Cannabidiol reduces intestinal inflammation through the control of neuroimmune axis

Abstract

Enteric glial cells (EGC) actively mediate acute and chronic inflammation in the gut; EGC proliferate and release neurotrophins, growth factors, and pro-inflammatory cytokines which, in turn, may amplify the immune response, representing a very important link between the nervous and immune systems in the intestine. Cannabidiol (CBD) is an interesting compound because of its ability to control reactive gliosis in the CNS, without any unwanted psychotropic effects. Therefore the rationale of our study was to investigate the effect of CBD on intestinal biopsies from patients with ulcerative colitis (UC) and from intestinal segments of mice with LPS-induced intestinal inflammation. CBD markedly counteracted reactive enteric gliosis in LPS-mice trough the massive reduction of astroglial signalling neurotrophin S100B. Histological, biochemical and immunohistochemical data demonstrated that S100B decrease was associated with a considerable decrease in mast cell and macrophages in the intestine of LPS-treated mice after CBD treatment. Moreover the treatment of LPS-mice with CBD reduced TNF-α expression and the presence of cleaved caspase-3. Similar results were obtained in ex vivo cultured human derived colonic biopsies. In biopsies of UC patients, both during active inflammation and in remission stimulated with LPS+INF-γ, an increased glial cell activation and intestinal damage were evidenced. CBD reduced the expression of S100B and iNOS proteins in the human biopsies confirming its well documented effect in septic mice. The activity of CBD is, at least partly, mediated via the selective PPAR-gamma receptor pathway. CBD targets enteric reactive gliosis, counteracts the inflammatory environment induced by LPS in mice and in human colonic cultures derived from UC patients. These actions lead to a reduction of intestinal damage mediated by PPARgamma receptor pathway. Our results therefore indicate that CBD indeed unravels a new therapeutic strategy to treat inflammatory bowel diseases.

Figure 1. Effect of CBD on glial cell hyperactivation in septic mice.

The intraperitoneal administration of CBD in septic mice significantly reduced S100B protein expression. Results are expressed as mean ± SEM of 3 experiments. ***p<0.001 vs. saline; °°°p<0.001 vs. LPS alone.

Figure 2. Effect of CBD on mast cell activation in septic mice.

(A) MC presence in intestinal tissue was evaluated in representative histological analysis of paraffined tissue stained with Toluidine blue. Fields are representative of 3 separate experiments. Original magnification, 100×.

Figure 3. Effect of CBD on mast cell derived.

(A) chymase protein expression, (B) MMP-9 protein expression. Results are expressed as mean ± SEM of 3 experiments ***p<0.001 vs. saline; °°°p<0.001 vs. LPS alone Representative Western blot analysis and relative densitometric analysis. Each bar in panel B shows the mean ± SD of 3 experiments. ***p<0.001 vs. saline; °°°p<0.001 vs. LPS alone.

Figure 4. Effect of CBD on macrophages activation in septic mice.

Immunohistochemical analysis for MAC3, a know marker of macrophages presence in inflamed tissue, revealed a reduced number of macrophages in intestinal tissues of CBD treated mice.

Figure 5. Effect of CBD in LPS-induced intestinal TNF-α expression in the mouse intestine.

Western blot analysis showing the effect of CBD on LPS induced TNF-α expression. Panel (a) shows TNF-α protein expression; (b) densitometric analysis of corresponding bands (optical density). Panel A is representative of n = 3 separated experiments. Each bar in panel B shows the mean ± SD of 3 experiments. ***p<0.001 vs. saline; °°°p<0.001 vs. LPS alone.

Figure 6. Effect of CBD on caspase-3 activation.

(A) Immunohistochemical analysis for cleaved-caspase-3 showed a reduced number of caspase3 positive cells in intestinal tissues of CBD treated mice in confront to LPS mice.

Figure 7. Effect of CBD in rectal biopsies of patients with ulcerative colitis (UC) in the remission phase.

(A) shows the effect of CBD on glial activation evaluated as S100B protein expression. Western blot and relative densitometric analysis evidenced that CBD significantly and in a concentration dependent manner reduced LPS- induced S100B. (B) shows the effect of CBD on LPS + INF-γ induced iNOS protein expression and (C) nitrate production. Results are expressed as mean ± SEM of 3 experiments ***p<0.001 vs. CTR; °p<0.05, °°°p<0.001 vs. LPS+INF-γ.

Figure 8. Effect of CBD in rectal biopsies of patients with ulcerative colitis (UC) in the acute phase.

(A) shows the effect of CBD on glial activation evaluated as S100B protein expression. Western blot and relative densitometric analysis evidenced that CBD significantly and in a concentration dependent manner S100B expression in biopsies of UC in acute phase. (B) shows the effect of CBD on iNOS protein expression and (C) nitrate production. Results are expressed as mean ± SEM of 3 experiments; °p<0.05, °°°p<0.001 vs. untreated biopsies.

Figure 9. Effect of CBD and GW9662 in rectal biopsies of patients with ulcerative colitis (UC) in the acute phase.

(A) shows the effect of CBD and GW9662 on glial activation evaluated as S100B protein expression. Western blot and relative densitometric analysis evidenced that GW9662 significantly counteracts the effect of CBD on S100B expression in biopsies of UC in acute phase. (B) shows the effect of GW9662 and CBD on iNOS protein expression and (C) nitrate production. Results are expressed as mean ± SEM of 3 experiments; °°°p<0.001 vs. untreated biopsies; **p<0.05, ***p<0.001 vs CBD.