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Anti-Inflammatory Activity in Colon Models Is Derived from Δ9-Tetrahydrocannabinolic Acid That Interacts with Additional Compounds in CannabisExtracts

Published in Volume: 2 Issue 1: July 1, 2017

Rameshprabu Nallathambi,1,† Moran Mazuz,1,† Aurel Ion,1 Gopinath Selvaraj,1 Smadar Weininger,1 Marcelo Fridlender,1 Ahmad Nasser,1 Oded Sagee,2 Puja Kumari,1 Diana Nemichenizer,1,3 Maayan Mendelovitz,1,3 Nave Firstein,4 Orly Hanin,4 Fred Konikoff,4,5 Yoram Kapulnik,1 Timna Naftali,4,5 and Hinanit Koltai1,*
1Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel.
2PLANTEX, Rishon LeZion, Israel.
3The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
4Department of Gastroenrterology and Hepatology, Meir Medical Center, Kfar Saba, Israel.
5Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
†The first two authors contributed equally.

*Address correspondence to: Hinanit Koltai, PhD, Agricultural Research Organization, Volcani Center, Rishon LeZion, Bet Dagan 7528809, Israel, E-mail: hkoltai@agri.gov.il
© Rameshprabu Nallathambi et al. 2017; Published by Mary Ann Liebert, Inc. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Introduction: Inflammatory bowel diseases (IBDs) include Crohn's disease, and ulcerative colitis. Cannabis sativapreparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in detail with in vitro and ex vivo colon models.

Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.

Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the anti-inflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner. Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell culture and colon tissue.

Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.


Inflammatory bowel diseases (IBDs), Crohn's disease (CD), and ulcerative colitis (UC) are characterized by chronic intestinal inflammation. Both diseases are chronic, relapsing, and associated with genetic predisposing backgrounds. Their onset and reactivation are triggered by environmental factors that transiently break the mucosal barrier. This may alter the balance between beneficial and pathogenic enteric bacteria and consequently stimulate immune responses. Both CD and UC patients have activated innate (macrophage, neutrophil) and acquired (T and B cell) immune responses (e.g., Sartor1).

Epithelial cells in the gastrointestinal (GI) tract act as barriers against the intrusion of potentially deleterious luminal substances and microorganisms from the intestinal lumen, and play an important role in inflammatory responses. They express a variety of proinflammatory cytokines, which are upregulated in IBD patients.2 Therapies aimed at downregulating intestinal inflammation utilize both mediator-specific and nonspecific immune suppression, but with potentially considerable side effects.3

Different preparations of marijuana (Cannabis sativa) have been used for the treatment of GI problems, such as GI pain, gastroenteritis, and diarrhea.4,5 C. sativa contains more than 60 terpenophenolic compounds termed phytocannabinoids (reviewed by Aizpurua-Olaizola et al.6). Of these, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), which were discovered about 50 years ago, have been defined as the most active.7–9 Also, it was suggested that combination that exists in the whole extract is more active than a single compound solely (e.g., Russo and Taming10). Cannabinoids have been previously shown to be immune modulators. They shift the balance of pro- and anti-inflammatory cytokines and act to suppress cell-mediated immunity in different physiological systems.11 For example, Δ9-tetrahydrocannabivarin (THCV) was demonstrated to inhibit nitrite production in macrophages and thereby to play an immunomodulatory role.12

Phytocannabinoids have been shown to exert their anti-inflammatory functions on the GI tract by activating receptors that are part of the endocannabinoid system, mainly the G-protein-coupled cannabinoid receptor type 2 (CB2).4,13 In the human colonic epithelial cell line HT29, a number of cannabinoid receptor agonists and antagonists, including the plant-derived THC, have been shown to inhibit tumor necrosis factor alpha (TNFα)-induced interleukin-8 (IL-8) release. This inhibition was antagonized by a CB2 receptor antagonist.13 Later, a third cannabinoid receptor, GPR55, was identified and found to affect GI inflammation.14,15 Cannabinoids have been shown to be effective in a mouse model of colitis.16 Cannabinoids have also been shown to promote wound healing in the GI tract via activation of cannabinoid type 1 (CB1) receptor.17 In addition, we have recently reported clinical data from IBD patients. In a retrospective study, we interviewed 30 CD patients who were licensed to use medical Cannabis,18 while in a prospective trial we randomized 20 CD patients to receive either Cannabis or placebo for their IBD.19 Both revealed beneficial effects.

Cannabis sativa extracts contain hundreds of different compounds. The activity of many synthetic or isolated cannabinoids and their receptor agonists or antagonists has been investigated and verified. However, there seems to be an advantage of the unrefined content of the flower extract versus an isolated compound in IBD. For example, standardized C. sativa extract with high content of CBD given after the inflammatory insult was shown in an animal model of GI inflammation to attenuate injury and motility, further supporting the rationale of combining CBD with other Cannabis compounds.20 Also, in three models of seizure, Cannabis-derived botanical drug substances exerted significant anticonvulsant effects and were of similar efficacy with purified cannabidivarin.21

Since we could not find any detailed characterization of the anti-inflammatory activity of the whole C. sativa extract on both colonic epithelial cells and tissue derived from IBD patient colon, we decided to study the anti-inflammatory activity of C. sativa extracts on these models.

IL-8 was previously shown to be an indicator for the level of IBD-related inflammation in both cell models and in IBD patients (e.g., Refs.13,22–25). Therefore, it was chosen in the present study as the main marker for IBD-related inflammation in cell and colon tissues.

Here we show that the anti-inflammatory activity of Cannabis extracts on colon cells derived from Δ9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7), while a combination of all fractions of Cannabis extract exerts an increased cytotoxic activity. Activity of the extract and the most active fraction was verified on colon tissue taken from IBD patients, and these were shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell culture and colon tissue.


Extraction of Cannabis
Fresh flowers of C. sativa strain AD were harvested from plants. They were either taken immediately for extraction and frozen at −80°C, or baked for 3 h at 150°C before extraction. Fresh and baked Cannabis flowers (2 g) were pulverized with liquid nitrogen and ground into fine powder. Absolute ethanol was added to each tube containing the powder at a sample-to-absolute ethanol ratio of 1:4 (w/v). The tubes were mixed thoroughly on a shaker for 30 min and then the extract was filtered through a filter paper. The filtrate was transferred to new tubes. The solvent was evaporated with a vacuum evaporator. The dried extract was resuspended in 1 mL of absolute methanol and filtered through a 0.45-μm syringe filter. The resuspended extract was diluted as to concentrations indicated in the Resultssection for each of the experiments and used for the treatment of cell cultures and biopsies in enzyme-linked immunosorbent assay (ELISA) experiments. The crude weight per milliliter for each extract was determined by drying 1 mL of the resuspended and filtered extract.

Chemical characterization
Standard preparation
The cannabinoid standards cannabigerol (CBG), CBD, cannabidiolic acid (CBDA), cannabinol (CBN), cannabigerolic acid (CBGA), THC, cannabichromene (CBC), and THCA were diluted to 10 ppm concentration with methanol and then subjected to high-performance liquid chromatography (HPLC) separation. For quantification of THC and THCA, the standards were dissolved in methanol at different concentrations from 5 to 40 ppm.

