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Abnormal Cannabidiol Attenuates Experimental Colitis in Mice, Promotes Wound Healing and Inhibits Neutrophil Recruitment

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Abnormal Cannabidiol Attenuates Experimental Colitis in Mice, Promotes Wound Healing and Inhibits Neutrophil Recruitment

Regina M Krohn et al. J Inflamm (Lond).

Abstract

Background: Non-psychotropic atypical cannabinoids have therapeutic potential in a variety of inflammatory conditions including those of the gastrointestinal tract. Here we examined the effects of the atypical cannabinoid abnormal cannabidiol (Abn-CBD) on wound healing, inflammatory cell recruitment and colitis in mice.

Methods: Colitis was induced in CD1 mice by a single intrarectal administration of trinitrobenzene sulfonic acid (TNBS, 4 mg/100 μl in 30 % ethanol) and Abn-CBD and/or the antagonists O-1918 (Abd-CBD), AM251 (CB1 receptor) and AM630 (CB2 receptor), were administered intraperitoneally (all 5 mg/kg, twice daily for 3 days). The degree of colitis was assessed macro- and microscopically and tissue myeloperoxidase activity was determined. The effects of Abn-CBD on wound healing of endothelial and epithelial cells (LoVo) were assessed in a scratch injury assay. Human neutrophils were employed in Transwell assays or perfused over human umbilical vein endothelial cells (HUVEC) to study the effect of Abn-CBD on neutrophil accumulation and transmigration.

Results: TNBS-induced colitis was attenuated by treatment with Abn-CBD. Histological, macroscopic colitis scores and tissue myeloperoxidase activity were significantly reduced. These effects were inhibited by O-1918, but not by AM630, and only in part by AM251. Wound healing of both HUVEC and LoVo cells was enhanced by Abn-CBD. Abn-CBD inhibited neutrophil migration towards IL-8, and dose-dependently inhibited accumulation of neutrophils on HUVEC.

Conclusions: Abn-CBD is protective against TNBS-induced colitis, promotes wound healing of endothelial and epithelial cells and inhibits neutrophil accumulation on HUVEC monolayers. Thus, the atypical cannabinoid Abn-CBD represents a novel potential therapeutic in the treatment of intestinal inflammatory diseases.

Keywords: Abnormal cannabidiol; Cannabinoids; Neutrophil recruitment; O-1918; TNBS-colitis; Wound healing.

