| BackgroundThe occurrence and development of neuropathic pain is closely related to the secondary inflammatory reaction after nerve injury.Effective treatment of neuroinflammatory response is a feasible direction for the treatment of neuropathic pain.Protectin D1 is an important member of Specialized pro-resolving mediators(SPMs),which has strong anti-inflammatory,anti-apoptosis and neuroprotective effects.Peroxisome proliferator-activated receptor-?(PPAR-?),members of the nuclear hormone receptor superfamily,are expressed in a variety of tissues and are a family of receptors closely related to inflammatory responses.It has been found that PD1 can relieve neuropathic pain by blocking the inflammatory response caused by nerve injury.Recent studies have shown that PD1 can inhibit the expression of inflammatory factors by activating PPAR-?.However,whether PD1 relieves neuropathic pain by activating PPAR-? to inhibit the expression of inflammatory factors remains unclear.ObjectiveIn this study,we established a rat SNI model,intrathecal injection of PD1 and PPAR-? specific inhibitor GW9662,and using Behavioral and Molecular Biological methods were used to explore whether PD1 alleviates neuropathic pain by activating PPAR-? to inhibit the expression of inflammatory factors.Methods 1.Changes of PPAR-? expression in spinal cord of SNI ratsMale SD rats were randomly divided into two groups(n = 20): selective injury group(SNI group)and control group(Sham group).The paw withdrawal threshold(PWT)on the operative side and contralateral side of the rats was measured 3 days before operation to determine the basic behavioral value(Baseline,BL).The PWT of the ipsilateral side and the contralateral side of the rats were measured on the 1st,3rd,5th,7th,10 th and 14 th day after operation.Three rats in each group were taken after the pain behavior was measured at 0 d,3 d,5 d,7 d and 14 d after operation.The expression of PPAR-? in the spinal cord was detected by Western-Blot.2.Effects of intrathecal injection of PD1,on mechanical hyperalgesia and expression of PPAR-?,TNF-? and IL-6 in spinal cord of SNI ratsMale SD rats with successful intrathecal catheterization were randomly divided into 5 groups(n = 11): Sham + Vehicle group,SNI + Vehicle group,SNI + PD1 100 ng group,SNI + PD1 300 ng group,SNI + PD1 900 ng group.PD1 or Vehicle,was injected intrathecally once a day from 30 min before operation to 7 days after operation.The paw withdrawal threshold(PWT)on the operative side and contralateral side of the rats was measured 3 days before operation to determine the basic behavioral value(Baseline,BL).The PWT of the ipsilateral side and the contralateral side of the rats were measured on the 1st,3rd,5th,7th,10 th and 14 th day after operation to observe the effect of PD1 on the behavior of rats after SNI.Three rats in each group were taken after the pain behavior test was completed 7 days after operation.The effects of intrathecal injection of PD1 on the expression of PPAR-?,TNF-? and IL-6 in spinal cord were detected by Western-Blot.Vehicle:10% DMSO?3.Effects of intrathecal injection of PPAR-? specific antagonist GW9662,on analgesic effect of PD1 and expression of PPAR-?,TNF-? and IL-6 in spinal cordMale SD rats with successful intrathecal catheterization were randomly divided into 6 groups(n = 11): Sham + Vehicle + Vehicle group,SNI + Vehicle + Vehicle group,SNI + PD1+Vehicle group,SNI + Vehicle + GW9662 200 ?g group,SNI + PD1 + GW9662 100 ?g group,SNI + PD1 + GW9662 200 ?g group.In the pretreatment group,GW9662 or Vehicle,a specific antagonist of PPAR-?,was injected intrathecally once a day from 30 min before operation to 7 days after operation.,and 15 min before PD1 injection.The PWT of the ipsilateral side and the contralateral side of the rats were measured on the 1st,3rd,5th,7th,10 th and 14 th day after operation to observe the effect of GW9662 and PD1 on the behavior of rats after SNI.Three rats in each group were taken after the pain behavior test was completed 7 days after operation.The effects of intrathecal injection of GW9662 on the expression of PPAR-?,TNF-? and IL-6 in spinal cord were detected by WesternBlot.Vehicle:10% DMSO. Results 1.Mechanical hyperalgesia induced and down-regulation of PPAR-? protein expression in spinal cord of rats by SNIThe model of selective injury of the branch of the sciatic nerve(spared nerve injury,SNI)induced mechanical hyperalgesia on the ipsilateral side of the rats.Behavioral results show PWT of ipsilateral side in SNI group began to decrease on the 1st day after operation,and maintained at a low level on the 7th day after operation.the duration was more than two weeks(figure-1.1 A).The results of Western-Blot showed that SNI induced the down-regulation of PPAR-? expression in rat spinal cord(figure-1.2).2.Intrathecal injection of PD1 alleviates mechanical hyperalgesia in SNI rats,resulting in up-regulation of PPAR-? protein expression and down-regulation of TNF-? and IL-6 expression in spinal cordIntrathecal injection of PD1 can reduce the mechanical hyperalgesia induced by SNI in rats.Compared with Vehicle group,PD1 treatment group increased PWT in a dose-dependent manner(figure-2.1A).The results of Western-Blot showed that intrathecal injection of PD1 could inhibit the up-regulation of TNF-? and IL-6 expression induced by SNI,and the down-regulation of PPAR-? expression(figure-2.2).3.Intrathecal injection of GW9662 could attenuate the analgesic effect of PD1,down-regulate the expression of PPAR-? protein and upregulate the expression of TNF-? and IL-6 in spinal cordIntrathecal injection of GW9662 attenuated the analgesic effect of PD1 in a dosedependent manner.Compared with the PD1 group,the PWT in the GW9662 pretreatment group decreased in a dose-dependent manner(Fig.-3.1A).The results of Western-Blot showed that intrathecal injection of GW9662 could inhibit the downregulation of TNF-? and IL-6 expression and the up-regulation of PPAR-? expression in rat spinal cord induced by PD1(Fig.-3.2).ConclusionPD1 may relieve neuropathic pain by activating PPAR-? to inhibit the expression of inflammatory factors. |
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