Sample preparation
For HPLC, the dry extract was resuspended in 1 mL methanol and filtered through a 0.45-μm syringe filter (Merck, Darmstadt, Germany). The filtered extract was diluted 10 times with methanol and then separated by HPLC.

HPLC separation
Sample separation was carried out in an UltiMate 3000 HPLC system coupled with WPS-3000(T) autosampler, HPG-3400 pump, and DAD-300 detector. The separation was performed on a Purospher RP-18 end capped column (250 mm×4.6 mm I.D.; Merck KGaA, Darmstadt, Germany) with a guard column (4 mm×4 mm I.D.). Solvent gradients were formed by isocratic proportion with 15% solvent A (0.1% acetic acid in water) and 85% solvent B (methanol) at a flow rate of 1.5 mL/min for 35 min. The compound peaks were detected at 220, 240, and 280 nm. The 220-nm peaks were taken for further processing. The extracts were fractionated into nine fractions according to the obtained chromatogram.

Mass spectrometry analysis
Analysis of the fractions was carried out using electrospray ionization (ESI) (quadrupole time-of-flight) 6545 (high resolution; Agilent). The mass spectrometry (MS) conditions were as follows: ESI-positive mode, m/z 50–1500, gas temperature 350°C, injection volume 5 μL, solvent composition 0.1% formic acid in water (46%), acetonitrile (50%), and water (4%; v/v).

Nuclear magnetic resonance analysis
1H and 13C spectra were recorded in a Bruker Avance-400 instrument (400.1 and 100.6 MHz, respectively) in CDCl3as the solvent, containing tetramethylsilane as an internal reference, at 300 K. In addition, three 2D experiments were performed: COSY (1H–1H correlation), HMQC (one-bond 1H–13C correlation), and HMBC (long-range 1H–13C correlation).

Determination of anti-inflammation and cytotoxic activities in HCT116, HT29 and CaCO2 cells
HCT116 (ATCC CCL-247), HT29 (ATCC HTB-38), and CaCO2 (ATCC HTB-37) colon cells were grown at 37°C in a humidified 5% CO2–95% air atmosphere. Cells were maintained in McCoy's 5a modified medium (HCT116 and HT29) and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (CaCO2). Cells were seeded, in triplicate, into a 24-well plate at a concentration of 50,000 cells per well in 500 μL of growing media and then incubated for 24 h at 37°C in a humidified 5% CO2–95% air atmosphere. When cell excitation was performed with TNFα (300 ng/mL recombinant human TNFα (PeproTech, Rocky Hill, NJ), cultures in each well were treated along with 50 μL plant extract, as described above. The supernatant was taken and the levels of IL-813 were measured 4 h after treatment using the commercial Human CXCL8/IL-8 DuoSet ELISA kit (R&D Systems, Minneapolis, MN). As a positive control, dexamethasone (Sigma-Aldrich, St. Louis, MO) at 200 and 400 μM final concentrations was used. The maximum involvement of the receptors (CB1, CB2, and GPR55) was examined by treating the cells with 20 μM of the CB1 receptor antagonist/inverse agonist rimonabant (Abcam, Cambridge, MA), CB2 receptor antagonist/inverse agonist SR144528 (Abcam), and GPR55 antagonist/inverse agonist CID16020046 (Sigma-Aldrich, Buchs, Switzerland). The whole extract from fresh flowers (C2F) or the active fraction (F7) was applied to cells along with TNFα 1 h after the antagonist treatment. Anti-inflammatory and cytotoxic activity of CBD (Restek, PA) was performed at different concentrations (16–252 μg/mL) on all three cell lines. Resazurin (R&D Systems) was used to check the cytotoxic effect of extracts. For this, 10% resazurin was added to each well of the treatments with different dilutions. Then, the plate was incubated for 2 h at 37°C in a humidified 5% CO2–95% air atmosphere. Supernatant (100 μL from each well) was transferred to a 96-well plate and the relative fluorescence at the excitation/emission of 544/590 nm was measured. Values were calculated as percentage of live cells relative to the nontreated (cells without TNFα and treatments) control after reducing autofluorescence of Alamar Blue without cells. For dose–response assays, data points were connected by nonlinear regression lines of the sigmoidal dose–response relationship. GraphPad Prism (version 6 for windows; GraphPad Software, Inc. San Diego) was used to produce dose–response curves and IC50 doses were calculated using nonlinear regression analysis.

Culture of biopsies
Three biopsies from both healthy and inflamed intestine of IBD patients were obtained from 29 patients with either CD or UC scheduled for colonoscopy as deemed necessary by their physician, Helsinki approval no. 0094-16. After obtaining informed consent, biopsies from inflamed and normal tissue were taken and placed in tissue culture media. On receiving the biopsies, phosphate-buffered saline was replaced with 75 μL dispase (StemCell Technologies, Cambridge, United Kingdom) and 150 μL collagenase 1A (StemCell Technologies) solution. Tubes were then incubated at 37°C for 1 h. After incubation, the tubes containing the biopsies were centrifuged at 8000 rpm (11,885 g) for 1 min. Then, the supernatant was removed and tissues were washed three times with Hank's balanced salt solution. After each wash, tubes were centrifuged as described above. Then, the tissues were placed on a small Petri dish and cut into 2–3 pieces with a clean scalpel. The pieces were then placed on Millicell hydrophilic polytetrafluoroethylene tissue culture inserts (Millipore, 30 mm, 0.4 μm). The inserts were placed in six-well plastic tissue culture dishes (Costar 3506) along with 1.5 mL of tissue culture medium (DMEM supplemented with 10% v/v heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL leupeptin, 1 mM phenylmethanesulfonylfluoride, and 50 μg/mL soybean trypsin inhibitor). When dexamethasone was included in the media, its concentration was 200 μg/mL. This was followed by treating the tissues with extracts as mentioned above, or leaving them untreated (control). The supernatants were taken after overnight incubation and used for determination of IL-8 and IL-6 cytokine profile by measuring the levels with a commercial ELISA kit. Levels of cytokines from inflamed, Cannabis-treated, and nontreated tissue were compared.

Quantitative real-time PCR
Cells were seeded into a six-well plate at a concentration of 1,500,000 cell/mL per well. After 24 h of incubation at 37°C in a humidified 5% CO2–95% air atmosphere, cells were treated with TNFα (final concentration of 1 ng/mL) and incubated overnight under the same conditions. Nontreated cells or cells treated only with TNFα served as negative and positive controls, respectively. Cells were then reincubated for 5 h with C2F (200 μg crude dry extract/mL) or F7 (from 200 μg crude dry extract/mL of C2F) at 37°C in a humidified 5% CO2–95% air atmosphere. The next day, cells were harvested and total RNA was extracted using TRI reagent (Sigma-Aldrich) according to the manufacturer's protocol. For biopsies, all treatments were added overnight, after which the biopsies were stored at −20°C in RNA Save solution (Biological Industries, Beit Haemek, Israel). RNA was extracted from frozen biopsies. Tissue samples were homogenized with an appropriate homogenizer in TRI reagent, as done for the cells. RNA (50 ng for biopsies: four UC patients and one CD patient) or 2.5 μg (for cells) was reverse-transcribed in a total volume of 20 μL using Maxima reverse transcriptase (Thermo Scientific, Boston, MA) according to the manufacturer's protocol. All primers were designed using Primer3Plus software. PCR was performed in triplicate using a Rotor-Gene 6000 instrument (Qiagen, Zurich, Switzerland) and Maxima SyGreen Mix (Thermo Scientific) according to the manufacturer's protocol. The expression of each target gene was normalized to the expression of GAPDH mRNA in the 2ΔΔCt and is presented as the ratio of the target gene to GAPDH mRNA, expressed as 2ΔCt, where Ct is the threshold cycle and ΔCt=(Ct Target gene − Ct GAPDH). Experiments were repeated three times. The primers were as follows: for COX2 (forward) 5′-ATTGACCAGAGCAGGCAGAT-3′ and (reverse) 5′-CAGGATACAGCTCCACAGCA-3′, and for MMP9 (forward) 5′-TTGACAGCGACAAGAAGTGG-3′ and (reverse) 5′-TCACGTCGTCCTTATGCAAG-3′.

