Efficacy of non-psychotropic Cannabis sativa L. standardized extracts in a model of intestinal inflammation

Abstract

Background: The use of Cannabis sativa L. (Cannabis) was reported by observational studies on inflammatory bowel diseases (IBD) patients. However, this indication is poorly supported by clinical trials. Several pre-clinical studies demonstrated the anti-inflammatory activity of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) at intestinal level. On the contrary, minor cannabinoids, such as cannabigerol (CBG), were less investigated. Moreover, several authors suggested that complex Cannabis extracts might display a higher efficacy in respect to pure cannabinoids against inflammatory disorders.

Methods: This study was aimed at investigating the role of Cannabis extracts, standardized in CBD and CBG content, in a model of in vitro-induced intestinal inflammation using CaCo-2 cells. Inflammatory mediators at transcriptional (PCR arrays) and protein level (ELISA assays) were investigated and correlated with enterocyte layer permeability. The two evaluated extracts, A and B, come from the mix of the same Cannabis varieties (Cannabis sativa L. Chemotype III and Chemotype IV), and are standardized in CBD and CBG at the same level, by changing the polarity of the primary extraction solvents.

Results: Pro-inflammatory cytokines involved in IBD, such as IL-1β and IFN-γ, induced the expression and the release of chemokines for lymphocytes (CXCL-9, CXCL-10, CCL20) in CaCo-2, while Cannabis extracts (100 µg/mL) or individual compounds (8 µM) showed inhibitory activity. After simulated digestion, extract A abrogated the release of CCL-20, while extract B abrogated the release of CXCL-9 and CXCL-10. The inhibition of CXCL-9 was demonstrated at transcriptional level also. The inhibitory activity paralleled with the content of CBD or CBG, acting at least in part through NF-κB impairment (-42% and - 66%, respectively). However, Cannabis extracts showed greater effect in the CaCo-2-THP-1 co-culture inflammation model compared to individual cannabinoids, thus partially recovering the epithelial barrier measured by transepithelial electrical resistance (TEER), and zonula occludens (ZO-1) expression.

Conclusions: Data collected within this study showed the importance of standardization and extraction method reproducibility through manufacturing and process control, besides demanding future investigations focusing on the effect of Cannabis extracts against intestinal inflammation, which show in this context effects higher than individual cannabinoids.

Keywords: CBG; Cannabis; Gut inflammation; IBD CBD.

Fig. 1

Effect of Cannabis extracts (Extract A, Extract B) and pure cannabinoids (CBD, CBG) on the release of CXCL-10 (a) and the activation of NF-κB (b) in colonocytes (CaCo-2). CXCL-10 release was measured by ELISA (24 h), while NF-κB driven transcription was measured by luciferase assay (6 h). Cells were treated with extracts (100 µg/mL) or pure molecules (8 µM) in addition to inflammatory stimuli (IL-1β/IFN-γ), which value was arbitrarily assigned to 100%. Sodium butyrate 2 mM was used as reference inhibitor of NF-κB activity (-55%) and CXCL-10 release (-30%). Data are expressed as average (%) ± SEM (n = 3). * p < 0.05; ***p < 0.001 (Kruskal-Wallis, and Brown-Forsythe and Welch test, respectively) vs. IL-1β/IFN-γ

Fig. 2

Scatter plots of the expression profile of inflammatory genes measured by PCR array in colonocytes (CaCo-2). Cells were treated for 24 h with Cannabis extracts (100 µg/mL) underwent to simulated digestion, in addition to inflammatory stimuli (IL-1β/IFN-γ). Plots of unstimulated control (a), Extract A Dig. (b), and Extract B Dig. (c) versus IL-1β/IFN-γ are reported, respectively. The center diagonal line indicates unchanged gene expression (black dots), while the outer diagonal lines indicate the selected fold regulation threshold. Genes with data points beyond the outer lines in the upper left and lower right corners are up-regulated (red dots) or down-regulated (green dots), respectively, by more than the fold regulation threshold in the y-axis Group relative to the x-axis Group. Scatter plots were automatically generated by GeneGlobe web portal analysis