Figures

Fig. 1
Fig. 1
The effects of Abn-CBD treatment on macroscopic damage score and MPO activity. TNBS-treated mice were given O-1918 (5 mg/kg) and/or Abn-CBD (5 mg/kg) twice daily for 3 days. Macroscopic damage score (a) and myeloperoxidase activity (b) were determined at the end of the experiment, 24 h after the last dose of drug. Abn-CBD significantly reduced macroscopic damage and MPO activity, and effect that was blocked by O-1918. Data were analyzed with one-way ANOVA with Tukey’s post-hoc test. *, p < 0.05 when compared to control and Abn-CBD + O-1918 (a and b) and when compared to O-1918 (b); n ≥ 10/group
Fig. 2
Fig. 2
The effects of Abn-CBD treatment in the presence of CB antagonists on macroscopic damage score and MPO activity. TNBS-treated mice were given AM251, AM630 and/or Abn-CBD (all at 5 mg/kg) twice daily for 3 days. Macroscopic damage score (a) and myeloperoxidase activity (b) were determined at the end of the experiment, 24 h after the last dose of drug. Abn-CBD treatment and Abn-CBD + AM251 or AM630 significantly decreased the damage score. Myeoloperoxidase activity was significantly decreased with Abn-CBD. Data were analyzed with one-way ANOVA with Tukey’s post-hoc test *, p < 0.05 and **, p < 0.01 when compared to controls; n = 4-5/group
Fig. 3
Fig. 3
The effects of Abn-CBD treatment on microscopic damage score and histology. TNBS-treated mice were given O-1918, AM251, AM630 (all at 5 mg/kg) and/or Abn-CBD (5 mg/kg) twice daily for 3 days. Microscopic damage score (a and b) and histological appearance of the hematoxylin/eosin stained tissue (c) were determined at the end of the experiment, 24 h after the last dose of drug. TNBS-treated mice displayed severe mucosal damage as well as an increase in lymphocyte and neutrophil infiltration. a Mucosal damage was significantly reduced by Abn-CBD treatment and reversed by addition of O-1918; *, p < 0.05, when compared to any other treatment group. b Mucosal damage was significantly reduced by Abn-CBD treatment and partly reversed by administration of AM251; *, p < 0.05, when compared to TNBS alone. Data were analyzed with one-way ANOVA with Tukey’s post-hoc test, n = 4-5/group. Scale bar in C. = 100 μm
Fig. 4
Fig. 4
The effects of Abn-CBD on neutrophil chemotaxis. Neutrophils were incubated with Abn-CBD and/or O-1918 and pipetted into the top chamber of 5 μm Transwells (2 × 105/well). Migration towards IL-8 (10 nM) was assessed after 3 h by counting adherent neutrophils at the bottom of the chamber (3 non-overlapping fields/well). The open bar depicts random migration without an IL-8 stimulus. Abn-CBD significantly reduced neutrophil chemotaxis, an effect that was blocked by O-1918. Data were analyzed with one-way ANOVA with Tukey’s post-hoc test. *, p < 0.05 when compared to control; n = 3/group
Fig. 5
Fig. 5
The effects of Abn-CBD on neutrophil recruitment assessed using a parallel plate flow chamber assay. HUVEC monolayers were activated with TNF-α (10 nM) 4 h prior to the experiment. a Abn-CBD was added to the HUVEC for 1 h and freshly isolated neutrophils were perfused over the monolayer for 4 min. b Using a different approach neutrophils were treated with Abn-CBD and O-1918 for 15 min and then perfused over the HUVEC monolayer, neutrophil accumulation was then assessed after 4 min of perfusion, and c transmigration of neutrophils through the monolayer was determined after 6 min. Neutrophil accumulation (after 4 min) and transmigration (after 6 min) was significantly decreased, when neutrophils were incubated with Abn-CBD. *, p < 0.05 and **, p < 0.01 when compared to control, Abn-CBD + O-1918 and O-1918; n = 5/group
Fig. 6
Fig. 6
Fluorescence micrographs of GPR18 immunoreactivity. Neutrophils on glass coverslips were fixed with 2 % paraformaldehyde in HBSS and stained with anti-GPR18 antibody (right panel) or isotype control (left panel), then washed and incubated with the secondary FITC-conjugated detection antibody (green). Cells were mounted with Hoechst 33258 (Sigma-Aldrich, Oakville, Ontario, Canada) (blue). No immunoreactivity was detected in the control cells, but most neutrophils expressed GPR18. Scale bar = 100 μm
Fig. 7
Fig. 7
The effects of Abn-CBD treatment on wound healing. HUVEC and LoVo cells were grown to confluence in 6 well plates. Then a scratch wound was made with a pipet tip. a HUVEC cells were incubated with the respective concentrations of Abn-CBD and O-1918. Wound area size was measured at 0 and 20 h. Abn-CBD significantly accelerated wound closure in HUVEC. Additional treatment with the O-1918 did not reverse this effect. b For incubation of LoVo cells, the effective dose for Abn-CBD (1 μM) was used. Wound area was measured at 0 and 20 h. Abn-CBD significantly accelerated wound closure. The Abn-CBD effect was reversed by addition of the antagonist O-1918. Data were analyzed with one-way ANOVA with Tukey’s post-hoc test. *, p < 0.05 when compared to any other treatment; n = 5-7/group
Fig. 8
Fig. 8
The effects of Abn-CBD treatment on wound healing. HUVEC and LoVo cells were grown to confluence in 6 well plates. Then a scratch wound was made with a pipet tip. Wound area was measured at 0 and 20 h. Scale bar = 100 μm. Abn-CBD treatment accelerated wound healing and O-1918 addition reversed the Abn-CBD effect (see Fig. 7 for detailed results)

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