Statistical analyses
Results are presented as mean±standard error of replicate analyses and are either representative of or include at least two independent experiments. Means of replicates were subjected to statistical analyses by the Tukey–Kramer test (p≤0.05) using the JMP statistical package and considered significant when p≤0.05.


C. sativa extracts from fresh flowers are highly active in reducing inflammation in colon cell lines

Anti-inflammation activity was determined for absolute ethanol extracts of fresh (C2F) and baked (C2B) flowers of C. sativa (Cs-AD var.). The activity was determined as the level of IL-8 in HCT116 colon cancer cell cultures pretreated with TNFα to induce IL-8 expression and then treated with C2F or C2B (Fig. 1A). Notably, IL-8 was used in several other studies, in HCT116 as well as other cell models and in IBD patients as an indicator for the level of IBD-related inflammation (e.g., Refs.13,22–25). At different concentrations of C2F and C2B (114–207 μg/mL), extracts significantly reduced IL8 levels when compared to TNFα. Under these conditions, dexamethasone (at concentrations of 200 and 400 μM) was inactive in reducing IL-8 level (Fig. 1A).

To determine that the reduction in IL-8 is due to anti-inflammatory rather than cytotoxic effect, cell viability was examined for the C2F and C2B treatments at different concentrations. At most examined concentrations C2B had significant cytotoxic activity, whereas C2F did not (Supplementary Figs. S1 and S2); slightly increased cytotoxicity at the higher concentration was determined for C2F in the CaCO2 cell line (Supplementary Fig. S2). Taken together, results demonstrated the strong, dose-dependent, anti-inflammation activity of C. sativa C2F, mostly absent in C2B.

Chemical composition of C. sativa extracts from fresh and baked flowers
HPLC chromatogram and main active compounds were determined for the anti-inflammation active extract, C2F, and for the inactive extract, C2B (Fig. 2; Table 1). Eight major cannabinoids were identified in the fresh and baked crude extracts at 220 nm. These peaks were identified as CBG, CBD, CBDA, CBN, CBGA, THC, CBC, and THCA, with retention times of 5.9, 6.4, 7.9, 10.9, 11.3, 13.1, 17.5, and 29.3 min, respectively, relative to the HPLC profile of cannabinoid standards (not shown). The levels of CBD, CBN, and THC were 36, 14, and 32 times higher in the C2B versus C2F extract. CBC was not identified in C2F but appeared in C2B. The levels of THCA and CBDA in C2B were reduced 1200 and 1.5 times, respectively, compared to those in C2F (Fig. 2; Table 1), due to decarboxylation of CBDA and THCA during heating (e.g., Smith and Vaughan26).

Identification of an active fraction of the fresh flower extract of C. sativa and determination of activity of combinations of fractions

C2F (at a concentration of 163 μg/mL) was fractionated (Supplementary Figs. S1 and S2).

Next, F1–F9 pool-excluding F7 and combined treatment of all fractions, including F7, at different concentrations, were examined on HCT116 cells for anti-inflammatory and cytotoxic activities (Fig. 4). As expected, F7 and C2F had similar anti-inflammatory activity, whereas F1–F9-excluding F7 treatment were inactive. However, once F7 was added to F1–F9-excluding F7 treatment, anti-inflammatory activity was retained (Fig. 4A). As for the cytotoxic activity, a marked induction of cytotoxicity was found for combined treatment of F1–F9-excluding F7 and addition of F7 at concentrations of 190 and 190 μg/mL, respectively, and even more profoundly, at concentrations of 163 and 190 μg/mL, for F1–F9-excluding F7 and F7, respectively (Fig. 4B).

These results suggest that F7 denotes anti-inflammatory activity in colon cell lines, whereas certain combinations of treatment with all fractions of the extract lead to a significant increase in the cytotoxic activity.

The active fraction of C. sativa extract contains mainly THCA
The chemical composition of the active fraction (F7) was analyzed by HPLC and ESI–MS. F7 was obtained as a broad peak in the HPLC chromatogram. To analyze its structure and verify its purity, it was analyzed at different dilutions, in comparison to a THCA standard. The results suggested that F7 is THCA (Fig. 5). ESI–MS results further confirmed that F7 contains THCA: C22H30O4 (358.214); m/z (MH+) 359.222, (MNa+) 381.203. 1H and 13C spectra were taken to verify the exact structure and determine the purity of F7. The nuclear magnetic resonance results showed that F7 is indeed THCA, at over 90% purity.

GPR55 receptor antagonist significantly reduces the anti-inflammatory activity of F7, whereas CB2 receptor antagonist significantly increases HCT116 cell proliferation

To determine whether activity of fractions of C2F in HCT116 cells is conferred via the CBs or GPR55 receptors, we determined the effect of CB1, CB2, and GPR55 receptor antagonists (rimonabant, SR144528 and CID16020046, respectively) on their anti-inflammatory or cytotoxic activities. CB1 and CB2 receptor antagonists did not change the anti-inflammatory activity of F7 or F1–F9 (Fig. 6A). However, addition of GPR55 antagonist led to a significant reduction in activity and to an increase in IL-8 levels in these treatments (Fig. 6A). Addition of GPR55 antagonist did not change IL-8 level in control (Fig. 6A). As for the cytotoxic activity, a significant reduction in F1–F9-excluding F7 with addition of F7 (163 and 190 μg/mL, respectively) activity was found for CB2 antagonist. However, CB2 antagonist increases cell number even in the control (Fig. 6B), suggesting that it counteracts the fractions' activity by inducing HCT116 cell proliferation (Fig. 6B).

Transcripts for CB1, CB2, and GPR55 were detected by qPCR in HCT116 cells. Expression of CB2 and GPR55 was significantly increased on treatment with TNFα in these cells (values are the steady-state level of gene expression in TNFα-treated vs. nontreated cells; Table 2).

CBD reduces inflammation only at lower concentration yet its cytotoxic activity is dose dependent
The fraction of C2F containing CBG, CBD, and CBDA (F2) did not show any anti-inflammatory activity in HCT116 cells in terms of IL-8 reduction (Supplementary Fig. S3A). Yet, no anti-inflammatory activity for CBD was determined for the higher CBD concentrations in HCT116 cells (Supplementary Fig. S3A). CBD was active in reduction of IL-8 levels in CaCO2 and HT29 cells (Supplementary Fig. S3A). However, treatments with CBD lead to a dose-dependent cell death in HCT116 and in CaCO2 cells, and to a lesser extent in HT29 cells (Supplementary Fig. S3B).

