Study On The Analgesic Mechanism Of Propentofylline And ERK1/2 In Spinal Glial Cells Of Rats With Incisional Pain

Objective:To investigate the role of extracellular signal-regulated kinase 1/2（ERK1/2）signaling pathway in rats with incisional pain and the molecular mechanisms of propentofylline on relieving postoperative pain.Methods:Seventy-eight male Sprague Dawley rats,weighting between 200 and250 g,were randomly divided into six groups:the blank control group（Blank group,n=6）,the incision pain group（IP group,n=18）,the normal saline group（NS group,n=15）,the propentofylline group（PPF group,n=18）,the dimethyl sulfoxide group（DMSO group,n=9）,the inhibitor group（U0126 group,n=12）).The rat model of incisional pain was prepared in all groups except the Blank group.Rats in NS group,PPF group,DMSO group and U0126 group were intrathecally injected with NS（10μl）,PPF（10μg）,10%DMSO（10μl）and U0126（10μg）respectively at 30 min before operation.The paw withdrawal mechanical threshold（PWTL）and paw withdrawal thermal latency（PWTL）were measured at 2,4,8,24 and 72 h after operation.The L_4-6-6 segments of spinal cord in rats were immediately collected after the behavioral measurement at the preset time points.The expression of NeuN,GFAP,Iba-1,t-ERK1/2 and p-ERK1/2 were detected by Western Blot.The expression of neuron,astrocyte,microglia,p-ERK1/2,TNF-αandCOX-2wereexposedby immunofluorescence.Furthermore,the co-expression of p-ERK1/2 with spinal neuron,astrocyte and microglia were also exposed by immunofluorescence.The data were collected and then analyzed statistically.Results:1.Behavioral measurement results:Compared with Blank group,PWMT and PWTL significantly decreased in IP group at all time points after operation.Compared with IP group,the PWMT and PWTL had no significant difference in NS group and DMSO group at all time points after operation;the PWMT and PWTL significantly increased in PPF group at all time points after operation;the PWMT and PWTL significantly increased in U0126 group at 2,4,8 and 24 h after operation,but had no significant difference at 72h after operation.2.Western Blot results:Compared with Blank group,the expression of NeuN,GFAP and Iba-1 increased in IP group at all time points after operation（P<0.05）;the expression of t-ERK1/2 had no significant difference at all time points after operation;the expression of p-ERK1/2 increased at 2,4 and 24 h after operation（P<0.05）,but had no significant difference at 72 h after operation.Compared with IP group,the expression of NeuN,GFAP and Iba-1 had no significant difference in NS group at 4 h after operation;the expression of t-ERK1/2 and p-ERK1/2 had no significant difference at all time points after operation.Compared with IP group,the expression of NeuN,GFAP,Iba-1,t-ERK1/2 and p-ERK1/2 had no significant difference in DMSO group at 4 h after operation.Compared with IP group,the expressions of NeuN,GFAP and Iba-1 decreased in PPF group at 4 h after operation（P<0.05）;the expression of t-ERK1/2 had no significant difference at all time points after operation;the expression of p-ERK1/2 was decreased at 2,4 and 24h after operation（P<0.05）,and had no significant difference at 72 h after operation.Compared with IP group,the expression of NeuN,GFAP,iba-1 and p-ERK1/2 decreased in U0126 group at 4 h after operation（P<0.05）;the expression of t-ERK1/2 had no significant difference at4 h after operation.3.Immunofluorescence results:Compared with Blank group,the expression of spinal neuron,astrocyte,microglia,p-ERK1/2,TNF-αand COX-2 increased in IP group increased after operation,when the cell body swelled and the protuberance increased in astrocyte and microglia.Compared with IP group,the expression of spinal neuron,astrocyte,microglia,p-ERK1/2,TNF-αand COX-2 decreased in PPF group and U0126 group at 4 h after operation,when the swell of cell body reduced and the protuberance decreased in astrocyte and microglia.4.Immunofluorescence result:The p-ERK1/2 were mainly expressed in the ipsilateral spinal cord dorsal horn and co-expressed with NeuN,GFAP and Iba-1 in IP group at 4 h after operation.Conclusions:1.The paw incision can induce the activation of spinal astrocyte and microglia,which can further regulate the neuronal excitability by intercellular information exchange;2.Spinal ERK1/2 signaling pathway promoted the development of incisional pain and expression of TNF-αand COX-2.After blocking the phosphorylation of ERK1/2,the expression of TNF-αand COX-2 decreased and the incisional pain was effectively reduced in the early stage.3.The ERK1/2 signaling pathway were co-expressed with spinal neuron,astrocyte and microglia when the activation was the strongest.4.The spinal mechanism of PPF on relieving incisional pain may partially relate to the inhibition of ERK1/2 signaling pathway in glial cells and decrease of TNF-αand COX-2.