripheral nerve and central nervous transmission center.The neurons on the DRG are false monopolar neurons.Each neuron on the ganglia emits an axon and forms two branches,which are distributed throughout the periphery(skin,visceral,etc.)to receive peripheral stimuliand pass to the center nervous system.The neurons on the DRG are the first neurons of the peripheral pain signal that pass on to the cerebral cortex,and are closely related to the mechanism of pain transmission.When the body is subjected to peripheral stimulation,the DRG first senses this change and activates the DRG neurons,causing peripheral sensitization,and then transmitted to the dorsal horn of the spinal cord,causing abnormal neuronal excitability of the dorsal horn of the spinal cord,Fiber LTP produces and causes central sensitization,which leads to the generation of pain in the brain.Although our previous results have shown that activation of the P2X7 receptor in the dorsal horn of the spinal cord is an important central mechanism of chronic pain after surgery,it is not clear about the peripheral mechanisms of CPSP production.Based on the above,we expect to explore the peripheral mechanism of postoperative pain chronic formation.In this study,SMIR model was used to simulate the effect of postoperative acute pain on postoperative persistent pain,to explore the mechanism of P2X7 receptor in postoperative chronic pain,and to explore the effect of P2X7 receptor on dorsal root ganglion by mediating the activation of ERK / MAPK signaling pathway in the process of chronic pain transition.Methods SD male adult rats measure 180 to 220 g were random divided into two groups:SMIR group and Sham group.SMIR group used Flatter’s skin / muscle incision and retraction(SMIR)to establish a chronic pain model after Surgery in rats.Sham uses sham surgery as a control.The mechanical threshold reaction(PWT)was measured at preoperative day 1(T0,baseline)and 1,3,7,12,22 and 32 days after operation,respectively.After the above-mentioned pain thresholds,The expression of P2X7 R,ERK / MAPK and TNF-α in the ipsilateral L2-4 DRGs were detected by Western Blot at different time points(n=6).P2X7 R and ERK were co-localization with double immunostaining(n=6).The cell types of P2X7 R and TNF-α were observed by use double immunostaining.In order to further confirm the relationship between P2X7 R,ERK / MAPK signal pathway and TNF-α release,we observed the changes of the expression of p-c Raf,p-MEK,p-ERK and TNF-α levels induced by SMIR after intraperitoneal injection of P2X7 R inhibitor BBG.The changes of P2X7 R and TNF-αrelease in L2-4 DRGs were observed in the model group with intrathecal injection of ERK inhibitor SCH772984.Results1.SMIR surgery induced a significant decrease in ipsilateral PWT Statistical analysis of the PWT values at each time point showed no significant difference in baseline PWT values between the two groups(P>0.05).In the SMIR group,PWT of the ipsilateral side was decreased from day 1(P<0.01)to day 22 after operation and return to the baseline level on day 32(P<0.001)after operation.The50% paw withdrawal threshold on the ipsilateral side of the SMIR group was significantly decreased,and the pain returned to baseline at 32 days after operation(P>0.05).On the day 12,PWT on the ipsilateral side reach the peak(P<0.001).There was no significant difference in PWT between the SMIR contralateral side and baseline(P>0.05).There was no significant difference in the PWT between the Sham group and their baseline(P>0.05).2.Up-regulation of P2X7 R on satellite glial cells in dorsal root ganglion after SMIR surgery Western blot results shown that the expression of P2X7 R in the ipsilateral L2-4dorsal root ganglion in the SMIR model group was significantly increased form day 1(P<0.05)to day 22(P <0.01),peaked at 7th day after operation(P <0.001),and then gradually reduced until day 32 returned to the preoperative level(P>0.05).To identify the cell types that express P2X7 R in DRGs after SMIR,we used double immunofluorescence labeling for P2X7 R and cell type specific markers :neuron specific marker NF200,CGRP,Ib4 and Satellite glial cells specific marker GFAP in the ipsilateral L3 DRG of rats on day 7 after operation.The results showed that P2X7 R were double-labeled with GFAP,but not with NF200,CGRP,and Ib4.The results suggest that P2X7 R is expressed on SGCs in the SMIR model rat dorsal root ganglion.3.ERK/MAPK signal pathway in dorsal root ganglia significantly activited in SMIR group Western blots results show that the expression of p-c Raf,p-MEK and p-ERK protein in the dorsal root ganglion of rats was significantly increased(P<0.001)on the first day after SMIR model establishment,and reached the peak at the day 12 after