| Object:To evaluate the role of spinal c-Jun N-terminal kinase(JNK) signaling pathway, and the link of JNK with the analgesia effect of PPF in a plantar incision rat model.Methods:One hundred and fifty adult male Sprague-Dawley rats, weighing200~250g, were randomly divided into 5 groups(n=30 each) using a random number table: IP group(incisional group),incisional surgery only underwent; DMSO group(10%DMSO group),10%dimwthy sufoxide 10μl was injected intrathecally at 30 min before surgery; NS group(0.9% saline group),0.9% saline 10μl was injected intrathecally at 30 min before surgery;SP group(SP600125 group), SP600125 25μg/10μl was injected intrathecally at 30 min before surgery; PPF group(Propentofylline group) propentofylline 10μg/10μl was injected intrathecally at 30 min before surgery. Six rats in each group were sacrificed, and the mechanical paw withdrawal threshold(MWT) and thermal paw withdrawal latency(TWL) were measured at 24 h pre- and 2,6,24,48,72 h postoperation. After measurement of the pain threshold at 24 h before and 6,24,48,72 h after establishment of the model, the lumbar segment of the spinal cord was removed for determination of the expression of TNF-α and phosphorylated JNK(p-JNK) by enzyme-linked immuno sorbent assay(ELISA) and immunofluorescence technique. Observed the co-expression of p-JNK with astrocyte, microglia and neuron at dorsal horn of spinal cord. 72 h after surgery, the expression of p-JNK were detected out by Western blot analysis.Results:1. The MWT was significantly lower, the TWL was shorter, at each time point after establishment of the model than at 24 h before in all group(P<0.05), but the SP group was no significant difference in 72h(P>0.05); compared with IP group, the threshold in SP group, PPF group increased significantly post-incision(P<0.05); as compared with SP group, a small MWT increased in PPF group at 2h(P<0.05), and TWL shorter recovery in PPF group at 6h after surgery(P<0.05). The two groups had similar trends at each other time point.2.The TNF-α in spinal cord: Compare to the preoperation, the level of TNF-α in spinal cord in each group has been the top in 6h, and then reduced from 24 h,but still at higher level at 72 h post-surgery(P<0.05); as compared with IP group, the TNF-α content in SP group and PPF group were down-regulated at each time point after surgery(P<0.05); and rats in PPF group secreted more TNF-α than SP group’s(P<0.05).3. The p-JNK location and expression in spinal dorsal horn: the expression of p-JNK in IP group and SP group was higher at 6 、 24 、 48 、 72 h postoperation than at 24 h preoperation(P<0.05);but was significantly reduced in SP group, as compared with IP group at same time point. The expression of p-JNK was observed at 6h after the incision, and arrived to the maximum at 72 h. And the p-JNK was co-expression with the marker of microglia(Iba-1) but not astrocyte’s nor neurons’ within the ipsilateral spinal dorsal horn.5. p-JNK expression was detected by Western blot at 72 h post-incision: Compare to properation, the expression of p-JNK after 72 h were up-regulation obviously in IP group and PPF group, but decreased in SP group(P<0.05); as compared with IP group, the p-JNK were down-regulation in SP group and PPF group(P<0.05);in PPF group the higher level of p-JNK were expressed in spinal cord, as compared with SP group(P<0.05).Conclusion:1. Spinal JNK signaling pathway is involved in the development and maintenance of incisional pain in rats, and the inhibitor of JNK pathway overturned the heat hyperalgesia and mechanical allodynia induced incision effectively;2. The expression of TNF-α could be down-regulation by inhibited the activation of JNK pathway;3. The analgesia effect of PPF in plantar incision rat model, which decreased the expression of TNF-α, was related to the inhibition of JNK pathway activation. |
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