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Background Our previous study showed that the μ-opioid receptor agonist fentanyl citrate inhibits endothelin-1-and bradykinin-mediated pain responses in mice orthotopically inoculated with melanoma cells. We also demonstrated that bradykinin induces endothelin-1 secretion in melanoma cells. However, the analgesic mechanisms of fentanyl citrate remain unclear. Thus, the present study was conducted to determine whether fentanyl citrate affects bradykinin-induced endothelin-1 secretion in B16-BL6 melanoma cells. Methods The amount of endothelin-1 in the culture medium was measured using an enzyme immunoassay. The expression of endothelin-1, kinin B 2 receptors, and μ-opioid receptors in B16-BL/6 melanoma cells was determined using immunocytochemistry. Results Fentanyl citrate inhibited bradykinin-induced endothelin-1 secretion. The inhibitory effect of fentanyl citrate on the secretion of endothelin-1 was attenuated by the μ-opioid receptor antagonist naloxone methiodide. The immunoreactivities of endothelin-1, kinin B 2 receptors, and μ-opioid receptors in B16-BL6 melanoma cells were observed. Conclusion These results suggest that fentanyl citrate regulates bradykinin-induced endothelin-1 secretion through μ-opioid receptors in melanoma cells.

Background Oxaliplatin, a platinum-based chemotherapeutic agent, induces acute cold allodynia and dysesthesia. Cold-sensitive transient receptor potential channels (TRPM8 and TRPA1) have been implicated as candidates to mediate oxaliplatin-induced cold allodynia and hyperalgesia, but precise roles of these channels remain unclear. In this study, we investigated the role of TRPM8 in oxaliplatin-induced cold allodynia. Methods Oxaliplatin was injected intraperitoneally in mice. Cold allodynia was evaluated by the acetone test. Expression levels of TRPM8 mRNA and protein were measured using reverse transcription-polymerase chain reaction and Western blotting, respectively. Results Oxaliplatin-induced cold allodynia was alleviated by the TRPM8 blockers N -(2-aminoethyl)- N -[4-(benzyloxy)-3-methoxybenzyl]- N ′-(1S)-1-(phenyl) ethyl] urea and TC-I 2014. Oxaliplatin increased the expression levels of TRPM8 mRNA and protein in the dorsal root ganglia and plantar skin, respectively. Prophylactic administration of the c-Myc inhibitor 10058-F4 prevented cold allodynia and the increase of TRPM8 mRNA after oxaliplatin injection. Conclusion These results suggest that oxaliplatin induces cold allodynia through the increase of c-Myc-mediated TRPM8 expression in primary sensory neurons.

Moutan Cortex and its major compounds have been shown to possess various biological activities, including anti-inflammatory properties. However, the effects of Moutan Cortex aqueous fraction (MCA) and its molecular mechanisms have yet to be elucidated. In this study, we attempted to evaluate the effects of MCA on mast cell-mediated allergy inflammation in vitro and in vivo compared with major Moutan Cortex compounds. Thus, we examined the anti-inflammatory effects of a water extract of Moutan Cortex by comparing the inhibition of β-hexosaminadase and tumor necrosis factor-α (TNF-α) release in an aqueous fraction with other major compounds of Moutan Cortex. The inhibitory mechanism of MCA was investigated by western blotting in IgE-mediated DNP-BSA-stimulated RBL-2H3 cells. We confirmed the pharmacological effects of MCA on compound 48/80-induced allergic reactions in a mouse model by assessing scratching behavior and passive cutaneous anaphylaxis (PCA)-like reaction. Consequently, MCA inhibited IgE-mediated DNP-BSA-induced β-hexosaminadase and TNF-α release via inactivation of p38, ERK, Akt, and NF-κB in RBL-2H3 cells. MCA reduced compound 48/80-induced PCA reaction and scratching behavior in mice. This inhibitory effect of MCA is more potent than major compounds of Moutan Cortex. In conclusion, our results suggest that MCA has more potential in the treatment of allergic inflammatory diseases compared to other major compounds of Moutan Cortex.

We developed a micro multipoint laser Doppler velocimeter (μ-MLDV) for noninvasive in-vivo measurements of blood flow and we presented the results of demonstrations performed on experimental animals. In this paper, we investigate the validity of power spectrum analysis for determining the flow velocity and the minimum power of the semiconductor laser in the μ-MLDV. Although average velocity is generally estimated from a peak position (fpeak) in the power spectrum, the power spectrum of blood flow included an additional component in the high-frequency region. The conventional method for determining the average velocity of flows of transparent artificial fluids, which involves determining the average velocity from fpeak, is unsuitable for in-vivo measurements of blood flow. The laser power was reduced from 140 to 30mW since 30mW was the minimum power at which images of blood flow velocity in microvessels could be obtained. About 30mW (power density of 15mW/mm2) is the maximum power which can be irradiated to humans. Further reduction in the laser power is necessary before this technique can be applied to humans.

Accumulating evidence indicates that purinergic P2X4 receptors (P2X4R: cation channels activated by extracellular ATP) expressed in spinal microglia are crucial for pathological chronic pain caused by nerve damage, suggesting a potential target for drug discovery. We identified NP-1815-PX (5-[3-(5-thioxo-4H-[1,2,4]oxadiazol-3-yl)phenyl]-1H-naphtho[1, 2-b][1,4]diazepine-2,4(3H,5H)-dione) as a novel antagonist selective for P2X4R with high potency and selectivity compared with other P2XR subtypes. In in vivo assay for acute and chronic pain, intrathecal administration of NP-1815-PX produced an anti-allodynic effect in mice with traumatic nerve damage without affecting acute nociceptive pain and motor function (although its oral administration did not produce the effect). Furthermore, in a mouse model of herpetic pain, P2X4R upregulation in the spinal cord exclusively occurred in microglia and intrathecal NP-1815-PX suppressed induction of mechanical allodynia. This model also showed K+/Cl− cotransporter 2 (KCC2) downregulation, which is implicated in dorsal horn neuron hyperexcitability; this downregulation was restored by intrathecal treatment with NP-1815-PX or by interfering with brain-derived neurotrophic factor (BDNF) signaling, a P2X4R-activated microglial factor implicated in KCC2 downregulation. Taken together, the newly developed P2X4R antagonist NP-1815-PX produces anti-allodynic effects in chronic pain models without altering acute pain sensitivity, suggesting that microglial P2X4R could be an attractive target for treating chronic pain.

Kyotorphin (L-Tyr-L-Arg), an analgesic dipeptide isolated from the bovine brain, and its analogue, L-Tyr-D-Arg (D-kyotorphin), have a naloxone-reversible analgesic effect. Both peptides (10−5 M) induced an approximately 4-fold increase of the basal release of Met-enkephalin from striatal slices. Therefore they may produce their analgesic effects through release of Met-enkephalin. The stronger in vivo effect of D-kyotorphin may be explained by its resistance to degradation.