Accepted

Neuropathic pain induced by peripheral nerve injury is a complex and chronic state that is accompanied by poor quality of life. However, whether PIM1 (proviral integration site 1) contributes to the development of nociceptive hypersensitivity induced by nerve injury remains unknown. The present study was designed to investigate the effects of PIM1 on spinal nerve ligation (SNL) induced pain hypersensitivity. Here, we found that PIM1 positive neurons in the dorsal root ganglion (DRG) were colocalized with nociceptive neuronal markers CGRP, IB4 and substance P and were upregulated after SNL surgery. Knockdown PIM1 in the DRG by AAV5-shPIM1 alleviated SNL-induced pain hypersensitivity. In neuroblastoma cells (neuro-2a), PIM1 regulated the expression of CXCR4 phosphorylated at ser339 (pCXCR4) as well as the CXCL12/CXCR4 pathway. In the DRG tissues, we found that PIM1 was co-expressed with CXCR4, and knockdown of PIM1 attenuated pCXCR4 (ser339) protein expression but had little effect on total CXCR4 protein expression after SNL surgery. These findings suggest that PIM1 contributes to nerve injury-induced nociceptive hypersensitivity. Based on these findings and the characteristics of PIM1, we speculate that PIM1 might be a viable therapeutic target for the treatment of neuropathic pain in the near future.

Deficiency of deleted in liver cancer 2 (DLC2), a novel domain to inhibit RhoA activity, plays an important role in inflammatory pain. However, the underlying mechanisms remain unclear. This study investigated the role of DLC2 and its downstream cascade of RhoA/ROCK in formalin-induced inflammatory pain using DLC2-knockout (DLC2) mice and compared them with DLC2 wild-type (DLC2) mice. Mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filament aesthesiometer and Hargreaves test, respectively. The spinal cord dorsal horn (L3-L5) was selected for molecular and cellular identification by Western blot and immunofluorescence. DLC2 mice showed increased mechanical allodynia and thermal hyperalgesia. Expression of ROCK1, ROCK2 and IL-1β was significantly higher in DLC2 mice. Intrathecal administration of RhoA inhibitor (C3 exoenzyme) or ROCK inhibitor (Y27632) significantly attenuated formalin-induced inflammatory hyperalgesia in DLC2 mice. ROCK2 and IL-1β expression were reduced by C3 exoenzyme or Y27632. Spinal p38 activation was also inhibited by C3 exoenzyme or Y27632. Double-labelling immunofluorescence demonstrated co-localization of DLC2 with spinal dorsal microglia. The number of activated microglia in the spinal dorsal horn was significantly higher in DLC2 mice, but was reduced by Y27632. These findings indicate that DLC2 deficiency increased formalin-induced inflammatory hyperalgesia through regulating RhoA/ROCK2, and IL-1β may be a downstream effector. Our results also suggest that RhoA/ROCK enhanced p38 activation plays an important role in formalin-induced inflammatory pain. The finding that DLC2 attenuated inflammatory pain through inhibiting RhoA/ROCK2 suggests that the DLC2/RhoA/ROCK2/p38/IL-β pathway may be a potential therapeutic target to reduce inflammatory pain.

Circular RNAs are non-coding RNAs, and are enriched in the CNS. Dorsal horn neurons of the spinal cord contribute to pain-like hypersensitivity after nerve injury in rodents. Here we show that spinal nerve ligation is associated with an increase in expression of circAnks1a in dorsal horn neurons, in both the cytoplasm and the nucleus. Downregulation of circAnks1a by siRNA attenuates pain-like behaviour induced by nerve injury. In the cytoplasm, we show that circAnks1a promotes the interaction between transcription factor YBX1 and transportin-1, thus facilitating the nucleus translocation of YBX1. In the nucleus, circAnks1a binds directly to the Vegfb promoter, increases YBX1 recruitment to the Vegfb promoter, thereby facilitating transcription. Furthermore, cytoplasmic circAnks1a acts as a miRNA sponge in miR-324-3p-mediated posttranscriptional regulation of VEGFB expression. The upregulation of VEGFB contributes to increased excitability of dorsal horn neurons and pain behaviour induced by nerve injury. We propose that circAnks1a and VEGFB are regulators of neuropathic pain.