Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

Pain is a worldwide public health problem and its treatment is still a challenge since clinically available drugs do not completely reverse chronic painful states or induce undesirable effects. Crotalphine is a 14 amino acids synthetic peptide that induces a potent and long-lasting analgesic effect on acute and chronic pain models, peripherally mediated by the endogenous release of dynorphin A and the desensitization of the transient receptor potential ankyrin 1 (TRPA1) receptor. However, the effects of crotalphine on the central nervous system (CNS) and the signaling pathway have not been investigated. Thus, the central effect of crotalphine was evaluated on the partial sciatic nerve ligation (PSNL)-induced chronic neuropathic pain model. Crotalphine (100 µg/kg, p.o.)-induced analgesia on the 14th day after surgery lasting up to 24 h after administration. This effect was prevented by intrathecal administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists. Besides that, crotalphine-induced analgesia was reversed by CTOP, nor-BNI, and naltrindole, antagonists of mu, kappa, and delta-opioid receptors, respectively, and also by the specific antibodies for β-endorphin, dynorphin-A, and met-enkephalin. Likewise, the analgesic effect of crotalphine was blocked by the intrathecal administration of minocycline, an inhibitor of microglial activation and proliferation. Additionally, crotalphine decreased the PSNL-induced IL-6 release in the spinal cord. Importantly, in vitro, crotalphine inhibited LPS-induced CD86 expression and upregulated CD206 expression in BV-2 cells, demonstrating a polarization of microglial cells towards the M2 phenotype. These results demonstrated that crotalphine, besides activating opioid and cannabinoid analgesic systems, impairs central neuroinflammation, confirming the neuromodulatory mechanism involved in the crotalphine analgesic effect.

Neurodegenerative diseases are one of the major causes of death worldwide, characterized by neurite atrophy, neuron apoptosis, and synapse loss. No effective treatment has been indicated for such diseases so far, and the search for new drugs is being increased in the last years. Animal venoms’ secretion/venom can be an alternative for the discovery of new molecules, which could be the prototype for a new treatment. Here, we present the biochemical characterization and activity of the extract from the box jellyfish Chiropsalmus quadrumanus (Cq) on neurites. The Cq methanolic extract was obtained and incubated to human SH‐SY5Y neurons, and neurite parameters were evaluated. The extract was tested in other cell types to check its cytotoxicity and was submitted to biochemical analysis by mass spectrometry in order to check its composition. We could verify that the Cq extract increased neurite outgrowth length and branching junctions, amplifying the contact between SH‐SY5Y neurons, without affecting cell body and viability. The extract action was selective for neurons, as it did not cause any effects on other cell types, such as tumor line, nontumor line, and red blood cells. Moreover, mass spectrometry analysis revealed that there are no proteins but several low molecular mass compounds and peptides. Three peptides, characterized as cryptides, and 14 low molecular mass compounds were found to be related to cytoskeleton reorganization, cell membrane expansion, and antioxidant/neuroprotective activity, which act together to increase neuritogenesis. After this evaluation, we conclude that the Cq extract is a promising tool for neuronal connection recovery, an essential condition for the treatment of neurodegenerative diseases.

This study explores the direct effects of amphibian skin secretions on human cells involved in joint diseases, aiming to identify species with potential for inflammatory modulation. Secretions were obtained from sixteen species distributed across Brazilian biomes and one European species. Following biochemical characterization, human chondrocytes, synoviocytes, and macrophages were treated with secretions for 24 h. The cytotoxicity and modulation of the IL-6, IL-8, TNF-α, and IL-1β release were assessed. Synoviocytes showed the greatest resistance to cytotoxic effects, though sensitivity varied by species. Secretions from Trachycephalus mesophaeus, Pipa carvalhoi, and Phyllomedusa bahiana exhibited the highest cytotoxicity. At non-cytotoxic concentrations, P. carvalhoi and Leptodactylus fuscus strongly induced IL-6 and IL-8 in chondrocytes and synoviocytes, with P. carvalhoi also stimulating IL-1β and TNF-α release in macrophages. Among Bufonidae species, particularly Rhinella jimi and Bufo bufo, were potent inducers of TNF-α and IL-1β in macrophages. Secretions lacking pro-inflammatory effects were further tested for anti-inflammatory activity. P. bahiana reduced TNF-α production in stimulated macrophages and IL-6 in synoviocytes, while Siphonops annulatus and T. mesophaeus reduced LPS-induced TNF-α in macrophages. Our data underscore the rich biodiversity of amphibians, supporting the bioprospecting of their cutaneous secretions. These data reveal substantial potential for uncovering bioactive compounds with pharmacological applications.