operation(P<0.001),And recovered to the preoperative level on day 32postoperatively(P> 0.05).The correlation between the expression of p-c Raf,p-MEK and p-ERK and the changes of PWT at different time points after SMIR is shown the expression of p-c Raf,p-MEK and p-ERK were significantly correlated with the changes of PWT(p-c-Raf(Pearson correlation;r=-0.965,P=0.002),p-MEK(Pearson correlation;r=-0.906,P=0.013),p-ERK(Pearson correlation;r=-0.887,P=0.018)).Immunofluorescence staining of P2X7 R and p-ERK of L3 DRG in the SMIR model group on day 7 postoperatively showed that P2X7 R and p-ERK were co-labeled.4.The release of TNF-α in dorsal root ganglia significantly up-regulated in SMIR group The results of Western blot showed that the L2-4 DRGs of the rats in the SMIR group increased at day 1 and peaked at day 12 after operation(P <0.001),and then gradually decreased until day 32 after surgery recover to the basic preoperative level(P>0.05).Immunofluorescence double staining showed that TNF-α was mainly expressed on DRG neurons.The neuronal specific markers such as large neuron specific marker NF200,C-peptide small neuron CGRP and C-fiber non-peptidic small neuron Ib4,TNF-α cells were significantl expressed on all,But not with Satellite glial cells specific marker GFAP.5.ERK-specific antagonist SCH772984 can relieve the pain of SMIR rats and down-regulate the release of TNF-α in dorsal root ganglion without affecting the expression of P2X7 receptor after SMIRThe results showed that the PWT value of the SCH772984 SMIR model group was significantly improved compared with that of the intrathecal injection of DMSO group.There was no significant difference in the PWT value between the SCH772984 SMIR model group and the sham operation group(P>0.05).Compared with the DMSO group,the PWT values in the SCH772984 group were significantly increased(P>0.001)from the day 1 to the day 22 after surgery,and the pain of the SCH772984 group was significantly alleviated.Western blot analysis of TNF-α in the ipsilateral L2-4 DRGs in the two groups of rats(SCH772984 and DMSO groups)on day 7 after SMIR showed that the expression of TNF-α in SCH772984 group and DMSO group was significantly higher than that in blank control group(P <0.001).Compared with DMSO group,the expression of TNF-α in SCH772984 group was significantly decreased(P<0.001).Western blot analysis of P2X7 R of the ipsilateral L2-4 DRGs in SCH772984 group and DMSO group on day 7 after SMIR showed that the expression of P2X7 R in SCH772984 group and DMSO group was significantly up-regulated compared with the blank control group(P <0.001).Compared with DMSO group,the expression level of P2X7 R in SCH772984 group had no significant difference(P>0.05).6.Intraperitoneal injection of BBG can inhibits mechanical allodynia,the activation of ERK/MAPK pathway and the up-regulation of TNF-α in DRGs induced by SMIR The results showed that PWT values of SMIR model rats with intraperitoneal injection of BBG were significantly improved compared with intraperitoneal injection of normal saline.BBG group PWT values compared with the sham-operated group review no significant difference(P>0.05).Compared with the NS group,the PWT values of the BBG group were significantly increased(P <0.001)from day 1 to day22.The pain of the SMIR model rats in the BBG group was significantly alleviated.Western blot analysis of p-c Raf,p-MEK,p-ERK and TNF-α in the ipsilateral L2-4 DRGs on day 7 after SMIR in the two groups(BBG and NS)showed that compared with the blank control group p-c Raf,p-MEK,p-ERK and TNF-α in the NS group were significantly higher(P<0.001)while in the BBG group were shown ameaningful decrease.In BBG group p-c Raf,p-MEK,p-ERK and TNF-α were significantly lower than those in NS group(P <0.001).Conclusion In this study,the rat skin/muscle incision traction(SMIR)model was used to simulate postoperative acute and chronic pain transformation,we found that1.Up-regulation of P2X7 R on satellite glial cells in dorsal root ganglion after SMIR surgery;ERK/MAPK signal pathway is activated;the release of TNF-α in dorsal root ganglia significantly up-regulated after SMIR surgery;2.ERK specific antagonist SCH772984 can relieve the pain of SMIR rats and down-regulate the release of TNF-α in dorsal root ganglion without affecting the expression of P2X7 receptor after SMIR;3.Intraperitoneal injection of BBG can inhibits mechanical allodynia,the activation of ERK/MAPK pathway and the up-regulation of TNF-α in DRGs induced by SMIR.So,the transformation from acute pain to chronic pain may be due to the activation of P2X7 receptor in the spinal dorsal root ganglion satellite glial cells,which activated the ERK/MAPK signaling pathway and mediates the overexpression of TNF-α in neurons.