Treatment with C. sativa extracts C2F and F7 leads to reduction in IL-8 levels in patient colon tissue
Since cell lines do not fully reflect the conditions in colon tissue, we further verified C2F and F7 inflammation-reducing activity in biopsies of colon tissue taken from IBD patients. Biopsies were maintained ex vivo and the levels of IL-8 and IL-6 were determined in nontreated versus C2F- and F7-treated tissue. Treatment with C2F reduced significantly both IL-8 and IL-6 levels compared to nontreated controls (n=29). These results confirmed the anti-inflammatory effect of C2F and F7 on colon tissues derived from IBD patients (Fig. 7).

Treatment of HCT116 cells and biopsies with C. sativa extracts C2F and F7 leads to reduction in MMP9 and COX2 expression

COX2 expression is induced in the large intestine of IBD patients (reviewed by Wang and DuBois28) and MMP9 is among the predominant proteinases expressed in the gut mucosa during active IBD, associated with disease severity.29 The steady-state levels of MMP9 and COX2 expression were examined as markers for inflammation level in HCT116 cells and colon biopsies of IBD patients (four UC and one CD). Expression of both COX2 and MMP9 was significantly induced in HCT116 cells treated with TNFα and significantly reduced by treatment with C2F and F7. F7 was more effective at reducing COX2 expression than C2F (Fig. 8A). In colon tissues, expression of both COX2 and MMP9 was downregulated by C2F and F7 treatments (Fig. 8B). As with the cell lines, F7 was more effective than C2F at reducing COX2 expression (Fig. 8B).


The present study suggests that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA. This conclusion is based on several lines of evidence. First, fresh flower ethanolic extracts of C. sativa led to reduction of IL-8 levels in HCT116, HT29, and CaCO2 colon cell lines, determined as reduction in IL-8 secretion. Second, fractionation of the fresh flower extract yielded only one fraction, F7, which retained activity. Third, chemical analysis showed that F7 contains mainly THCA. Also, a combination of F7 with the other F1–F9 fractions led to anti-inflammatory activity in HCT116 cells similar to that of the whole extract. Furthermore, under the examined conditions, F7 did not show cytotoxic activity, further suggesting that under the examined conditions, F7 activity is anti-inflammatory and does not derive simply from cell death.

As previously indicated, THC is considered to be one of the most active compounds in C. sativa. However, other phytocannabinoids, including CBG, CBN, and THCV, are now known to have therapeutic effects.10 As for THCA, other cell-based experiments have demonstrated its immunomodulatory, anti-inflammatory, neuroprotective, and antineoplastic activity (reviewed in Moreno-Sanz30). For example, in lipopolysaccharide-activated U937 macrophages and peripheral blood macrophages, THCA-A inhibits the release of TNFα and COX1 and COX2 expression in a dose-dependent manner and prostaglandin production.31,32

In agreement, C2F and F7 were found to inhibit the expression of COX2. COX1 and COX2 catalyze the production of prostaglandins from arachidonic acid, and prostaglandins are known to be important in mediating the inflammatory process. COX2 is an immediate early response gene induced mainly at sites of inflammation in response to inflammatory stimuli, whereas it is normally absent from most cells. It is induced in the large intestine of IBD patients and in inflamed tissues of an IL-10-deficient mouse model of IBD (reviewed by Wang and DuBois28). Reduction of COX2 has been suggested as a major target for the treatment of IBD (e.g., El Miedany et al.33). In agreement, in in vitro enzyme-based COX1/COX2 inhibition assay and in HT29 cell line, COX1 and COX2 enzyme activity was inhibited by THCA, however, only in the high millimolar concentration range.32

Our results showed that C2F and F7 inhibit the expression of MMP9 as well. MMP9 is among the predominant proteinases expressed in the gut mucosa during active IBD and it is associated with disease severity.29 In addition, MMP9 levels are significantly higher in active IBD and in UC compared to CD.34 Indeed, in UC patients, fecal MMP9levels are significantly correlated with several measurement parameters, such as Mayo score, endoscopic score, and serum C-reactive protein levels, suggesting that it is a good selective marker for evaluation of disease activity in UC patients.35 The inhibition of MMP9 expression by C2F and F7 in both cell lines and IBD patients (three UC and one CD) provides another indication of their possible efficacy against colon inflammation.

Perhaps even more significant, inhibition of COX2 and reduction of prostaglandin production have been proposed to play an important role in the inhibition of colon cancer development.28 Importantly, COX2 expression is elevated in up to 90% of colorectal carcinomas and is correlated with poor prognosis, whereas it has been shown in mice that for cancer therapy, selective COX2 inhibitors may enhance the activity of antiangiogenic agents against pre-established metastases.36

In this light, our findings that a certain combination of F7 with the other fractions highly induces cell death suggest interaction between THCA and other C. sativa compounds for this activity. Interactions between C. sativa-derived compounds were notified before for different activities.10 Practically, using certain combinations of C. sativacompounds may further potentiate the use of C. sativa as an anticancer drug.

Phytocannabinoids as well as endocannabinoids bind to target receptors and activate various signaling pathways thereby affecting several biological processes. The main receptors for endocannabinoids are CB1 and CB2 (reviewed by Chiurchiù et al.37). CB1 and CB2 are expressed in different tissues and cells. CB1 is mainly expressed in the nervous system and is involved in the regulation of cognitive, memory, motor functions, and analgesia. CB2 is expressed by the cells of the immune system and is associated with the modulation of different immune functions (reviewed by Chiurchiù et al.37). CB2 was also found in additional tissues, including epithelial cells of the colon.4,37Upregulation of CB2 is associated with chronic inflammation of the nervous system37 and modulation of intestinal inflammation.4

We found that CB2 receptor antagonist led to an increase in the percentage of live cells even in the absence of C. sativa treatment, suggesting that CB2 activity may be negatively involved with cell proliferation. However, CB2 was shown to be expressed with great intensity in epithelial cells of colorectal cancer tumor and correlated with tumor growth and disease progression. Moreover, its expression was suggested to be a marker for poor prognosis.38 Also, Romano et al. found that Cannabis extract with high content of CBD inhibits colorectal cancer cell proliferation and attenuates colon carcinogenesis. This activity involved CB1 and CB2 receptor activation.39

GPR55, activated by phytocannabinoids and endocannabinoids, is widely expressed in several tissues, including the GI and in different human innate and adaptive immune cells.40 Our results suggest that the anti-inflammatory activity of F7 is affected by GPR55 receptor antagonists, suggesting that the anti-inflammatory activity of F7 is mediated, at least partially, via GPR55. In contrast, the findings that GPR55 negatively affects the ability of monocytes to phagocytose, enhances IL-12 and TNFα production, and that pharmacological blockade of GPR55 reduces intestinal inflammation in mice by reducing leukocyte migration and activation, positioning GPR55 with a proinflammatory role.14,40

Our results also show a dose-dependent cytotoxic activity for CBD in all three examined cell lines, in agreement to other studies suggesting CBD to be cytotoxic (e.g., ChoiPark et al.41). However, we found anti-inflammatory activity for CBD only for the low concentrations, and in a bell-shaped rather than dose-dependent manner. At high CBD concentrations, even a proinflammatory effect was detected. Similar results for bell-shaped anti-inflammatory activity were found for CBD in an animal model of inflammation.42 Yet, CBD is still considered to be a strong anti-inflammatory agent.27 Our results suggest that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.

