Phytocannabinoids are secondary metabolites of
The following reagents were used for conducting the experiments: phytocannabinoid extract as Rich Hemp Oil/THC-free with CBD content of 887.17 mg/g/ (residual phytocannabinoids: CBG < 0.3 mg/g, CBN < 0.3 mg/g, CBC < 0.3 mg/g and CBD-A < 0.3 mg/g - Folium Biosciences, Weesp, the Netherlands); acetylcholine chloride, water soluble dexamethasone and diclofenac sodium salt (Sigma Chemicals Co, St. Louis, MO, USA); CaCl2, (Merck, Darmstadt, Germany); NaH2PO4 (Fluka Chemie, Buchs, Switzerland); and NaCl, KCl, MgSO4, NaHCO3 and glucose (Avantor, Gliwice, Poland). The incubation of the strips was conducted in modified Krebs–Henseleit Solution (MK-HS) containing NaCl (123.76 mM), KCl (5 mM), CaCl2 (2.5 mM), MgSO4 (1.156 mM), NaHCO3 (14.5 mM), KH2PO4 (2.75 mM) and glucose (12.5 mM) at 37°C and in a constant pH range (7.35–7.45) maintained by carbogen bubbling (95% O2 + 5% CO2). The CBD was dissolved in dimethyl sulfoxide and used at a solvent concentration which did not influence the spontaneous activity and reactivity of the strips (0.5%).
The tissues were isolated from male Wistar rats weighing approximately 250 g obtained from the Center for Experimental Medicine of the Medical University of Białystok, a registered laboratory animal breeder. The animals were euthanised using sedation by gradual introduction of carbon dioxide into the animal holding chamber and cervical dislocation. The procedure was carried out according to the current regulations and guidelines of the National Ethics Committee and complied with Annex IV to Directive 2010/63/EU on the protection of animals used for scientific purposes. Since all research activities were carried out post mortem, no Local Ethics Committee approval was required for the experiment.
Directly after opening the abdominal cavity, fragments of the descending colon were excised and placed in MK-HS at 37°C. After removing the digestive content by gentle washing of the intestinal lumen, the surrounding tissues were dissected. Next the strips were prepared in such a way as to be approximately 15 mm long and in the physiological tubular shape.
The colon preparations were incubated in the chambers of a Schuler Organ Bath set (Hugo-Sachs Elektronik Harvard Apparatus, March-Hugstetten, Germany) in isometric conditions under a load of 0.01 N. The registration of the data was performed through a force transducer and bridge amplifier (DBA, F30 type 372, Hugo-Sachs Elektronik) and PowerLab data acquisition system (ADInstruments, Dunedin, New Zealand). The graphical records were analysed in the Chart v7.0 and LabChart Reader v8.1.1 programmes (ADInstruments) and Excel for Windows XP (Microsoft, Redmond, WA, USA).
After placing the colon strips in the organ bath chambers, they were preincubated for 90 min to stabilise muscle activity in
The reaction of colon strips to ACh was established as the change of muscle tension from its state before to its state after application of the substances. The strength of the muscle contraction produced by ACh in the presence of CBD, DCF, DEX and a combination of CBD and DEX or DCF was related to the muscle reaction to ACh acting alone (this control being defined as 100%). The obtained graphical data were calculated as area under the curve. The data are expressed as mean values (n = 5) ± standard deviation. In the statistical analysis, a one-way analysis of variance with post-hoc Tukey’s test was used to compare the mean values between the investigated groups and one sample
Before starting the planned experiment, the effective concentration of all tested substances was determined in separate preliminary experiments in which the dose-effect relationship was tested in terms of the effect on intestinal motility. The effective concentrations were as follows: ACh 1 μM, CBD 80 μM, DCF 100 μM and DEX 100 μM.
When administered at the beginning of the experiment proper, acetylcholine caused a strong intestinal contraction. After changing the buffer solution in the incubation chamber (flushing), the intestine displayed spontaneous contractile activity. Applying CBD followed by ACh caused a statistically significantly weaker contraction than that observed at the start of the incubation. The area under the curve for the intestinal contraction in the presence of CBD was 26.5 ± 8.6% of the contraction at the beginning of the incubation. Fig. 1 shows an example of intestinal motility changes observed after administration of ACh and CBD.
Diclofenac slightly attenuated the contractile response of the intestine to ACh administration. The area under the curve for the intestinal contraction caused by ACh in the presence of DCF was 80 ± 17.6% of the response observed at the start of the incubation, which did not differ statistically significantly from the area under the curve of contraction caused by the administration of ACh alone. Administration of CBD and DCF in the second phase of the third treatment scheme almost completely suppressed the contractile response to ACh. The area under the curve for this contraction was 5.1 ± 11% of the response observed at the start of the incubation. Fig. 2 shows an example of changes in spontaneous intestinal contractility after administration of DCF and CBD.
When administered at the beginning of the incubation, ACh caused a strong intestinal contraction. After changing the buffer solution and administering DEX, the intestine displayed spontaneous contractile activity, which intensified throughout the time of incubation. Administration of ACh in the presence of DEX caused a statistically significantly larger contraction than that observed at the beginning of the incubation. The area under the curve for this contraction is 121.9 ± 18.7% of the initial contraction. After changing the medium and administering DEX and CBD, the ACh-induced contraction was statistically significantly smaller than the initial contraction, being 67.6 ± 16.8% of that. Fig. 3 shows representative changes in intestinal motility after administering DEX and CBD.
