Anti-inflammatory effects of oral cannabidiol in rat models

Animals

A total of 48 male Sprague Dawley rats (300-320 g, Nomura Siam International, Thailand) were used. The animals were housed in the Laboratory Animal Canter Thammasat University under standard conditions of strict hygienic conventional temperature (22±1°C), relative humidity (30-70%), light (130-325 Lux), and a light/dark cycle (12 h/12 h). Rats were kept under laboratory conditions for one week prior to the experiments. The protocols and methods used in this study were approved by the Institutional Animal Care and Use Committee of Thammasat University, Thailand (Protocol Approval No. 010/2021).

We complied with the standard practices and guidelines to treat rats respectfully and compassionately. After six hours post-carrageenan injection, rats were sacrificed with isoflurane and confirmed with cardiac puncture. All tissues and blood samples were collected after being sacrificed with isoflurane by veterinarian staff at Animal Laboratory Animal Center, Thammasat University, Thailand. The center employs trained veterinarians knowledgeable about animal behavior and welfare to ensure the sacrifice is conducted efficiently and with minimal stress to the rat.

Chemicals and drugs

Cannabidiol powder was purchased from Suranaree University of Technology, Thailand, and was dissolved in sunflower oil. Lambda-carrageenan, hematoxylin and eosin (H&E) staining solution, absolute ethanol, acetic acid, and ethylenediaminetetraacetic acid were purchased from Sigma-Aldrich, Milano, Italy. Diclofenac and formaldehyde were purchased from Tokyo Chemical Industry, Tokyo, Japan.

Carrageenan-induced edema

Carrageenan-induced edema was conducted using the procedure by Morris (2003).¹⁶ The original protocol of Morris (2003) used a mouse animal model, and indomethacin (Non-steroidal anti-inflammatory drugs; NSAIDs) was used as a positive control in the experiment. However, we modified the Morris (2003) protocol to use a rat animal model and diclofenac (Non-steroidal anti-inflammatory drugs; NSAIDs) as a positive control. Rats were orally administered with either olive oil placebo (olive oil) (10 mg/kg), diclofenac (10 mg/kg), or CBD (5, 10, 20, and 40 mg/kg). Paw edema was induced by injection of 0.1 mL carrageenan (1% w/v in saline) into the plantar of the right hind paw. Paw edema was measured using a plethysmometer (Ugo Basile, Varese, Italy). After carrageenan injection, the volume of paws was recorded at 0, 1, 2, 3, 4, 5, and 6 h. Paw volume was expressed as the difference in volume between before and after carrageenan induces inflammation.

Histopathology analysis

After six hours post-carrageenan injection, rats were sacrificed with isoflurane and confirmed with cardiac puncture. The right hind paw of each rat was cut and fixed in a 10% neutral buffered formalin solution for 12 h. Each sample was decalcified with 10% EDTA solution, dehydrated with ethanol, and embedded in paraffin. Each sample was cut into 2 μm thick sections and stained with H&E. A senior pathologist performed the histologic analysis with a light microscope.

The degree of inflammation was assessed by choosing the area of maximal infiltration with inflammatory cells in each case and grading the density of inflammatory cells into three tier grades 1, 2, and 3. The criteria for grading inflammation started from grade 1, showing sparse inflammatory cells. In contrast, grade 3 shows distinct high cellularity of inflammatory cells, and in grade 2, the number of inflammatory cells lies between these two grades. The assessment was performed blindly without knowing the information regarding each specimen.

Blood and tissue samples preparation

Plasma samples were used for serotonin, prostaglandin E₂, chemokine, and cytokine assays, whereas tissue samples were used for cyclooxygenase activity assay. Samples were collected after rats were euthanized using isoflurane. Blood samples (6 mL) were collected in EDTA K2/gel tubes (BD Vacutainer BD Diagnostic, Milan, Italy) via cardiac puncture. Plasma samples were collected by centrifugation for 5 min at 10,000 revolutions per minute. The rats’ plasma samples were stored at -20°C until used. For tissue samples preparation, samples were prepared according to the COX activity assay kit instructions. Briefly, the claws of rat paws were cut and then washed three times with phosphate-buffered saline buffer (PBS). Tissue samples were ground in liquid nitrogen and then homogenized in a lysis buffer with protease inhibitors using a pestle. The supernatant was collected after centrifugation at 12,000×g for 3 min at 4°C.

Serotonin assay

Serotonin levels in serum samples were measured by a competitive enzyme-linked immunosorbent assay (ELISA) using a commercial test kit (Abcam, UK #ab133053) following the manufacturer’s instruction. Briefly, serum samples or serotonin standards were prepared and mixed into an alkaline phosphatase (AP)-conjugated serotonin solution. A specific antibody to serotonin was then added to each well of the 96-well plate, which was pre-coated with goat anti-rabbit antibody. A mixture solution was incubated at room temperature (RT) for two hours with plate shaking before washing three times with washing buffer. A substrate solution for AP (n p-nitrophenyl phosphate disodium salt; pNpp) was added and incubated for one hour. Followed by a stop reaction step by adding a stop solution before measuring absorbance at 405 nm using a microplate reader (SpectraMax M5 microplate reader, Molecular device). The concentrations of serotonin in samples were calculated from the standard curve.