Fig. 3

Scatter plots of the expression profile of inflammatory genes measured by PCR array in enterocytes (differentiated CaCo-2). Cells were treated for 24 h with Cannabis extracts (100 µg/mL) subjected to simulated digestion, in addition to inflammatory stimuli (IL-1β/IFN-γ). Plots of unstimulated control (a), Extract A Dig. (b), and Extract B Dig. (c) versus IL-1β/IFN-γ are reported, respectively. The center diagonal line indicates unchanged gene expression (black dots), while the outer diagonal lines indicate the selected fold regulation threshold. Genes with data points beyond the outer lines in the upper left and lower right corners are up-regulated (red dots) or down-regulated (green dots), respectively, by more than the fold regulation threshold in the y-axis Group relative to the x-axis Group. Scatter plots were automatically generated by GeneGlobe web portal analysis

Fig. 4

Effect of Cannabis extracts underwent to simulated digestion (Extract A Dig., Extract B Dig.) and cannabinoids (CBD, CBG) on the release of CXCL-10 (a), CCL-20 (b), and CXCL-9 (c) in colonocytes (CaCo-2). Chemokine release was measured by ELISA (24 h). Cells were treated with extracts (100 µg/mL) or pure molecules (8 µM) in addition to inflammatory stimuli (IL-1β/IFN-γ), which value was arbitrarily assigned to 100%. Data are expressed as average (%) ± SEM (n = 3). *p < 0.05; ** p < 0.01; ***p < 0.001 (Kruskal-Wallis test) vs. IL-1β/IFN-γ

Fig. 5

Effect of Cannabis extracts underwent to simulated digestion (Extract A Dig., Extract B Dig.) and cannabinoids (CBD, CBG) on epithelial integrity measured by TEER in enterocytes (differentiated CaCo-2). Cells were treated with extracts (100 µg/mL) or pure molecules (8 µM) for 24–48 h, according to two inflammatory settings, respectively: IL-1β/IFN-γ (a) and LPS/IFN-γ-induced THP-1 co-culture (b). Sodium butyrate 2 mM was used as reference inducer of epithelial integrity (+ 119 Ω and + 219 Ω, respectively). Data are expressed as normalized TEER variation (ΔΩ = Ωt_24/48-Ωt₀). Data from independent experiments (n = 4) were reported as ΔΩ ± SEM vs. stimulus, to which was arbitrarily attributed the value of 0. *p < 0.05; ** p < 0.01; ***p < 0.001 (Kruskal-Wallis test) vs. stimulus

Fig. 6

Effect of Cannabis extracts underwent to simulated digestion (Extract A Dig., Extract B Dig.) and cannabinoids (CBD, CBG) on the release of IL-1β in macrophages (THP-1). IL-1β release was measured by ELISA (24 h). Cells were treated with extracts (10 µg/mL) or pure molecules (0.8 µM) in addition to inflammatory stimuli (LPS/IFN-γ), which value was arbitrarily assigned to 100%. Apigenin 20 µM was used as reference anti-inflammatory compound (-77%). Data are expressed as average (%) ± SEM (n = 3). **p < 0.05, ***p < 0.001 (Kruskal-Wallis test) vs. LPS/IFN-γ

Fig. 7

Effect of Cannabis extracts underwent to simulated digestion (A Dig., B Dig.) and cannabinoids (CBD, CBG) on the expression and organization of ZO-1, measured by immunofluorescence in enterocytes (differentiated CaCo-2). Cells were co-cultured with human macrophages (THP-1) stimulated with LPS/IFN-γ and treated with extracts (100 µg/mL) or pure molecules (8 µM) for 48 h. Representative images (60x magnification, 20 μm bar scale) report cell nuclei (DAPI, blue color) and membrane-associated ZO-1 (red color), in a separate and merged form