Animal Studies

Metabotropic glutamate receptor 5 (mGluR5) has been reported to contribute to inflammatory pain. The intracellular C-terminal domain has a Homer-binding motif that can form an mGluR5/Homer complex. Phosphorylation of mGluR5 at the Homer binding domain enhances the mGluR5/Homer interaction and modulates intracellular signal transduction. However, the characteristics of this interaction have not been fully elucidated in inflammatory pain. We aimed to evaluate the effects of CFA-induced phosphorylation of mGluR5 at the Homer binding domain on the mGluR5/Homer interaction. Von-frey filaments and thermal latency were used to monitor the development of inflammatory pain. Spinal mGluR5 phosphorylation at Ser and mGluR5/Homer crosslinking were detected. Mutant mGluR5 that could not be phosphorylated at Thr or Ser was evaluated in inflammatory pain. CFA-induced inflammatory pain resulted in obvious phosphorylation at Ser of mGluR5. Moreover, increased phosphorylation at the Homer-binding motif enhanced crosslinking between mGluR5 and Homer. Mutations at Thr and Ser of mGluR5 blocked the development of CFA-induced inflammatory pain. Overall, our findings showed that disruption of the phosphorylation of mGluR5 Thr and Ser alleviated CFA-induced inflammatory pain.

Pepducins are cell-penetrating, membrane-tethered lipopeptides designed to target the intracellular region of a G protein-coupled receptor (GPCR) in order to allosterically modulate the receptor's signaling output. In this proof-of-concept study, we explored the pain-relief potential of a pepducin series derived from the first intracellular loop of neurotensin receptor type 1 (NTS1), a class A GPCR that mediates many of the effects of the neurotensin (NT) tridecapeptide, including hypothermia, hypotension and analgesia. We used BRET-based biosensors to determine the pepducins' ability to engage G protein signaling pathways associated with NTS1 activation. We observed partial Gα and Gα activation at a 10 µM concentration, indicating that these pepducins may act as allosteric agonists of NTS1. Additionally, we used surface plasmon resonance (SPR) as a label-free assay to monitor pepducin-induced responses in CHO-K1 cells stably expressing hNTS1. This whole-cell integrated assay enabled us to subdivide our pepducin series into three profile response groups. In order to determine the pepducins' antinociceptive potential, we then screened the series in an acute pain model (tail-flick test) by measuring tail withdrawal latencies to a thermal nociceptive stimulus, following intrathecal (i.t.) pepducin administration (275 nmol/kg). We further evaluated promising pepducins in a tonic pain model (formalin test), as well as in neuropathic (Chronic Constriction Injury) and inflammatory (Complete Freund's Adjuvant) chronic pain models. We report one pepducin, PP-001, that consistently reduced rat nociceptive behaviors, even in chronic pain paradigms. Finally, we designed a TAMRA-tagged version of PP-001 and found by confocal microscopy that the pepducin reached the rat dorsal root ganglia post i.t. injection, thus potentially modulating the activity of NTS1 at this location to produce its analgesic effect. Altogether, these results suggest that NTS1-derived pepducins may represent a promising strategy in pain-relief.

Osteoarthritis (OA) is the most prevalent joint disorder associated with severe chronic pain. Although synovial inflammation is well correlated with pain severity, the molecular mechanism responsible for OA pain remains unclear. Here, we show that extracellular miR-21 released from synovial tissue mediates knee OA pain in surgical OA model rats. miR-21 was the most abundant among increased microRNAs (miRNAs) in the synovial tissue. miR-21 was released into extracellular space from the synovial tissue and increased in the synovial fluid. A single intra-articular injection of miR-21 inhibitor exerted long-term analgesia of knee OA pain, whereas miR-21 injection in naive rats caused knee joint pain. miR-21 mutant, which lacks the Toll-like receptor (TLR) binding motif, but not in the seed sequence, did not cause joint pain, suggesting a non-canonical mode of action different from translational repression. Consistent with this, the algesic effect of miR-21 was blocked by antagonizing TLR7. The TLR7 antagonist also exerted a long-lasting analgesic effect on knee OA pain. Therefore, extracellular miR-21 released from synovial tissue mediates knee OA pain through TLR7 activation in surgical OA model rats. Extracellular miRNA in the joint may be a plausible target for pain therapy, providing a novel analgesic strategy for OA.

Platinum-based chemotherapy-induced peripheral neuropathy is one of the most common causes of dose reduction and discontinuation of life-saving chemotherapy in cancer treatment; it often causes permanent impairment of quality of life in cancer patients. The mechanisms that underlie this neuropathy are not defined, and effective treatment and prevention measures are not available. Here, we demonstrate that SIRT2 protected mice against cisplatin-induced peripheral neuropathy (CIPN). SIRT2 accumulated in the nuclei of dorsal root ganglion sensory neurons and prevented neuronal cell death following cisplatin treatment. Mechanistically, SIRT2, an NAD+-dependent deacetylase, protected neurons from cisplatin cytotoxicity by promoting transcription-coupled nucleotide excision repair (TC-NER) of cisplatin-induced DNA crosslinks. Consistent with this mechanism, pharmacological inhibition of NER using spironolactone abolished SIRT2-mediated TC-NER activity in differentiated neuronal cells and protection of neurons from cisplatin-induced cytotoxicity and CIPN in mice. Importantly, SIRT2's protective effects were not evident in lung cancer cells in vitro or in tumors in vivo. Taken together, our results identified SIRT2's function in the NER pathway as a key underlying mechanism of preventing CIPN, warranting future investigation of SIRT2 activation-mediated neuroprotection during platinum-based cancer treatment.

The activation of the AMP activated protein kinase (AMPK) exerts antinociceptive effects in acute and neuropathic pain models. Mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c), a mitochondrial-derived peptide, regulates many biological activities via activating AMPK. However, the role of MOTS-c in the formalin-induced inflammatory nociception remains unclear. In this study, we investigated the role of MOTS-c in the formalin-induced inflammatory nociception. The antinociceptive effect of MOTS-c was assessed by recording the time spent licking paw. The anti-inflammatory effect of MOTS-c was evaluated by detecting the inflammatory cytokine level changes in the mouse serum. Western blot was used to detect the changes of protein phosphorylation levels in the mouse spinal cord. Changes of c-fos expression in the spinal cord were assessed by immunohistochemistry. Our results showed that the intraperitoneal administration of MOTS-c reduced the time spent on licking in phase 2 in a dose-dependent manner in the formalin test. The antinociceptive effects of MOTS-c (50 mg/kg, i.p.) were attenuated by the AMPK antagonist compound C (10 mg/kg, i.p.). MOTS-c (50 mg/kg, i.p.) significantly reduced pro-inflammatory cytokine levels and elevated the level of anti-inflammatory cytokine in mouse serum. In addition, MOTS-c treatment significantly increased AMPKα phosphorylation level and suppressed formalin-induced extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinases (JNK), and P38 activation and c-fos expression in the mouse spinal cord. These results suggest that systemic administration of MOTS-c exerts antinociceptive and anti-inflammatory effects, at least partially, through activating AMPK pathway and inhibiting MAP kinases-c-fos signaling pathway in the mouse formalin test.