Prostaglandin E₂ (PGE₂) assay

Prostaglandin E₂ in serum samples was tested using a competitive ELISA kit (Abcam, UK, #ab133021). Briefly, samples or standards were mixed with alkaline phosphatase-conjugated PGE₂ in each well of the 96-well plate. A specific antibody to PGE₂ was added and incubated at RT for two hours with shaking. Then, the plate was washed with washing solution three times. An enzyme (pNpp substrate) was added and incubated for 45 min before stopping the reaction with the kit’s stop solution reagent. Absorbance at 405 nm was measured using a microplate reader. The concentrations of PGE₂ in samples were calculated from the standard curve.

Cytokines and chemokine level

To understand an immune response after carrageenan-induced edema, serum 14 cytokines (namely G-CDF/CSF-3, GM-CSF, IFN-gamma, IL-10, IL-12p70, IL-13, IL-17A, IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, and TNF-alpha) and eight chemokines (namely Eotaxin, Gro-alpha/KC, IP-10, MCP-1, MCP-3, MIP-1alpha, MIP-2, and RANTES) were measured using the commercial bead array assay (ThermoFisher Scientific, #EPX220-30122-901). Samples were prepared and tested according to manual instructions. Briefly, samples or standards were added to each well of the 96-well plate. Then, a mixture of antibodies-coated beads was added and incubated at RT for two hours. After incubation, the unbound beads were removed, and beads were washed twice with a washing buffer. Next, a rat anti-cytokine/chemokine detecting antibodies cocktail was added. The reaction was incubated at RT for one hour. The washing was repeated before phycoerythrin-conjugated streptavidin was added and incubated for 30 min. Then, beads were washed twice before readout the signal using a MAGPIX detector (Luminex, Austin, TX).

COX-1 and COX-2 activity assay

The cyclooxygenase (COX) activity in tissue serum samples was quantitated using a commercial test kit (Abcam, UK, #ab204699). Samples were tested following the manufacturer’s instructions. Briefly, samples were mixed with a COX probe and COX factor. Then, the mixture solution was added to celecoxib or SC560 solution to measure COX-1 or COX-2 activity, respectively. After that, the arachidonic acid solution was added to each mixture solution and immediately measured fluorescence (Excitation at 535 nm and Emission at 587 nm) in a kinetic mode (15-s interval, 30 min). The protein concentration of each sample was measured by a Bradford protein assay method. The amount of resorufin in samples was calculated from the standard curve. The COX reactivities were calculated from the amount of resorufin per reaction time and the amount of protein in the sample. The COX reactivity was reported in pmol/min/mg or μU/mg.

Statistical analysis

Each group was constituted of at least eight rats. All results were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM) using Graphpad Prism version 9 (Graphpad Software, San Diego, CA). The experiments were compared using analysis of variance (ANOVA) and Tukey’s post hoc test for multiple comparisons. The correlation between data sets was analyzed using the Spearman r-test and plotted by simple linear regression. The Mann-Whitney U test used analysis of the variance. A p-value of < 0.05 was considered to indicate a statistically significant difference.

Cyclooxygenase (COX) activity, prostaglandin E₂ (PGE₂), and serotonin levels

The COX enzymes, a catalyzer for converting arachidonic acid to prostaglandin, have two isoforms: COX-1 and COX-2. The enzymes also are a common target for anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs).¹⁸ To determine COX activity, after six hours of the carrageenan induction, tissue samples from oral CBD (40 mg/kg) and control groups were collected, and COX-1 and COX-2 levels were determined by the ELISA method. The results showed that COX-1 (Figure 4A) and COX-2 (Figure 4B) activity were not significantly different among placebo, diclofenac, and the treated-CBD groups. The COX-1 and COX-2 activity results agreed with the level of PGE₂ (Figure 4C). Serotonin, a pro-inflammatory-like neurotransmitter,¹⁹ in serum isolated from the same group of samples and controls was also measured after six hours of carrageenan injection. Serotonin levels in the diclofenac and oral CBD (40 mg/kg) groups were significantly lower than the placebo (p < 0.01; p < 0.001), respectively. Moreover, the results between the diclofenac and oral CBD groups were not different, indicating that the anti-inflammatory efficacy of CBD is probably equal to that of diclofenac (Figure 4D).

Figure 4. COX activity, PGE₂, and serotonin levels in different interventions, including Placebo (10 mg/kg), Diclofenac (10 mg/kg), and oral CBD (40 mg/kg), were measured by ELISA at six hours after carrageenan injection.

One-way ANOVA tested these analyses of variance following the Tukey post hoc test (**p < 0.01; ***p < 0.001, ns; not significant).

Serum cytokine and chemokine levels

The level of serum cytokines and chemokines from rats that received different interventions, including placebo, diclofenac, and oral CBD (40 mg/kg), were determined using a bead array technique. As the results (Figure 5), four out of 18 cytokines and chemokines showed significantly different levels between CBD and either placebo or Diclofenac which were MCP-1 (Figure 5N), MCP-3 (Figure 5O), MIP-2 (Figure 5Q), and RANTES (Figure 5R). The others were not significantly different between the compared groups. However, the IL-17A level of the CBD group had a trend of lowered IL-17A when compared with placebo (Figure 5I).

Figure 5. Serum cytokine and chemokine levels in different interventions, including; Placebo, Diclofenac (10 mg/kg), and oral CBD (40 mg/kg: CBD OR).

Those serum cytokines and chemokines were collected from the cardiac at six hours and detected by bead-based flow cytometry. The Mann performed comparisons between the groups–Whitney U test, p-value < 0.05 would be considered statistical significance.