Diclofenac inhibited and DEX stimulated ACh-induced intestinal contractions to respective 80.0 ± 17.6% and 121.9 ± 18.7% of the control baseline level (Fig. 4 a). The presence of CBD in the incubation buffer solution always diminished the intestinal contractile response to ACh. Independent administration of CBD caused intestinal contractile response to drop to 26.5 + 8.6% of the contraction induced by ACh alone. In the presence of DCF, CBD caused the strongest inhibition of intestinal contractile response to ACh. In this case the contraction was only 5.1 ± 11.3% of the control value. In the presence of CBD and DEX, ACh caused contractions of 67.6 ± 16.8% of the ACh-only contraction. This inhibition was significantly smaller than the inhibition caused by CBD alone. Fig. 4 b compares the intestinal response to administration of CBD, CBD + DCF and CBD + DEX.
Despite the interest in cannabinoids, knowledge of how they influence the gastrointestinal tract is still incomplete. More and more evidence suggests that the endocannabinoid system is crucial in modulating gastrointestinal physiology, influencing satiety, immune function, secular secretion, visceral sensation, vomiting (inhibitively), mucosal integrity and gastrointestinal motility (11). Due to the increasing availability of cannabis products, including high-purity CBD oils, CBD is becoming commonly used for the purposes of reducing anxiety, pain and inflammation. A group of drugs with a similar use exploiting their analgaesic and anti-inflammatory properties are NSAIDs. Both CBD and NSAIDs are often used outside of medical supervision, which may make their concurrent usage likely. Simultaneous use of CBD and steroid drugs is also probable. The potential for indiscriminate use of these therapeutics in combination makes it important to understand the interactions of CBD with these two classes of anti-inflammatory drugs. Commonly used examples of steroidal and nonsteroidal preparations were selected for study.
Cannabidiol inhibited spontaneous intestinal motor activity and attenuated the intestinal response to ACh to 26.6% of the control value. This finding is consistent with the research by Layman and Milton, which described an attenuation of intestinal response to acetylcholine in the guinea pig colon after administration of CBD (3). Izzo
In this study it was observed that DEX enhanced spontaneous intestinal motor activity and strengthened ACh-induced colonic contraction. The short duration of the experiment and rapid effect make the assumption safe that that the mechanism of action of DEX was not related to transcription-level events characteristic of glucocorticoids. The observed effect must stem from transcription-independent mechanisms. The rapid nongenomic effects of glucocorticoids may be connected to the activation of the protein kinase C and phosphoinositide signalling system (21), their effect on calcium availability for contractile proteins (13), and many other mechanisms, including the activation of mitogen-activated protein kinase and adenyl cyclase (12). Many fast-acting effects of glucocorticoids are related to the limiting of cytokine activity (6). Enhanced contractility may be due to an increase of calcium-sensitivity of contraction (14). The above mechanisms cannot be ruled out as causes for the increased intestinal motility in the experiment.
Interestingly, the rapid onset of action of glucocorticoids can also be explained by their inhibition of cytosol phospholipase A2 and subsequent diminished arachidonic acid release (6). Such a mechanism is very probable in this experiment, because enhancement of intestinal contractile activity due to increased calcium-sensitivity of contraction is much faster than the gradual process which was observed after administration of DEX. On the basis of these data, it might be suggested that the inhibition of arachidonic acid release by DEX blocks the synthesis of endogenous cannabinoids. Thus, the component inhibiting intestinal motility is switched off. When DEX is co-administered with CBD, this component is bolstered by CBD.
The presented experiment did not find any significant changes in spontaneous intestinal contractility after the administration of DCF; however, it clearly attenuated intestinal response to ACh and enhanced the inhibitory effect of CBD. The mechanism underlying this phenomenon is difficult to explain. As an NSAID, DCF works through inhibiting cyclooxygenase (COX) activity, thereby decreasing prostaglandin concentrations. In humans, it has been observed that DCF does not affect upper gastrointestinal tract motility (1). Likewise, indomethacin (a drug from the NSAID group) does not affect gastric emptying in humans (2). Contrasting observations in dogs suggest that indomethacin may stimulate intestinal motility and delay gastric emptying. This effect was not observed with meloxicam (also a drug from the NSAID group) (16). Based on their observations, Gustafsson
In the experiment described in this article, DCF was not found to enhance intestinal contractility, and in fact was found to decrease the contractile response to ACh. This suggests that changes in intestinal motility induced by NSAIDs may be varied and contingent on many factors, such as the basal activity of prostaglandin cocktails, and may exert a multitude of effects on the organism. It can be assumed that the mechanism of action of DCF in this experiment is related to COX inhibition and lowered concentrations of prostaglandins stimulating gastrointestinal motility. It is worth noting that COX inhibition causes a decrease in arachidonic acid utilisation for prostaglandin synthesis, thus making more acid available for the synthesis of leukotrienes or endogenous cannabinoids. Since leukotrienes are associated with smooth muscle contractions (20), it can be surmised that the presence of DCF mainly causes an increase in endogenous cannabinoid synthesis, which has an inhibitory effect on gastrointestinal tract contractility. It is also worth noting that in this experiment, intestinal motility inhibition after co-administration of CBD and DCF (a lowering of contractile activity by 95% of the control value) was equal to the sum of inhibition produced by CBD (a 74% drop relative to the control value) and DCF (a 20% drop relative to the control value). This is an example of additive synergism, which is most often observed in co-administration of drugs with identical mechanisms of action. Because CBD’s mode of action originates in the stimulation of transmission specific to endogenous cannabinoids (CB receptor agonism), DCF’s mechanism of action should be similar.
The obtained results indicate that DCF enhances the inhibitory effect of CBD on gastrointestinal tract motility. This could imply that most NSAIDs will exert a similar effect. The interaction discovered is significant for the practical therapeutic use of CBD and NSAIDs, because strong inhibition of gastrointestinal motility is usually deleterious to the organism. It suggests that the observed interactions are related to the synthesis of endogenous cannabinoids, which must play a significant role in the regulation of colonic motility in the rat.