Preparation of Snake Neurotoxin Nanocapsules and the Analgesic Mechanism of P38 Mitogen-Activated Protein Kinase Signal Pathway Combining Snake Neurotoxin Nanocapsules with Gabapentin

This study aimed to explore the analgesic effect of snake neurotoxin combined with gabapentin (Gab) on neuropathic pain in rats with chronic compression injury (CCI) of the sciatic nerve based on the nanotechnology. Firstly, various solutions were prepared to obtain the inner water phase, the oil phase, the outer water phase, and the dilution phase. Poly(lactic-co-glycolic) Acid (PLGA) and polyethylene glycol-poly(lactic-co-glycolic) acid (PEG-PLGA) were added to the prepared oil phase solution to obtain the PLGA snake neurotoxin nanocapsule and PEG-PLGA snake neurotoxin nanocapsule, respectively. After the nanocapsules were obtained, a rat CCI model was further modelled, and the reactive oxygen species (ROS) content in the rat brain tissue was analyzed and tested by the kit, and the optimal physical conditions for preparing the nanocapsules were tested. In order to test the effect of nanocapsules on the p38 mitogen-activated protein kinase (MAPK) signaling pathway, the rats were divided into Control group, Sham group, CCI group, Gabapentin (Gab) group, and PEG-PLGA snake neurotoxin nanocapsule + Gab group. The rats in different groups were given abdominal injections to compare relevant indicators of signal pathway. In the experiment, neuropathic pain was related to changes in ROS content, and snake neurotoxin nanocapsules could reduce the ROS content; PLGA snake neurotoxin nanocapsules and PEG-PLGA snake neurotoxin nanocapsules had encapsulation efficiencys of 24.7% and 22.8% and drug loading of 3.28% and 3.02%, respectively, and the particle sizes of prepared nanocapsules were 760 nm~1,150 nm. Besides, the phase transition temperature of about 50 °C and the light time of 1 h can accelerate the release of nanocapsules to the greatest extent; and the snake neurotoxin could inhibit the activation of p38 MAPK signaling pathway so as to play the analgesic effects on neuropathic pain.

αM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of αM-MIIIJ induced paralysis in goldfish (Carassius auratus) but not mice. Intracellular recording from skeletal muscles of fish (C. auratus) and frog (Xenopus laevis) revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 μM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of IACh by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of X. laevis, C. auratus, and D. rerio. (Note, we observed that αM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC50s ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that αM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.