An important advantage of using THCA (present in F7) is its lack of narcotic activity. This is due to two pharmacological traits: decreased central nervous system penetration due to the presence of a carboxylic group and a probable inability to convert to THC in vivo.30 Hence, the use of C. sativa extract that contains THCA could have major implications for the medical use of THCA, the relevant fraction in combination with other fractions or, alternatively, raw, unheated Cannabis preparations. Such preparations might promote more precise treatment with medical Cannabis for IBD patients, maximizing Cannabis's therapeutic gain while minimizing the undesirable psychoactive side effects. Our results further underscore the need to allocate the right treatment with the right compounds for each medical indication treated by Cannabis.


Research was performed under the authorization of IMCA (Israel Medical Cannabis Agency) for research in medical cannabis.


O.S. was paid as an employee of PLANTEXT, Israel. For other authors, no competing financial interests exist.


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Cite this article as: Nallathambi R, Mazuz M, Ion A, Selvaraj G, Weininger S, Fridlender M, Nasser A, Sagee O, Kumari P, Nemichenizer D, Mendelovitz M, Firstein N, Hanin O, Konikoff F, Kapulnik Y, Naftali T, Koltai H (2017) Anti-inflammatory activity in colon models is derived from Δ9-tetrahydrocannabinolic acid that interacts with additional compounds in Cannabis extracts, Cannabis and Cannabinoid Research 2:1, 167–182, DOI: 10.1089/can.2017.0027.

Abbreviations Used
cannabinoid receptor type 1

cannabinoid receptor type 2



cannabidiolic acid


cannabigerolic acid


Crohn's disease



Dulbecco's modified Eagle's medium

enzyme-linked immunosorbent assay

electrospray ionization

fraction 7


high-performance liquid chromatography

inflammatory bowel disease



mass spectrometry


retention time

standard error


Δ9-tetrahydrocannabinolic acid


tumor necrosis factor alpha

ulcerative colitis

Last edited:
THCA is so bloody simple to make! I did it accidentally and thought I'd fluffed something up... So - tincture like normal - alcohol, (not decarbed) herb, agitate, filter and then evap. Scrape the powder of'n yer drying platter - side note: go to a flew market and get a glass base from an old 70s vintage microwaves. The ones large enough to nukrowave a side of beef on. Large, with low sides. Keep liquidy things in, and yet easy to scrape.

Anyhoo - scrape away for THCA.

A fellow vet buddy suffers quite badly from crohns and colitis, and has been on cannabis therapy for the past 2 years now. He's able to cut back on a few meds, but between the GI issues, plus pain from ankylosing spondylitis, his bad days are pretty bad. I'm going to get him to give this a shot for a week or two and see how it shakes out...
Do you ever get the feeling that all things cannabis-based are somehow about to explode? People will start getting somehow magically 'better', while others will continue to munch pills because thats how it's always been done. Those of us that have been involved for even a short period of time have seen significant mind shifts from people who would otherwise be following the usual path. Military and RCMP vets are great at giving it a shot because a fellow vet suggested it, and vets don't lie about things like health and meds to fellow vets. And we tell other people. Someopne referred to us as 'The Green Wave', and I'm absolutely fine with that :)
Cannabidiol restores intestinal barrier dysfunction and inhibits the apoptotic process induced by Clostridium difficile toxin A in Caco-2 cells
Stefano Gigli, Luisa Seguella, Marcella Pesce,
Eugenia Bruzzese, Alessandra D’Alessandro, Rosario Cuomo, Luca Steardo, Giovanni SarnelliGiuseppe Esposito
First Published March 13, 2017

Clostridium difficile, cannabinoids, cannabidiol, clostridium difficile toxin A, intestinal permeability
1 It is known that Clostridium difficile produces two enterotoxins, named Clostridium difficile toxin A and B (TcdA and TcdB, respectively) that, in turn, are responsible for the extensive colonic mucosal damage, causing severe diarrhoea, colitis, shock and death in most severe cases.2,3 TcdA is the major cause of Clostridium difficile enterotoxicity. TcdA is a glucosyltransferase, that once internalised into the host cell via receptor-mediated endocytosis, inactivates small GTPases.4 Among these proteins, RhoA, a small GTPase member of the Rho subfamily that is a critical regulator of actin cytoskeleton and tight junction assembly, is the primary target of TcdA.5 TcdA-induced inactivation of RhoA results in the transition from guanosine triphosphate (GTP)-bound form (active) to guanosine diphosphate (GDP)-bound form (inactive), leading to an alteration of cellular structure and tight junction integrity, and consequently to increased epithelial barrier permeability; this process is also sustained by the acute inflammation of colonic mucosa and contributes to the leaky gut and massive ions’ secretion.6 Due to its role in the mucosal homeostasis and functions, the targeting of RhoA may represent an innovative pharmacological strategy for the treatment of CDI.

In the last decade, cannabinoids extracted from the marijuana plant (Cannabis sativa) and synthetic cannabinoids have shown numerous beneficial effects on gastrointestinal (GI) functions.7 Non-psychotropic phytocannabinoid cannabidiol (CBD) is one of the most interesting compounds, since it exerts a wide range of beneficial pharmacological actions on GI functions, ranging from antioxidant to antinflammatory activities.8,9 Unlike psychoactive cannabinoids such as tetrahydrocannabidiol (THC), CBD has little binding affinity to cannabinoid receptors (either CB1 and CB2); whereas, by acting on peroxisome proliferator-activated receptor gamma (PPARγ)10 and 5-hydroxytryptamine (5HT)-1 A receptors,11 it displays antinflammatory and antioxidant effects.12 Unlike other phytocannabinoids, CBD has been shown to act as a non-competitive negative allosteric modulator of CB1 receptors.13 Notably, CBD is able to restore in vitro intestinal permeability increased by ethylenediaminetetraacetic acid (EDTA) or pro-inflammatory stimuli.14,15 So far, no evidence has been produced about the putative protective role exerted by CBD in CDI. To further this aim, the present study was addressed at evaluating the in vitro effects of CBD on TcdA-induced apoptosis in Caco-2 cells and at investigating the effects of CBD and its mechanism of action.

16 Caco-2 cells were purchased from European Collection of Cell Cultures (ECACC, Public Health England, Porton Down, Salisbury, UK). Cell medium, chemicals and reagents used for cell culture, and TcdA were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Instruments, reagents, and materials used for Western blot analysis were obtained from Bio-Rad Laboratories (Milan, Italy). CBD and AM251 (CB1 receptor antagonist) were purchased from Tocris Cookson, Inc. (Ballwin, Missouri, USA). The antibodies rabbit anti-zonula occludens-1 (ZO-1), rabbit anti-occludin, rabbit anti-bax and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were procured from Cell Signalling Technology (Danvers, Massachusetts, USA). Mouse anti-ZO-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Mouse monoclonal antibody anti-active RhoA by New East Bioscience (Pennsylvania, USA) has been used. Fluorescein isothiocyanate-conjugated anti-rabbit antibody and Texas red conjugated anti-mouse antibody were purchased from Abcam (Cambridge, UK) and horseradish peroxidase (HRP) was obtained from Dako (Milan, Italy).

