**1. Introduction**

Crotalphine, a structural analogue to an antinociceptive peptide that was first identified in the crude venom from the South American rattlesnake *Crotalus durissus terrificus*, induces a potent and long-lasting antinociceptive effect, mediated by the activation of peripheral opioid receptors [1]. Unlike opioids, treatment with crotalphine for several days does not induce tolerance and withdrawal symptoms. Crotalphine is not an opioid receptor agonist, however, this peptide induces the release of dynorphin A that activates peripheral kappa opioid receptors (KOR) [2]. More recently, attempting to identify the direct molecular targets of crotalphine in pain pathways, it was demonstrated that this peptide partially activates and desensitizes the TRPA1 ion channel at subnanomolar concentrations and this effect is critical for the peptide's analgesic effect [3].

Mitogen-activated protein kinases (MAPKs) transduce a multiple extracellular stimulus into intracellular effects by modifying the transcription, as well as inducing posttranslational changes in target proteins. There are three major members in the MAPK family: extracellular signal-regulated kinases (ERK) 1/2, C-Jun N-terminal kinase (JNK)

**Citation:** de Freitas, B.G.; Hösch, N.G.; Pereira, L.M.; Barbosa, T.C.; Picolo, G.; Cury, Y.; Zambelli, V.O. PKCζ-Mitogen-Activated Protein Kinase Signaling Mediates Crotalphine-Induced Antinociception. *Toxins* **2021**, *13*, 912. https://doi.org/10.3390/ toxins13120912

Received: 30 October 2021 Accepted: 6 December 2021 Published: 20 December 2021

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and p38, which represent three different signaling pathways [4]. The emerging evidence suggests that MAPKs are involved in nociception and in the development of side effects of drugs, such as morphine. In this regard, peripheral inflammation or nerve injury are followed by spinal activation of ERK1/2, JNK and p38 that seems to increase neuronal excitability and up-regulate transcriptional factors involved in nociception [5]. However, the ERK1/2-signaling activation is essential for morphine and KOR agonists-mediated analgesia in rodents, suggesting that ERK activation may also positively affect the pain outcome [6,7].

It is known that crotalphine increases the level of activated ERK and JNK in cultured sensory neurons from the dorsal root ganglia (DRG) and the ERK levels increase is dependent on the activation of KOR and the protein kinase Cζ (PKCζ) [8]. However, whether the PKCζ-MAPK signaling pathway is involved in crotalphine-induced antinociception has not yet been evaluated in vivo. Considering that crotalphine has a potent and long-lasting analgesic effect in rodents, identifying the intracellular signaling mechanisms responsible for the peptide effects may provide insights to guide the development of better analgesics. Thus, using biochemical and pharmacological strategies, we sought to investigate the role of MAPK in crotalphine-induced antinociception.
