Neosaxitoxin Inhibits the Expression of Inflammation Markers of the M1 Phenotype in Macrophages

Abstract

(1) Background: Neosaxitoxin (NeoSTX) has been used as a local anesthetic, but its anti-inflammatory effects have not been well defined. In the present study, we investigate the effects of NeoSTX on lipopolysaccharide (LPS)-activated macrophages. (2) Methods: Raw 264.7 and equine PBMC cells were incubated with or without 100 ng/mL LPS in the presence or absence of NeoSTX (1µM). The expression of inflammatory mediators was assessed: nitric oxide (NO) content using the Griess assay, TNF-α content using the ELISA assay, and mRNA of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) using a real-time polymerase chain reaction. (3) Results: NeoSTX (1 μM) significantly inhibited the release of NO, TNF-α, and expression of iNOS, IL-1β, and TNF-α in LPS-activated macrophages of both species studied. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by NeoSTX. Additionally, NeoSTX deactivated polarized macrophages to M1 by LPS without compromising its polarization towards M2. (4) Conclusions: NeoSTX inhibits LPS-induced release of inflammatory mediators from macrophages, and these effects may be mediated by the blockade of voltage-gated sodium channels (VGSC).

Keywords: Nav channels; inflammation; macrophages; neosaxitoxin.

Figure 1

Cell viability. RAW 264.7 cells were treated with Neosaxitoxin (NeoSTX) at concentrations of 0.1, 1, 10, and 100 nM, and 1 and 10 µM for 24, 48, and 72 h.

Figure 2

Characterization of the Nav isoforms in a macrophage model. (A) Expression of Navs in primary culture. RT–PCR product for isoforms of Nav 1.4, 1.5, and 1.6 in equine PBMC (Mϕ). Positive controls (striated muscle, heart, and brain) and negative control (liver) are shown. (B) Representative histogram of flow cytometry in equine PBMC for anti-mouse anti-Nav 1.6 antibody. (C) Expression of Nav 1.6 in murine macrophages. Representative histogram of flow cytometry in RAW 264.7 cells for rabbit anti-mouse Nav 1.6. (D) Confocal microscopy in RAW 264.7 cells for FITC isotype anti-rabbit antibody, DAPI (blue). Confocal microscopy in RAW 264.7 cells for anti-Nav 1.6 antibody (green), DAPI (blue). (E) Effect of NeoSTX on the expression of the equine Nav 1.5 and equine Nav 1.6 mRNA. Equine PBMC were cultured with LPS (100 ng/mL) for 18 h. The expression of the equine Nav 1.6 mRNA was quantified by RT–PCR. β-Actin was used as a control gene. * p ≤ 0.05 (n = 4).

Figure 2

Characterization of the Nav isoforms in a macrophage model. (A) Expression of Navs in primary culture. RT–PCR product for isoforms of Nav 1.4, 1.5, and 1.6 in equine PBMC (Mϕ). Positive controls (striated muscle, heart, and brain) and negative control (liver) are shown. (B) Representative histogram of flow cytometry in equine PBMC for anti-mouse anti-Nav 1.6 antibody. (C) Expression of Nav 1.6 in murine macrophages. Representative histogram of flow cytometry in RAW 264.7 cells for rabbit anti-mouse Nav 1.6. (D) Confocal microscopy in RAW 264.7 cells for FITC isotype anti-rabbit antibody, DAPI (blue). Confocal microscopy in RAW 264.7 cells for anti-Nav 1.6 antibody (green), DAPI (blue). (E) Effect of NeoSTX on the expression of the equine Nav 1.5 and equine Nav 1.6 mRNA. Equine PBMC were cultured with LPS (100 ng/mL) for 18 h. The expression of the equine Nav 1.6 mRNA was quantified by RT–PCR. β-Actin was used as a control gene. * p ≤ 0.05 (n = 4).

Figure 3

Representative histogram of flow cytometry in RAW cells 264.7 for rabbit anti-NeoSTX antibody. The cells were cultured with NeoSTX (1 µM) for 24 h. (A) Non-permeabilized cells, (B) permeabilized cells.

Figure 4

NeoSTX inhibits the expression of markers of inflammation in macrophages. (A) RAW 264.7 cells were pre-treated with NeoSTX (1 µM) or lidocaine (Lido) (20 µg/mL) for 24 h and subsequently exposed to LPS (100 ng/mL) for 18 h. The production of NO and TNF-α in the culture supernatant was quantified using the Griess and ELISA technique, respectively, and the expression of mouse TNF-α mRNA by RT–PCR. (B) Equine PBMC cells were pretreated with NeoSTX (1 µM) or lidocaine (20 µg/mL) for 24 h and subsequently exposed to LPS (100 ng/mL) for 18 h. The production of equine NO was quantified using the Griess technique and equine TNF-α using the ELISA technique in the culture supernatant. (C) The expression of the equine TNF-α, equine iNOS, and equine IL-1β mRNA was quantified by RT–PCR (C). β-Actin was used as a control gene. * p < 0.05 ; ** p < 0.001 (n = 4).

Figure 4

NeoSTX inhibits the expression of markers of inflammation in macrophages. (A) RAW 264.7 cells were pre-treated with NeoSTX (1 µM) or lidocaine (Lido) (20 µg/mL) for 24 h and subsequently exposed to LPS (100 ng/mL) for 18 h. The production of NO and TNF-α in the culture supernatant was quantified using the Griess and ELISA technique, respectively, and the expression of mouse TNF-α mRNA by RT–PCR. (B) Equine PBMC cells were pretreated with NeoSTX (1 µM) or lidocaine (20 µg/mL) for 24 h and subsequently exposed to LPS (100 ng/mL) for 18 h. The production of equine NO was quantified using the Griess technique and equine TNF-α using the ELISA technique in the culture supernatant. (C) The expression of the equine TNF-α, equine iNOS, and equine IL-1β mRNA was quantified by RT–PCR (C). β-Actin was used as a control gene. * p < 0.05 ; ** p < 0.001 (n = 4).

Figure 5

Effect of NeoSTX on M2 phenotype. RAW 264.7 cells were cultured with NeoSTX (1 µM) or lidocaine (20 µg/mL) for 24 h. The expression of the equine ARG1 and equine IL-10 mRNA was quantified by RT–PCR. β-Actin was used as a control gene.

Figure 6

NeoSTX reverses the expression and production of cytokines of the M1 phenotype. (A) RAW 264.7 cells were cultured with LPS (100 ng/mL) for 18 h and subsequently exposed to NeoSTX (1 µM) or lidocaine (20 µg/mL) for 24 h. The production of mouse NO and mouse TNF-α was quantified using the Griess and ELISA techniques, respectively, and the expression of the TNF-α mouse mRNA by RT-PCR (A). (B) Equine cells were cultured with LPS (100 ng/mL) for 18 h and subsequently exposed to NeoSTX (1 µM) or lidocaine (20 µg/mL) for 24 h. The production of equine NO and equine TNF-α was quantified using the Griess and ELISA techniques, respectively. (C) Equine iNOS, equine TNF-α and equine IL-1β mRNA expression were determined by RT–PCR. β-Actin was used as a control gene. * p < 0.05, ** p < 0.001 (n = 4).