| Backgrund:Neuropathic pain caused by trauma, infection, neuropathy, metabolic and inflammatory diseases, drug side effects, severely affects the life quality of patients. The mechanism behind neuropathic pain is quite complex, involving neurons nociceptor sensitization, disorder of ions and protein coupled receptors, non neuronal cells(such as activation of glial cells),immune cell infiltration, and release of factors such as ATP, glutamate, reactive oxygen species, proinflammatory, cytokines, chemokines, bradykinin, substance P, prostaglandin, neurotrophic factors.RAGE (the recepotr for advanced glycation end products) is a kind of surface member Ig superfamily protein receptor. Recent studies have shown that RAGE participated in various diseases in the nervous system. The main mechanism is to increase the level of oxidative stress and immune inflammatory reaction. The accumulation of RAGE ligands promote activation of RAGE in glial cells, which increase the level of inflammatory, such as IL-6,TNF-alpha and IL-1beta. Release of peroxide and proinflammatory cytokine can lead to neuronal apoptosis. ROS and proinflammatory cytokine lead to neurotoxicity and cause nerve function disorder as well as a vicious cycle. RAGE is also involved in peripheral nerve injury. RAGE can regulate migration and infiltration of macrophage around injury nerve in distal to promote axon regeneration. This helps nerve reconstruction and recovery of conduction fuction. Axonal demyelination after long-term overexpression of RAGE lead to axonal transduction dysfunction. It is considered that RAGE-NF-kB axis is the most important signal pathway mediated by peripheral nerve injury. RAGE gene knockout significantly reduced NF-kB activation and increased the number of sensory conduction fibers compared with the control groups. The sensory desensitization of peripheral nerve can be blocked by inhibitors of RAGE signal.Over expression of RAGE can aggravate peripheral nerve desensitization. Due to the important role of RAGE in glial cell activation and inflammatory factor release, we deem that it is critical to clarify the role of RAGE to reveal the underlying mechanisms of neuropathic pain.As the role of RAGE in neuropathic pain research is unclear, the model of spinal nerve ligation of rats was established to observe the expression of RAGE in dorsal root ganglia and dorsal horn of the spinal cord. Evaluating the effect of intrathecal anti-RAGE neutralizing antibody treatment on neuropathic pain and testing the expression of NF-κB,TNF-α,IL-1β, GFAP, Wntl in the dorsal root ganglion (DRG) at 7 day after surgery to explore the mechanism of RAGE in the development of neuropathic pain in SNL rat. This study can provide new ideas for clinical treatment of neuropathic pain, and provide reference for damage of acute inflammatory mechanisms.Methods:SNL model was established with adult male Sprague-Dawley rats(200-250g) by L5 spinal nerve ligation. The expression of RAGE in DRG and spinal cord was tested on Id,3d,7d after surgery by immunofuorescence and westernblot. SD rats were randomly divided into 3 groups(n=10 each):drug group, intrathecal injection with anti-RAGE body after SNL surgery; vehicle group, intrathecal injection with phosphate buffered saline (PBS) after SNL surgery; sham group, underwent the same surgical procedure except that the left L5 spinal nerve was not ligated and intrathecal injected with phosphate buffered saline (PBS). Paw withdrawal thresholds(PWT) and paw thermal withdraw latency(PTWL) of each group were tested before and 1,3,5,7 day after opreation. The dorsal root ganglion were removed for westernblot of NF-κB, GFAP, Wntl and for determination of TNF-a and interleukin-1β by ELISA on 7d after surgery.Results:The expression of RAGE in L5 DRG increased on 3d and 7d after SNL surgery compared with preoperation control group by immunofuorescence intensity, colacalized on neurons and satellite glial cells(SGCs) in DRG The expression of RAGE in L5 DRG increased on Id,3d,7d after SNL surgery compared with preoperation control group by westernblot(P<0.05). There was no statistic difference of RAGE expression in spinal dorsal horn before and after surgery(P>0.05) There was no statistic difference of PWT and PTWL before and after surgery in sham group(P>0.05). Compared with the sham group, the PWT and PTWL were significantly lower in vehicle group and drug group after SNL surgery; Compared with vehicle group, PWT and PTWL were higher in drug group on 3d,5d,7d after SNL surgery(P<0.05). Compared with the sham group, the expression of NF-κB(0.80±0.04 vs.0.31 ± 0.02, P<0.05) and GFAP(1.46 ± 0.04 vs.0.32 ± 0.08, P<0.05) were significantly upregulated in vehicle group; but Wntl (0.41■0.04 vs. 2.47 ±0.12, P<0.05) was decreased in DRG 7d after SNL surgery. Compared with the sham group, the expression of NF-κB (0.51±0.03 vs.0.31 ± 0.02, P<0.05) and GFAP(0.69 ± 0.06 vs.0.32 ± 0.08, P<0.05) were significantly upregulated in drug group; but Wntl (0.93 ± 0.17 vs.2.47 ±0.12, P<0.05) was decreased in DRG 7d after SNL surgery. Compared with the vehicle group, the expression of NF-κB (0.51 ± 0.03 vs.0.80 ± 0.04, P<0.05) and GFAP (0.69 ± 0.06 vs.1.46 ± 0.04, P<0.05) were significantly lower in vehicle group, while Wntl (0.93 ± 0.17 vs.0.41 ± 0.04, P<0.05) was increased in DRG 7d after SNL surgery. The level of TNF-α and IL-1β in vehicle group were significantly higher than the sham group (109.9 ± 4.97 vs.40.18 ± 3.13,168.77 ± 6.34 vs.58.12 ± 3.11, respectively, P<0.05). The level of TNF-α and IL-1β in drug group were significantly higher than the sham group (70.53 ± 6.57 vs.40.18 ± 3.13; 88.24 ± 3.6 vs.58.12 ± 3.11, respectively, P<0.05). Intrathecal injection with anti-RAGE body decreased the level of TNF-α (70.53 ± 6.57 vs.109.9 ± 4.97, P<0.05) and IL-1β (88.24 ± 3.6 vs.168.77 ± 6.34, P<0.05) in DRG 7d after surgery.Conclusions:The expression of RAGE protein increased in DRG after SNL; Intrathecal anti-RAGE body injection can reduce allodynia and inhibit activation of SGCs. In addition, it can suppress the expression of TNF-α, IL-1β and NF-κB but promote expression of Wntl. All these results support the role of RAGE in DRG to the involvement of neuropathic pain. |
PDF Full Text Request