| ObjectiveMicroRNA-146a, an important modulator in inflammatory immune reaction can regulate the expression of IRAKI and TRAF6, which are proteins in the TLR4 signaling pathway. It has been proved that TLR4 signaling pathway played a key role in the pathogenesis of neuropathic pain. We aim to investigate the expression of miR-146a and the activation of TLR4 signaling pathway in L4-6 dorsal root ganglions of bilateral chronic constriction injury (bCCI) rats, and to explore the regulatory mechanism of miR-146a in neuropathic pain by studying the effect of miR-146a on rats pain behavior and the expression of its target gene IRAKI and TRAF6.Methods1. To evaluate the expression of miR-146a and TLR4 signaling pathway in DRG of bCCI rats. Thirty-six female SD rats weighting 200-250g were divided randomly into bCCI group in which 12 rats received bilateral chronic constriction surgery, sham group and naive group. Behavioral test were performed on the day before surgery and on day 1,3,7,14 and 21 after surgery. RT-qPCR was used to test the mRNA expression of miR-146a, IRAKI, TRAF6 and TLR4.Western Blot was performed to explore the protein expression of TLR4, MyD88, TRIF, IRAK1, TRAF6,NF-κB and p-NF-κB. Immunohistochemistry was used to identify distributions of target proteins in DRG. ELISA was used to test the level of IL-6 and TNF-ain DRG of rats.2. To investigate the effect of miR-146a on bCCI rats pain behavior and its molecular mechanism. Twenty-four female SD rats weighing 200-250g were randomly divided into agomir-146a group (n=6), antagomiR-146a group (n=6) and negative control group (n=12). Rats in all groups received bilateral chronic sciatic nerve constriction injury (bCCI). Equal volume of agomiR-146a, antagomiR-146a or negative control was administered intrathecally on the surgery day and on day 3,5,7 and 10 after surgery. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured to identify the behavioral changes before operation, on day 1,3,7 and 14 after surgery. L4-L6 dorsal root ganglions were harvested on day 14 after surgery. RT-qPCR was used to test the mRNA expression of miR-146a, IRAKI and TRAF6. Western Blot was performed to explore the protein expression of IRAK1 and TRAF6. Primary DRG neurons were incubated overnight with agomiR-146a, antagomiR-146a or negative control reagent separately. Calcium imaging was used to study LPS stimulated [Ca2+]1 change in DRG neurons.Results1. Pain-related behavioral tests scores of bCCI rats were significantly decreased compared with sham group and naive group rats at each time point after surgery (P<0.05). Compared with sham group and naive group, the expression of miRNA-146a was greatly decreased, whereas both mRAN and protein level of IRAK1 and TRAF6 were significant increased after surgery in bCCI rat group (P<0.05). Immunohistochemistry results showed that IRAK1 and TRAF6 were located on DRG neurons. IRAK1-IR was more significantly colocalized with the peptidergic calcitonin gene related peptide (CGRP)(52.3%) and less colocalized with the nonpeptidergic isolectin B4 (IB4) (26.1%) neurons, whereas TRAF6-IR was more greatly colocalized with IB4 (63%) and less colocalized with CGRP (21%).2. Compared with sham group and naive group, the expression of TLR4 mRNA was significantly increased after surgery in bCCI rat group. Western Blot result showed that TLR4, MyD88, TRIF, NF-κB and p-NF-κB on protein level increased in DRG of bCCI rat postoperatively (P<0.05). Immunohistochemistry result showed that TLR4 was located on DRG neurons. ELISA result showed that the level of IL-6 and TNF-ain DRG of bCCI rats were significantly increased compared with naive and sham group rats.3. Compared with control group, an increased of PWTL was observed from day 3 after surgery whereas PWMT increased only on day 7 and 14 after surgery in agomiR-146a group (P<0.05). Furthermore, the expression of miRNA-146a was greatly increased, whereas IRAKI mRNA and TRAF6 mRNA were significantly decreased on day 14 after surgery in the agomiR-146a group (P<0.05). Western Blot result showed that the protein level of IRAKI and TRAF6 decreased in DRG of bCCI rat in the agomiR-146a group on day 14 postoperatively (P<0.05). There is no significant difference between control group and antagomiR-146a group in rats’pain behavior. However, the expression of miRNA-146a was greatly descreased, whereas IRAKI mRNA and TRAF6 mRNA were significantly increased on day 14 after surgery in the agomiR-146a group (P<0.05). Calcium imaging results revealed that the percentage of positive reaction rate to LPS stimulation in each group:5.4%(NC group),1.59%(agomiR-146a group) and 12.5%(antagomiR-146a group).Conclusion1. Decreased expression of miR-146a in DRG was involved in neuropathic pain by targeting IRAK1 and TRAF6.2. The TLR4 signaling pathway in the dorsal root ganglion was significantly activated in bCCI rats.3. Intrathecal agomiR-146a treatment can attenuate neuropathic pain of bCCI rats. Therefore, agomiR-146a may be a novel target for neuropathic pain. |
PDF Full Text Request