Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica

Abstract

Acid-sensing ion channels (ASICs) have been known as sensors of a local pH change within both physiological and pathological conditions. ASIC-targeting peptide toxins could be potent molecular tools for ASIC-manipulating in vitro, and for pathology treatment in animal test studies. Two sea anemone toxins, native Hmg 1b-2 and recombinant Hmg 1b-4, both related to APETx-like peptides, inhibited the transient current component of human ASIC3-Δ20 expressed in Xenopus laevis oocytes, but only Hmg 1b-2 inhibited the rat ASIC3 transient current. The Hmg 1b-4 action on rASIC3 as a potentiator was confirmed once again. Both peptides are non-toxic molecules for rodents. In open field and elevated plus maze tests, Hmg 1b-2 had more of an excitatory effect and Hmg 1b-4 had more of an anxiolytic effect on mouse behavior. The analgesic activity of peptides was similar and comparable to diclofenac activity in an acid-induced muscle pain model. In models of acute local inflammation induced by λ-carrageenan or complete Freund's adjuvant, Hmg 1b-4 had more pronounced and statistically significant anti-inflammatory effects than Hmg 1b-2. It exceeded the effect of diclofenac and, at a dose of 0.1 mg/kg, reduced the volume of the paw almost to the initial volume. Our data highlight the importance of a comprehensive study of novel ASIC-targeting ligands, and in particular, peptide toxins, and present the slightly different biological activity of the two similar toxins.

Keywords: APETx-like peptides; acid-sensing ion channels; anxiolytic effect; inflammation; pain killers; sea anemones (Heteractis magnifica).

Figure 1

(a) Map of the pET32a(+)-Hmg 1b-4 expression plasmid. The sequence encoding the Hmg 1b-4 and enterokinase site was introduced using the restriction enzyme recognition sites KpnI and XhoI. (b) The schematic representation of the expected fusion proteins Trx-Hmg 1b-4 and Hmg 1b-4 sequences are shown.

Figure 2

Purification of the toxin after fusion protein digestion by enteropeptidase by RP-HPLC (a) on a Jupiter C5 column (150 mm × 4.6 mm) in the presence of 0.1% TFA, and a linear gradient from 0 to 60% MeCN over 60 min at a flow rate 1 mL/min; and (b) on a Luna Phenyl–Hexyl column (250 mm × 4.6 mm) in the presence of 0.1% TFA, and at a linear gradient from 20 to 50% over 30 min at a flow rate of 1 mL/min. Black box indicates the elution time of the toxin. The inset shows MALDI analysis of the pure toxin.

Figure 3

Electrophysiological study of recombinant Hmg 1b-4 on rASIC1a (a) and rASIC3 (b) channels expressed in X. laevis oocytes. Current traces generated by a pH drop of 7.4 to 5.5 are shown for the control application (black line) and the cells pre-incubated with 1 µM of Hmg 1b-4 (red line).

Figure 4

Inhibitory activity of peptides Hmg 1b-2 (a) and Hmg 1b-4 (b) toward hASIC3-Δ20. Acid-induced currents were evoked by a pH drop from 8.0 to 5.5. Representative current traces (left panel) and the concentration–response curve (right panel) for the inhibitory effect on the transient current are presented. Data are fitted using the Hill equation, and the resulting values of the fitting parameters are IC₅₀ 14.6 ± 0.9 µM (n_H of 1.09 ± 0.06) and IC₅₀ 14.1 ± 2.5 µM (n_H of 1.1 ± 0.2) for Hmg 1b-2 and Hmg 1b-4, respectively. Each point indicates the means ± SEM (n = 5).

