Phytocannabinoids Act Synergistically with Non-Steroidal Anti-Inflammatory Drugs Reducing Inflammation in 2D and 3D In Vitro Models

Abstract

Lung inflammation is associated with elevated pro-inflammatory cytokines and chemokines. Treatment with FCBD:std (standard mix of cannabidiol [CBD], cannabigerol [CBG] and tetrahydrocannabivarin [THCV]) leads to a marked reduction in the inflammation of alveolar epithelial cells, but not in macrophages. In the present study, the combined anti-inflammatory effect of FCBD:std with two corticosteroids (dexamethasone and budesonide) and two non-steroidal anti-inflammatory drugs (NSAID; ibuprofen and diclofenac), was examined. Enzyme-linked immunosorbent assay (ELISA) was used to determine protein levels. Gene expression was determined by quantitative real-time PCR. Inhibition of cyclo-oxygenase (COX) activity was determined in vitro. FCBD:std and diclofenac act synergistically, reducing IL-8 levels in macrophages and lung epithelial cells. FCBD:std plus diclofenac also reduced IL-6, IL-8 and CCL2 expression levels in co-cultures of macrophages and lung epithelial cells, in 2D and 3D models. Treatment by FCBD:std and/or NSAID reduced COX-1 and COX-2 gene expression but not their enzymatic activity. FCBD:std and diclofenac exhibit synergistic anti-inflammatory effects on macrophages and lung epithelial cells, yet this combined activity needs to be examined in pre-clinical studies and clinical trials.

Keywords: NSAID; cannabinoids; chemokine; corticosteroids; epithelial cells; inflammation; interleukin; lung; macrophages.

Figure 1

Synergistic interactions between FCBD:std with (a) ibuprofen, (b) budesonide, (c) dexamethasone or (d) diclofenac on KG1 cell IL-8 levels following combined treatments. The Bliss independence-drug interaction model was used to calculate synergy. Synergy is determined when the experimental value of IL-8 reduction is higher than the calculated value. In the Y axis is the delta between the experimental and the calculated values. In Supplementary Table S2, different letters indicate significantly different levels of the experimental (observed) values from all pairs’ combinations according to Tukey–Kramer honest significant difference test (n = 3; HSD; p ≤ 0.05).

Figure 2

Synergistic interactions between FCBD:std with (a) ibuprofen, (b) budesonide, (c) dexamethasone or (d) diclofenac on IL-8 level of A549 cells following combined treatments. The Bliss independence-drug interaction model was used to calculate synergy. Synergy is determined when the experimental value of IL-8 reduction is higher than the calculated value. In the Y axis is the delta between the experimental and the calculated values. In Supplementary Table S2, different letters indicate significantly different levels of the experimental (observed) values from all pairs’ combinations according to Tukey–Kramer honest significant difference test (n = 3; HSD; p ≤ 0.05).

Figure 3

Effect of treatments with FCBD:std and (a) ibuprofen (Ibu), (b) budesonide (Bud), (c) dexamethasone (Dexa) or (d) diclofenac (Diclo) on IL-8 levels in co-cultures of A549 and differentiated KG1 cell lines. Control is vehicle control (1% methanol, 0.5% DMSO, 0.2% methanol + 0.5% DMSO and 1% DMSO for Ibu, Bud, Dexa and Diclo, respectively). TNFα is solvent control + TNFα. Means ± SE (n = 3) are shown. Levels of means with different letters are significantly different from all pairs’ combinations according to Tukey–Kramer honest significant difference (HSD; p ≤ 0.05).

Figure 4

The effect of treatments with FCBD:std and diclofenac (Diclo) or ibuprofen (Ibu) on IL-6 levels in co-cultures of A549 and differentiated KG1 cell lines. Control is vehicle control (1% methanol; 1% DMSO vehicle control is not significantly different from 1% methanol, not shown). TNFα is solvent control + TNFα. Means ± SE (n = 3) are shown. Levels of means with different letters are significantly different from all pairs’ combinations according to Tukey–Kramer honest significant difference (HSD; p ≤ 0.05).

Figure 5

Determination of the RNA steady-state level using quantitative PCR-based in co-culture of A549 and differentiated KG1 cell line of (a) IL-6, (b) IL-8 or (c) CCL2 after treatment for 2 h with FCBD:std or ibuprofen (Ibu) or diclofenac (Diclo) and their combinations, relative to control. Quantitative PCR determined gene transcript values as a ratio between the target gene versus β-actin as a reference gene. Using the 2^ΔΔCt method, values were calculated relative to the average expression of target genes in treated versus control. Control (1% methanol) treatment served as solvent (vehicle) control. TNFα is solvent control+TNFα. Means ± SE (n = 3) are shown. Levels of means with different letters are significantly different from all pairs’ combinations according to Tukey–Kramer honest significant difference (HSD; p ≤ 0.05).

Figure 6

Determination of the RNA steady-state level using quantitative PCR-based in co-culture of A549 and differentiated KG1 cell line of (a) COX-1, (b) COX-2 after treatment with FCBD:std or ibuprofen (Ibu) or diclofenac (Diclo) and their combinations, for 2 h relative to control. Quantitative PCR determined gene transcript values as a ratio between the target gene versus β-actin as a reference gene. Using the 2^ΔΔCt method, values were calculated relative to the average expression of target genes in treated versus control. Control (1% methanol) treatment served as solvent (vehicle) control. TNFα is solvent control+ TNFα. Means ± SE (n = 3) are shown. Levels with different letters are significantly different from all pairs’ combinations by the Tukey–Kramer honest significant difference test (HSD; p ≤ 0.05).

Figure 7

Confocal Images of 3D co-cultures. (a,b) 8-µm PET membrane seeded with A549 cells, angle (a) and side (b) views (Bar = 50 µm); (c) Monolayer of A549, top view (Bar = 50 µm); (d) ECM seeded with macrophages, side view (Bar = 50 µm); (e) Macrophages in ECM, top view (Bar = 50 µm). (f) Ciliated A549 cells on the membrane, arrows point to ciliate structures (Bar = 20 µm). Nuclei are stained by Hoechst (blue stain) and membrane by Dil perchlorate (red stain). Merged includes both nuclei and membrane staining—illustration of the 3D printed model in Supplementary Figure S2.

Figure 8

The effect of treatments with FCBD:std and/or ibuprofen (a,b) and FCBD:std and/or diclofenac (c,d) on IL-8 (a,c) or IL-6 (b,d) levels in a 3D co-culture model of A549 monolayer and differentiated KG1 cell lines in ECM. Control is vehicle control (1% methanol and 1% DMSO for Ibu and Diclo, respectively). TNFα is solvent control + TNFα. Means ± SE (n = 3) are shown. Levels of means with different letters are significantly different from all pairs’ combinations according to the Tukey–Kramer honest significant difference test (HSD; p ≤ 0.05).