Cannabidiol Isolated From Cannabis sativa L. Protects Intestinal Barrier From In Vitro Inflammation and Oxidative Stress

Abstract

The relevance and incidence of intestinal bowel diseases (IBD) have been increasing over the last 50 years and the current therapies are characterized by severe side effects, making essential the development of new strategies that combine efficacy and safety in the management of human IBD. Herbal products are highly considered in research aimed at discovering new approaches for IBD therapy and, among others, Cannabis sativa L. has been traditionally used for centuries as an analgesic and anti-inflammatory remedy also in different gastrointestinal disorders. This study aims to investigate the effects of different C. sativa isolated compounds in an in vitro model of intestinal epithelium. The ability of treatments to modulate markers of intestinal dysfunctions was tested on Caco-2 intestinal cell monolayers. Our results, obtained by evaluation of ROS production, TEER and paracellular permeability measurements and tight junctions evaluation show Cannabidiol as the most promising compound against intestinal inflammatory condition. Cannabidiol is able to inhibit ROS production and restore epithelial permeability during inflammatory and oxidative stress conditions, suggesting its possible application as adjuvant in IBD management.

Keywords: Cannabis sativa; cannabidiol; intestinal barrier dysfunction; intestinal inflammation; intestinal permeability; transepithelial electrical resistance.

GRAPHICAL ABSTRACT

FIGURE 1

HPLC-DAD chromatogram of C. sativa scCO₂ extract. Tentative identification of the main cannabinoids are reported in the Figure. CBDA, cannabidiolic acid; CBGA, cannabigerolic acid; CBD, cannabidiol; CBN, cannabinol; THCA, tetrahydrocannabinolic acid; THC, tetrahydrocannabinol; CBC, cannabichromene. *: identification was confirmed by co-injection with reference standards.

FIGURE 2

Chemical structures of the cannabinoids isolated from C. sativa scCO₂ extract. THC, tetrahydrocannabinol; THCA, tetrahydrocannabinolic acid; CBD, cannabidiol; CBDA, cannabidiolic acid.

FIGURE 3

Effect of a 24 h treatment with THC (A), THCA (B), CBD (C), and CBDA (D) 0.01-0.1-1-10 μg/mL on Caco-2 cells viability. Results are the mean ± SEM of n = 3 experiments and are expressed as percentage of absorbance of treated cells related to control. **p < 0.01 treatment vs. control.

FIGURE 4

Effect of a 24 h treatment with THC (A), THCA (B) CBD (C), CBDA (D) (0.01-0.1-1-10 μg/mL), on Caco-2 ROS production in basal condition. Data are expressed as mean ± SEM of fluorescence intensity (FI) of treated cells related to control. n = 3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 treatment vs. control, Standard ANOVA procedures.

FIGURE 5

Effect of a 24 h treatment with THC (A), THCA (B) CBD (C), CBDA (D) (0.01-0.1-1-10 μg/mL), on Caco-2 ROS production after oxidative simulation with H₂O₂ (E). Data are expressed as mean ± SEM of fluorescence intensity (FI) of treated cells related to control. n = 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 treatment vs. control, unpaired Student’s t-test.

FIGURE 6

Effects of THC (A) and CBD (B) 0.1–1 μg/mL on transepithelial electrical resistance in Caco-2 cells monolayer. Data are expressed as mean ± SEM percentage of baseline TEER value of n = 3–5 experiments, *p < 0.05 treatment vs. control, unpaired Student’s t-test.

FIGURE 7

Effects of THC (B) and CBD (C) 0.1–1 μg/mL on transepithelial electrical resistance in Caco-2 cells monolayer stimulated with H₂O₂ (A). Data are expressed as mean ± SEM percentage of baseline TEER value of n = 3–5 experiments. **p < 0.01 oxidative stimulus vs. control; §p < 0.05; §§p < 0.01 treatment vs. oxidative stimulus, unpaired Student’s t-test.

FIGURE 8

Effects of THC (B) and CBD (C) 0.1–1 μg/mL on transepithelial electrical resistance in Caco-2 cells monolayer stimulated with INFγ+TNFα (A). Data are expressed as mean ± SEM percentage of baseline TEER value of n = 3–5 experiments. *p < 0.05 inflammatory stimulus vs. control; §p < 0.05; §§§p < 0.001 treatment vs. oxidative stimulus, unpaired Student’s t-test.

FIGURE 9

Effects of THC and CBD (0.1–1 μg/mL) on Caco-2 cell monolayers paracellular permeability in basal condition (A), oxidative stress induced by H₂O₂ (B) and inflammatory conditions induced by INFγ-TNFα treatment (C). Data are shown as mean ± SEM percentage of basal fluorescent intensity (n = 3). *p < 0.05; **p < 0.01 treatment vs. control; §p < 0.05 treatment vs. stressor stimulus, unpaired Student’s t-test.

FIGURE 10

Effect of CBD and THC extracts (0.1 and 1 μg/mL) on occludin and ZO-1 expression in Caco-2 cells. (A) Representative images of the effect of CBD-THC treatment on tight junction proteins in Caco-2 cell monolayers. (B) Representative images of the effect of CBD and THC on tight junction proteins in inflammatory conditions (INFγ+TNFα stimulation). Images were collected by confocal laser-scanning microscope LSM800 and software ZEN 2.1, magnification 60X and are representative of three experiments.