Figure 5

Effect of Hmg 1b-2 and Hmg 1b-4 administered intravenously on normal mouse behavior in the open-field test: time of mouse activity or immobility (a), time spent in the central or border zone (b), distance traveled by animals (c), mean movement speed of animals (d), peeps into the holes (e) and number of vertical stances (f). Control animals received saline in identical volumes. Results are presented as the mean ± SD (n = 7–8). The significance of differences was estimated via a one-way ANOVA, followed by Tukey’s test versus saline group, with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Effect of Hmg 1b-2 and Hmg 1b-4 administered intravenously on normal mouse behavior in the open-field test: time of mouse activity or immobility (a), time spent in the central or border zone (b), distance traveled by animals (c), mean movement speed of animals (d), peeps into the holes (e) and number of vertical stances (f). Control animals received saline in identical volumes. Results are presented as the mean ± SD (n = 7–8). The significance of differences was estimated via a one-way ANOVA, followed by Tukey’s test versus saline group, with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

The anti-anxiety activity of Hmg 1b-2 and Hmg 1b-4 administered intravenously in the elevated plus maze test: time spent in the closed arms, open arms and central area (a); number of entries to the closed arms, open arms and central area (b); number of hanging-down reactions (c); and vertical stances (d). Control animals received saline in identical volumes. Results are presented as the mean ± SD (n = 7–8). The significance of differences was estimated via a one-way ANOVA followed by Tukey’s test versus the saline group, with * p < 0.05 and ** p < 0.01.

Figure 7

Analgesic activity of Hmg 1b-2 and Hmg 1b-4 administered intravenously in the model of the acid-induced muscle pain: number of writhings (a), time of writhings (b); and the hot plate test: latent time of front paw licking (c), latent time of hind paw licking (d). A saline buffer as a negative control and diclofenac at a dose of 1 mg/kg as a positive control were administered intravenously, and metamizole sodium at a dose of 500 mg/kg as a positive control was administered intraperitoneally. Results are presented as the mean ± SD (n = 7–8). The significance of differences was estimated via a one-way ANOVA followed by Tukey’s test versus the saline group, with ** p < 0.01 and *** p < 0.001.

Figure 8

λ-Carrageenan-induced inflammation model. Time-dependent effect of Hmg 1b-2 (a,c) and Hmg 1b-4 (b,d) administered intravenously 30 min before the induction of the inflammation on the paw volume (a,b) and on the volume growth index (%) (c,d), respectively. A saline buffer as a negative control and diclofenac at a dose of 1 mg/kg as a positive control were administrated intravenously. Results are presented as the mean ± SD (n = 7–8). The significance of differences was estimated via a one-way ANOVA followed by a Tukey’s test versus the saline group, with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 8

λ-Carrageenan-induced inflammation model. Time-dependent effect of Hmg 1b-2 (a,c) and Hmg 1b-4 (b,d) administered intravenously 30 min before the induction of the inflammation on the paw volume (a,b) and on the volume growth index (%) (c,d), respectively. A saline buffer as a negative control and diclofenac at a dose of 1 mg/kg as a positive control were administrated intravenously. Results are presented as the mean ± SD (n = 7–8). The significance of differences was estimated via a one-way ANOVA followed by a Tukey’s test versus the saline group, with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 9

Complete Freund’s adjuvant-induced inflammation model. Time-dependent effect of Hmg 1b-2 (a,c) and Hmg 1b-4 (b,d) administered intravenously 30 min before the inflammation induction on the paw volume (a,c) and the volume growth index (%) (b,d), respectively. A saline buffer as a negative control and diclofenac at a dose of 1 mg/kg as a positive control were administered intravenously. Results are presented as the mean ± SD (n = 7–8). The significance of differences was estimated via a one-way ANOVA, followed by Tukey’s test versus the saline group, with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 10

Sequence alignment of rASIC1a (Uniprot ID P55926), rASIC3 (Uniprot ID O35240), mASIC3 (Uniprot ID Q6X1Y6) and hASIC3 (Uniprot ID Q9UHC3). The putative regions involved in the Hmg 1b-4 binding are shown, and the residues that tentatively contribute to the rASIC1a-Hmg 1b-4 complex formation are colored yellow. The residues that distinguished rodent ASIC3 from human ASIC3 are in green boxes, and three proton-sensing residues are marked with red asterisks.