Cell culture and experimental conditions
Caco-2 cells were grown at 37℃ with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) in addition with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 2 mM L-glutamate, and 1% non-essential amino acids. Caco-2 cells were plated at a density of 1 × 106 cells/well in six-well plates and incubated for 24 h. Every 24–48 h the medium was replaced with fresh medium to confluence. After reaching confluence, the cells were washed three times with phosphate-buffered saline (PBS), detached with trypsin/EDTA, plated in six-well plates some containing and on polyethylene-terephtalate (PET) filter inserts (Falcon Becton-Dickinson, 0.4 mm pore diameter, area 4.21 cm2, pore density 2 ± 0.2 106/cm2) to measure the transepithelial electrical resistance (TEER), and allowed to adhere for an appropriate time. Caco-2 cells were randomly divided into the following groups: vehicle, 30 ng/ml TcdA, 30 ng/ml TcdA plus CBD at 10−9, 10−8 and 10−7 M CBD and 30 ng/ml TcdA plus 10−7 M CBD plus 10−7 M CB1 receptor antagonist AM251. The concentrations of CBD and AM251 were selected on the basis of previous reports14,15 and our preliminary experiments (Supplementary Material, Figure 1, data not shown); in brief, cells were treated with different concentrations of CBD and/or AM251 for 24 h and then incubated at 37℃ in the presence of TcdA for 24 h.

Caco-2 (TEER) was measured using the EVOM volt-ohm meter (World Precision Instruments Germany, Berlin, Germany) according to the method described by Wells and colleagues.17

In brief, cells were used for experimentation between 14–21 days and each epithelial cell layer with a TEER value greater than 1000 Ω × cm2, was considered to have tight adhesion. At this point, cell monolayers were treated according to experimental protocol described above and TEER measurements were performed at different time points (2, 3, 5, 7, 12, 18 and 24 h, respectively). TEER values were measured at a current of 20 mA, corrected for background, resistance value without cells, and normalised by multiplying the determined resistance by effective membrane growth area, 4.71 cm2.

TEER(Ω×cm2)=(Totalresistance-blankresistance)(Ω)×Area(cm2)" role="presentation" style="display: inline; line-height: normal; font-size: 16px; word-spacing: normal; word-wrap: normal; white-space: nowrap; float: none; direction: ltr; max-width: none; max-height: none; min-width: 0px; min-height: 0px; border: 0px; padding: 0px 2px 0px 0px; margin: 0px; position: relative;">TEER(Ω×cm2)=(Totalresistance−blankresistance)(Ω)×Area(cm2)TEER(Ω×cm2)=(Totalresistance-blankresistance)(Ω)×Area(cm2)

Cytotoxicity assay
The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay was used to determine Caco-2 cell proliferation and survival.18 At least (5 × 104 cells/well) were plated in 96-well plates and allowed to adhere for 3 h. Then DMEM was replaced with fresh medium and then cells were treated according to the different experimental protocols (see above). After 24 h, 25 μl MTT (5 mg/ml MTT in DMEM) was added to the cells and the mixture was incubated for further 3 h at 37℃. Subsequently, the cells were lysed and the dark blue crystals were solubilised using a 100 μl solution containing 50% N,N-dimethylformamide and 20% (w/v) sodium dodecyl sulphate (SDS) (pH 4.5). The optical density (OD) of each well was determined using a microplate spectrophotometer equipped with a 620 nm filter (PerkinElmer, Inc.; Waltham, Massachusetts, USA).

Western blot analysis
Twenty-four hours after treatment, the cells (1 × 106/well) were washed with ice-cold PBS, were harvested into Separate Eppendorf tubes for different treatment groups and collected by centrifugation at 180 g for 10 min at 4℃. The cell pellet, obtained after centrifugation, was re-suspended in 100 µl ice-cold hypotonic lysis buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulphonylfluoride, 1.5 µg/ml soybean trypsin inhibitor, 7 µg/ml pepstatin A, 5 µg/ml leupeptin, 0.1 mM benzamidine and 0.5 mM dithiothreitol (DTT)) and incubated on ice for an additional 15 min.

The suspension was rapidly passed through a syringe needle five to six times to lyse the cells and then centrifuged for 15 min at 13,000 × g to obtain the cytoplasmic fraction. The cytoplasmic fraction proteins were used to determine the protein concentration with Bradford assay and mixed with non-reducing gel loading buffer (50 mM Tris (hydroxymethyl) aminomethane (Tris),10% SDS, 10% glycerol, 2 mg bromophenol/ml) at a 1:1 ratio. The solutions were then boiled for 3 min, centrifugated at 10,000 g for 10 min and 50 µg of each homogenate was used for electrophoresis using 12% discontinuous polyacrylamide mini gels. Proteins were then transferred to nitrocellulose membranes that were saturated by incubation with 10% non-fat dry milk in 1X PBS overnight at 4℃ and then incubated with rabbit anti-ZO-1 (1:1000), rabbit anti-occludin (1:1000), mouse anti-active RhoA (1:1000), rabbit anti-bax (1:1000) and rabbit anti-GAPDH (1:1000) antibodies. After being extensively washed in TBS 1X with 0.1% Tween 20, membranes were then incubated for 2 h at room temperature with the specific secondary antibodies conjugated to HRP anti-mouse (1:2000) or anti-rabbit (1:3000). Immune complexes were identified by enhanced chemiluminescence detection reagents (Amersham Biosciences, Milan, Italy) and the blots were analysed by scanning densitometry (GS-700 Imaging 143 Densitometer; Bio-Rad, Segrate, Italy). Results are expressed as OD; (arbitrary units; mm2) and normalised against the expression of the housekeeping protein GAPDH.

For these experiments, Caco-2 cells were cultured onto coverslips until confluence, and then treated according to the different above-described protocols. Cells were then fixed for 30 min in 4% formaldehyde, washed with ice-cold PBS and permeabilised with 0.3% Triton-X100 in PBS for one hour. Subsequently, 2% bovine serum albumin (BSA) was used to block the nonspecific binding sites. The cells were then incubated overnight with mouse anti-ZO-1 (1:100), or rabbit anti-occludin antibody (1:100), following PBS washing and further incubated in the dark for half an hour with the appropriate secondary antibody (fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or Texas red conjugated anti-mouse). After final PBS washing, the cells were analysed using a microscope (Nikon Eclipse 80i), and images were captured with a high-resolution digital camera (Nikon Digital Sight DS-U1). Texas Red was excited at a wavelength of 568 nm and collected through a long pass filter (590LP). FITC was excited with a wavelength of 488 nm and collected with a narrow band filter (515–540BP). Texas Red and FITC were assigned to the red and green channels respectively of the generated RGB channel image.

Statistical analysis
Results are expressed as mean ± standard error of the mean (SEM) of four or five experiments and each experiment was performed in triplicate. Statistical analysis was performed using parametric one-way analysis of variance (ANOVA) and Bonferroni’s post-hoc test was used for multiple comparisons. Values of p < 0.05 were considered significant.

