| BackgroundThe clinical therapy of patients who present with neuropathic pain is a common problem in medical care. Because of its complex mechanism, the neuropathic pain is still a challenge in clinical practice. After nervous system is damaged, inflammatory cytokines inevitably activates and the inflammatory medium is released which causes inflammation reaction. Excessive inflammatory reaction is hurtful and stimulating to the neurons. The neurons sensitivity is changed, which involves in the occurrence and maintenance of neural plasticity and neuropathic pain.JAK (Janus kinase, JAK)/STAT (signal transducers and activators of transcription, STAT) signaling pathway which is activated by cytokines is found in recent years. JAK/STAT pathway is the key signaling pathway mediating inflammation and immune response, which occupies an important role in synaptic plasticity, neural degeneration and memory formation in central nervous system (CNS). Cytokines can lead the activation of JAK/STAT signaling pathways, and cytokine signal eventually causes the target gene expression. Suppressor cytokine signal (suppressors of cytokine signaling, SOCS)is a kind of negative regulation of protein families related to signal transduction pathways with cytokines and growth factors, which is a negative feedback to JAK/STAT pathway. Moreover, SOCS adjusts and involves in inflammation and apoptosis. However, the complex regulation of JAK/STAT pathway and the significance of inhibiting this pathway during the development of neuropathic pain remain elusive.Our previous study found that miR-203expression level was significantly lower (9tines and10times respectively) in spinal cord dorsal horn tissue of bilateral sciatic nerve ligation (bCCI) neuropathic pain model compared with control group and normal rats. However, the mechanisms of miR-203involving in neuropathic pain is unclear. It is reported that miR-203can inhibit the expression of SOCS3which is the feedback of the JAK/STAT signaling.In order to evaluate the contribution of the JAK2/STAT3pathway to neuropathic pain and the potentiality of this pathway as a therapeutic target, we examined the effects of the STAT3inhibitor WP1066by intrathecal administration in a rat model of bilateral chronic constriction injury (bCCI). So as to investigate whether changing the level of rno-miR-203in the spinal dorn can alleviate the pain behavior in our study setting.Methods1. To verify the JAK2/STAT3pathway activation in the bCCI neuropathic pain rats60SD female rats were randomly divided into3groups, Naive group, Sham group and the bCCI group. The rats had no intervention in Naive group. The sciatic nerve was exposed without ligation in Sham group rats. The bilateral sciatic nerves were ligated in the bCCI group. After the rat model of bCCI was established, pain-related behavior tests were performed before and on day3,7,14and21after surgery respectively to observe the reaction to mechanical stimulation, thermal stimulation and cold stimulation L4～L6dorsal spinal cord was harvested at different time points. RT-PCR and Western blot were performed to explore the activation of JAK2/STAT3pathway. Immunohistochemical was used to do qualitative analysis on the pathway.2. Application of JAK2/STAT3inhibitors WP1066to observe the influence of neuropathic pain and its molecular mechanism18SD female rats were randomly divided into three groups, WP1066group, DMSO group and W-N group. WP1066(10μl,10mmol/L in DMSO) or the same capacity of DMSO were applied through the intrathecal tube on the day before bCCI surgery, on the day of surgery, on day3and5after bCCI surgery in the rats of WP1066group and DMSO group respectively; The rats were only with intrathecal catheterization but with same intervention drug in W-N group as in the WP1066group. Behavior tests were performed before and on day3,5,7,10,14after surgery to observe the reaction to mechanical stimulation, thermal stimulation and cold stimulation. L4-L6dorsal spinal cord was harvested on day14, followed by RT-PCR and Western blot experiments to investigate the activation of the JAK2/STAT3pathway.3. Intrathecal injection of rno-miR-203overexpressed virus vector to observe the pain behavior in the bCCI neuropathic pain ratsTo observe the analgesic action of rno-miR-203,10μl (4E+8TU/mL) lentiviral mediated mo-miR-203over-expressed vector was injected into intrathecal in the bCCI neuropathic pain model [rno-miR-203(+) group] while the same volume negative virus vector was injected in the negative group[mo-miR-203(-) group], behavior tests were performed before surgery and on day3,5,7,10,14after bCCI surgery to observe the reaction to mechanical stimulation, thermal stimulation and cold stimulation. On day14after bCCI surgery, L4-L6spinal cord dorsal horn was harvest to do immuno fluorescence and RT-PCR to verify the expression level of rno-miR-203.Results1. Pain-related behavioral scores in the bCCI rats were significant reduced as compared to the Sham-operated and Naive group starting from7day postoperatively (P<0.05). SOCS3mRNA and STAT3mRNA were significantly increased on day7and14in the bCCI rats, accompanied by JAK2mRNA with a similar time course. Western blot analysis showed that JAK2and P-STAT3has been significantly increased since3days after bCCI. JAK2peaked on day14while P-STAT3peaked on day7and gradually decreased thereafter. The SOCS3protein peaked on day3. Immunohistochemical showed that JAK2and STAT3, SOCS3expressed in microglial cells when IBA1as the marker of microglia.2. When WP1066was administered intrathecally, the pain-related behavior changes were significantly improved in the bCCI rats as compared to DMSO group. WP1066could significantly inhibit the JAK2, STAT3and SOCS3mRNA in rats with bCCI. What’s more, WP1066could significantly decrease the ratio of JAK2, SOCS3and phosphorylation of STAT3(P-STAT3) protein expression on day14after bCCI.3. Pain-related behavioral scores in the bCCI rats were significant reduced in the rno-miR-203(+) group starting from the day7after bCCI surgery as compared to the mo-miR-203(-) group (P<0.05). The rno-miR-203expression level was qualitative verified by immuno fluorescence and quantitative verified by RT-PCR. The rno-miR-203expression level increased in the rno-miR-203(+) group compared with the level in the rno-miR-203(-) group.Conclusion1. Our results suggest that the JAK2/STAT3pathway in the spinal dorsal horn was significantly activated in a bCCI neuropathic pain rat model.2. WP1066specifically inhibits the STAT3signaling pathway and partially alleviates pain-related behavioral changes in the bCCI neuropathic pain model, which may serve as a novel therapeutic strategy against neuropathic pain. 3. Rno-miR-203can improve the pain behavior of bCCI neuropathic pain rats. |
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