Figure 1(a), TcdA exposure induced a significant and time-dependent reduction of TEER (by −35, −46, −57, −69, −78, −81 and −86%, at 2, 3, 5, 7, 12, 18 and 24 h, respectively; p < 0.01 at 2 h and p < 0.001 at all other time-points). Starting from 2 h after toxin challenge, the effect of TcdA on electrical resistance was significantly and concentration-dependently counteracted by CBD treatment (Figure 1(a)); TEER values at 2, 3, 5, 7, 12, 18 and 24 h were indeed significantly increased by 15, 33, 56, 73, 119, 133 and 225% in CBD 10−9 M-treated cells (p = ns at 2 h and p < 0.01 at all other time-points), while CBD 10−8 M and CBD 10−7 M treatments yield to a significant increase of TEER values by 31, 52, 90, 133, 219, 239, 361%, and 38, 65, 114, 193, 300, 372 and 538%, respectively (p < 0.01 at 2 and 3 h and p < 0.001 at all other time points for CBD 10−8 M; p < 0.01 at 2, 3 and 5 h and p < 0.001 at all other time-points for CBD 10−7 M).

Figure 1. Effect of cannabidiol (CBD) on transepithelial electrical resistance (TEER) and barrier integrity of Clostridium difficile toxin A (TcdA)-exposed Caco-2 cells. (a) 24 h Time course TEER changes following treatment (n = 4); (b) immunofluorescent staining showing the effects of TcdA on zonula occludens-1 (ZO-1) and occludin co-expression at 24 h. Nuclei were stained by DAPI (scalebar = 25 µm); (c) immunoreactive bands corresponding to ZO-1 and occludin expression at 24 h following the TcdA challenge; (d) relative densitometric analysis of immunoreactive bands (arbitrary units normalised against the expression of the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein; n = 5). Results are expressed as mean ± standard error of the mean (SEM) of experiments performed in triplicate. ***p < 0.001 and **p < 0.01 vs vehicle group; °°°p < 0.001, °°p < 0.01 and °p < 0.05 vs TcdA group. DAPI (4',6-diamidino-2-phenylindole).

Interestingly, the effect of CBD on the TcdA-induced TEER reduction was completely abolished in the presence of the CB1 antagonist, AM251 (p < 0.01, Figure 1(a)).

Immunofluorescence analysis, showed that 10−7 M CBD, markedly reversed the TcdA-induced decrease of both occludin and ZO-1 co-expression in cultured cells, thus restoring the epithelial barrier architecture (Figure 1(b)). This finding was confirmed by quantitative analysis showing that TcdA-reduced expression of occludin and ZO-1 (0.3 ± 0.1 and 0.2 ± 0.1 vs 1.0 ± 0.1 fold-change in the vehicle group, respectively; all p < 0.001) was significantly and concentration dependently restored by CBD at the doses of 10−9 M (occludin: 2.1 ± 0.2 vs 1.0 ± 0.3 fold-change in TcdA-treated cells, p < 0.01; ZO-1: 2.6 ± 0.5 vs 1.0 ± 0.5 fold-change in TcdA-treated cells, p < 0.05), 10−8 M (occludin: 2.6 ± 0.3 vs 1.0 ± 0.3 fold-change in TcdA-treated cells, p < 0.001; ZO-1: 4.4 ± 0.4 vs 1.0 ± 0.5 fold-change in TcdA-treated cells, p < 0.001) and 10−7 M (occludin: 3.1 ± 0.3 vs 1.0 ± 0.3 fold-change in TcdA-treated cells, p < 0.001; ZO-1: 5.5 ± 0.5 vs 1.0 ± 0.5 fold-change in TcdA-treated cells, p < 0.001) (Figure 1(c) and (d)). Once again, AM251 significantly inhibited the CBD-mediated rescue of ZO-1 and occludin proteins (all p < 0.001) (Figure 1(b)–(d)).

CBD inhibits TcdA-induced apoptosis and cells’ toxicity
As shown in Figure 2(a), a significant decrease in Caco-2 cell viability was observed at 24 h following the TcdA challenge (−70% as compared to vehicle group assumed as 100% viable cells, p < 0.001). Under the same experimental conditions, CBD caused a significant and concentration-dependent inhibition of TcdA-induced cytotoxicity, resulting in an increased cells’ viability (by 61, 133 and 328% at 10−9, 10−8 and 10−7 M, respectively, vs TcdA group (p < 0.05, p < 0.01 and p < 0.001, respectively).

Figure 2. Effect of cannabidiol (CBD) on Clostridium difficile toxin A (TcdA)-induced cells toxicity and apoptosis. (a) 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) cell viability absorbance at 24 h (n = 5); (b) immunoreactive bands corresponding to RhoA GTP and Bax expression at 24 h following the TcdA challenge; (c) relative densitometric analysis of immunoreactive bands (arbitrary units normalised against the expression of the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein; n = 5). Results are expressed as mean ± standard error of the mean (SEM) of experiments performed in triplicate. ***p < 0.001 and vs vehicle group; °°°p < 0.001, °°p < 0.01 and °p < 0.05 vs TcdA group. GTP: guanosine triphosphate.

Exposure to TcdA significantly reduced the expression of RhoA GTP (0.2 ± 0.1 vs 1.0 ± 0.3 fold-change in the vehicle group, p < 0.001) and increased the expression of the pro-apoptotic Bax protein (10.5 ± 1.2 vs 1.0 ± 0.5 fold-change in the vehicle group, p < 0.001) (Figure 2(b) and (c)); these effects were significantly restored by CBD, that at 10−9, 10−8 and 10−7 M increased the expression of RhoA GTP (1.8 ± 0.4, 3.3 ± 0.5 and 4.5 ± 0.5 vs 1.0 ± 0.3 fold-change in TcdA-treated cells; p < 0.05, p < 0.001 and p < 0.001, respectively) and decreased the expression of Bax (0.7 ± 0.1, 0.6 ± 0.1 and 0.2 ± 0.1 vs 1.0 ± 0.1 fold-change in TcdA-treated cells; p < 0.05, p < 0.001 and p < 0.001) (Figure 2(b) and (c)). As shown for the TcdA-impaired barrier function the protective effects of CBD on cells toxicity were completely abolished in the presence of AM251 (all p < 0.001) (Figure 2(a)–(c)).

19 since it is associated with significant morbidity, 5% infection-related mortality and an overall mortality of 13–20 %.3,20 There is an urgent need for new drugs able to improve CDI outcome, maximising the recovery of patients.

Due to its ability to inhibit Rho GTP activation,4,21 TcdA has been postulated as the main enterotoxin involved in gut mucosal disruption,5,22 leaky gut and loss of cell-to-cell integrity, leading to massive apoptosis.23,24 The inhibition of TcdA effects might thus represent the key for a targeted therapy of CDI.

In this perspective, cannabinoids might display a wide range of protective effects on the GI epithelial barrier, due to their antinflammatory, anticancer and antioxidant properties.25,26 Among the almost 113 active phytocannabinoids isolated from Cannabis sativa plant, CBD is one of the most interesting compounds considered for medical use, as different clinical reports showed its almost complete lack of side effects in humans.27 Remarkably, CBD is a non-psychotropic cannabinoid (unlike Δ9-THC) and does not interfere with psychomotor learning and psychological functions.28

In this study we have demonstrated, for the first time, that CBD is able to preserve mucosal integrity and to reduce cellular permeability in in vitro cultured Caco-2 cells, counteracting the effects of TcdA. CBD, indeed, caused a concentration-dependent increase of transepithelial resistance, significantly preventing the enterotoxin-evoked damage. Moreover, CBD caused a marked inhibition of cell death in TcdA-exposed cells, due to a concentration-dependent up-regulation of both occludin and ZO-1 protein, two of the main cell-to-cell tight junction proteins.29Furthermore, CBD caused a significant RhoA GTP rescue that raised in parallel with the inhibition of pro-apoptotic Bax protein expression; these combined effects likely account for the restoration of the TcdA-induced intestinal barrier dysfunction and apoptosis.

CBD effects were, at least partially, mediated by critical involvement of the CB-1 receptor, since they were almost completely abolished in the presence of the specific CB-1 receptor antagonist AM251.

Although different receptors have been proposed to mediate CBD activity,11,30 it has been postulated that CBD may represent a non-competitive negative allosteric modulator of CB1 receptors.13 Consequently, the presence of a specific CB1 antagonist markedly impairs CBD activity, as previously demonstrated by different studies.14,15 Accordingly, CBD was able to contain cellular damage in the in vitro model of mucosal disruption, as it occurs in our experimental conditions. Our results indicated that CBD is able to increase RhoA GTP expression, via the selective involvement of CB-1 receptors. However, CBD exhibits both antioxidant and antinflammatory properties, labelled as generic neuroprotective functions,30mediated by a number of different pathways and cellular effectors, that have been only partially recognised so far. These so-called ‘entourage’ effects are not to be excluded a priori when considering the potential therapeutic effects of this compound in CDI.

One can speculate that this entourage activity might synergistically cooperate with CB-1 dependent negative allosteric modulation, further enhancing the protective effects on gut epithelial cells; preventing the cytotoxic effects of reactive oxygen species products and pro-inflammatory cytokines,31,32 released in the mucosa following TcdA stimulus.

In recent decades, CBD has been proposed as an effective therapeutic option in a variety of GI pathologies, ranging from inflammatory bowel disease8 to colon cancer,33 inflammatory hypermotility in mice34 and intestinal sepsis.35 The results of our preliminary report indicate that CBD might be an intriguing candidate in CDI treatment, as well.

Although to be confirmed in vivo, the multifaceted activities exerted by CBD might prevent the cytotoxic damage in CDI and from a translational standpoint, given its lack of any significant toxic effect in humans, may ideally represent an effective adjuvant treatment in this high-mortality and morbidity rate condition.

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We may finally know why marijuana helps people with chronic gut problems

As John Mayer tells us (and tells us, and tells us), your body is a wonderland. When it comes to microbial life, this holds especially true for your gut. There, hundreds of residential species eat, breed, and excrete waste. Somehow, your intestines manage to thrive with this zoo inside them—for the most part. In some cases things aren’t so wonderful: your gut starts attacking itself in an autoimmune response that’s bad for microbes and host alike.

People with this condition, known as inflammatory bowel diseases like Crohn’s disease or ulcerative colitis, face a chronic problem. Current treatment options are laden with side effects and require constant tweaking to remain effective. Some of those people have turned to marijuana for treatment—but their stories about how it has helped them have remained just that, stories, until now. A new study from University of Massachusetts and University of Bath researchers is the first to demonstrate the physical process by which cannabis affects IBD, opening up the possibility of creating new drugs to treat these chronic ailments.

Although numerous IBD patients use cannabis products to help treat their illness, and the phenomena has been subject to some medical research, nobody knew exactly how the medically active parts of marijuana (known as cannabinoids) had an anti-inflammatory effect on irritated bowels before this study. Ironically, however, the researchers weren’t even looking for this precise answer; they just happened upon it in the course of trying to understand how the healthy intestine regulates itself.

In the gut, a thin layer of epithelial cells mediates between our bodies and the microbial “zoo” living within. Beth McCormick of the University of Massachusetts has been studying the role these cells play in regulating the gut microbiome for well over a decade, and the starting point for this current research was her prior discoveryof a chemical pathway by which epithelial cells help neutrophils, a kind of white blood cell, to cross into the gut and eat up some of the microbes. But that was clearly only half of the answer. In order to produce balance, something else had to stop too many neutrophils from getting in and killing peaceful microbes and even the gut itself—leading to IBD.

The answer, reported in the new study out Monday in the Journal of Clinical Investigation, is a different pathway, also in the epithelial cells of the gut lining. That chemical pathway produces substances that prevent neutrophils from getting through the epithelial cells and into the gut. And it turns out those substances, in mice at least, are endocannabinoids. These fatty substances bind to the same chemical receptors as the cannabinoids found in, well, cannabis. Patients missing this secondary pathway “were more likely to develop ulcerative colitis,” McCormick says.

Although the current research is in mice, it points to a possible result in humans as well. It would help explain why cannabinoids seem to provide relief for people with IBD, because they perform basically the same regulatory function as the endocannabinoids would if the body were producing them itself. More research, of course, is needed, but McCormick says it opens up the possibility of creating new IBD treatments that work on the new pathway—including, perhaps, therapeutic agents extracted from marijuana.

And that’s not all, says Vanderbilt University gastroenterologist Richard Peek, who wasn’t involved in the new study. McCormick’s findings “may not just be specific to the intestine,” Peek says. Epithelial cells are found on the surfaces of organs throughout the body, so this mechanism of action may exist in other systems as well, he says. That would change our understanding of autoimmune responses elsewhere in the body, too.

This is good news for the 1.6 million Americans who currently have IBD. But given how common a treatment cannabis is for IBD, some might ask why researchers didn’t look for its mechanism of action in the gut before. That’s partially because cannabis research tends to be politicized, says Peek. He thinks that this discovery may open up new possibilities for the legalization of medical marijuana. For McCormick, their “unbiased approach” was the key to finding this result: they weren’t looking to explain cannabis’s mechanism of action, they just found it. “Sometimes, as they say in the field, the blind squirrel finds the nut,” she says.
Thank you for posting.
My dear daughter has Crohns. She nearly died from it 10 years ago (so much bleeding she required 12 blood transfusions leading up to surgery where she died and was revived on the operating table). All that is on offer for treatment is Remicade and Methotrexate. She was engaged to a lovely man, who also had Crohns, developed a leukemia like illness from the medications, had a bone marrow transplant, rejected it and died. This doesn't make it easier for her to take the medication, but she does what needs to be done. I will pass on this information to her, and hope it is of some use.
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I refuse to take biological drugs and that’s where I’m at with my doctor now. The side effects scare me. Nothing else is working from the doctor. Thank god for cannabis. I have been battling some major fatigue lately along with some crazy nausea. Some mornings I try to not vape, then realize how much it helps in the long run for the day. Knocks out fatigue and the nausea almost instantly. Fuck this IBD, it won’t win.
I refuse to take biological drugs and that’s where I’m at with my doctor now. The side effects scare me. Nothing else is working from the doctor. Thank god for cannabis. I have been battling some major fatigue lately along with some crazy nausea. Some mornings I try to not vape, then realize how much it helps in the long run for the day. Knocks out fatigue and the nausea almost instantly. Fuck this IBD, it won’t win.
I can't say I blame you there, @Kurtdiggler. My daughter has been taking Humera for over 10 years, and her doctor has explained it should already have stopped working for her, and she should be prepared for it to stop. The side effects are horrendous. I wish she'd try cannabis, but she views it as a moral evil. She seems to think it won't come back because she's had an illeostomy. She doesn't blink at taking Humera, and insists it's "safer" than Remicade. I'm not sure what she'll do when Humera doesn't work, but it is her choice.

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