Altered endocannabinoid system gene expression in inflammatory bowel disease mucosa: New perspectives in inflammatory bowel disease management

Abstract

BACKGROUND

Inflammatory bowel disease (IBD) is a broad classification including various chronic inflammatory gastrointestinal conditions that comprises two main disorders: Crohn’s disease (CD) and ulcerative colitis (UC). The key components of the endocannabinoid system (ECS) are highly expressed within the gastrointestinal tract, playing a crucial role in maintaining homeostasis and providing protection against intestinal inflammation.

AIM

To investigate possible impairment of the genes belonging to ECS by analyzing their expression levels in IBD patients and controls.

METHODS

The paired biopsies of endoscopically inflamed (IM) and noninflamed (NIM) colonic mucosa from 30 IBD-diagnosed patients (17 UC and 13 CD), and the colonic mucosa from 17 non-IBD controls, were collected and analyzed. The messenger RNA expression level of cannabinoid receptor (CNR) 1, CNR 2, diacylglycerol lipase alpha, diacylglycerol lipase beta, fatty acid amide hydrolase (FAAH), G protein-coupled receptor (GPR) 18, GPR55, monoglyceride lipase, peroxisome proliferator-activated receptor gamma (PPARG), and transient receptor potential cation channel, subfamily V, member 1 (TRPV1) was determined by quantitative polymerase chain reaction.

RESULTS

Six out of the 10 investigated genes were found to be dysregulated in at least one comparison. Specifically, in IBD patients, FAAH, PPARG, and TRPV1 were significantly downregulated in IM compared to NIM (FAAH, P = 0.012; PPARG, P = 0.001; TRPV1, P = 0.032) and in IM compared to controls (FAAH, P < 0.001; PPARG, P < 0.001; TRPV1, P = 0.002). An opposite trend was reported for CNR2 and GPR55, which showed an upregulation in IM compared to NIM (CNR2, P = 0.005; GPR55, P = 0.001).

CONCLUSION

We found a significant impairment of the ECS in IBD patients. Further analyses on larger cohorts are needed for a better understanding of the potential of cannabinoids in managing IBD.

Key Words: Endocannabinoid system; Inflammatory bowel disease; Crohn’s disease; Ulcerative colitis; Gene expression

Core Tip: The anti-inflammatory benefits of cannabinoids could serve as a potential new treatment for patients suffering from inflammatory bowel disease (IBD). In this study, we analyzed the expression level of 10 genes belonging to the endocannabinoid system in colonic mucosa from IBD patients vs controls. CNR2, FAAH, GPR18, GPR55, PPARG, and TRPV1 genes were dysregulated in the entire IBD cohort. Stratifying patients into Crohn's disease and ulcerative colitis subgroups, we found that the same 6 genes were dysregulated in at least one comparison. Further analysis of the endocannabinoid system could bridge the gap between preclinical findings and its role in gastrointestinal diseases.

INTRODUCTION

Inflammatory bowel disease (IBD) comprises two idiopathic conditions: Crohn’s disease (CD) and ulcerative colitis (UC), affecting millions of people worldwide[1]. These conditions are associated with inflammation and mucosal damage in the gastrointestinal tract, leading to symptoms like abdominal pain, diarrhea, and weight loss[2,3]. While the exact cause of IBD remains elusive, recent studies have shed light on the potential role of the endocannabinoid system (ECS) in the management and treatment of this disease, suggesting that targeting this system could reduce inflammation and potentially ameliorate the symptoms of this condition[4]. Treatment lines in IBD target multiple inflammatory pathways that are constantly expanding. Despite significant advances that facilitate long-term management, a curative treatment line has not been identified[5]. The anti-inflammatory effects of cannabinoids have been studied in multiple pathologies, such as multiple sclerosis, human immunodeficiency virus, and various oncological complications[6-8]. To fully utilize cannabis, it is critical to identify the active ingredients and comprehend the cellular and molecular processes underlying the plant’s anti-inflammatory properties[9].

The ECS is a complex signaling system found within the human body that has complex actions mainly in the nervous and immune systems, but also in other organs, being implicated in many processes related to the physiological response to internal or environmental stressors[10]. It consists of three main elements: (1) Endogenous cannabinoids, two of the most well-studied are anandamide (AEA) and 2-arachidonoylglycero[11]; (2) Cannabinoid receptors (CNR), which can be “classical”, such as CNR1 and CNR2, and “non-classical”, such as transient receptor potential (TRP) channels, peroxisome proliferator-activated receptors (PPARs), and other G protein-coupled receptors (GPRs)[12]; and (3) Regulating enzymes, two of them being represented by the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)[13].

CNR1 is present in the gastrointestinal tract, and its activation has been shown to modulate intestinal motility and reduce excessive secretion, contributing to maintaining homeostasis[4]. Additionally, this receptor is present in nerve fibers in the gut, and its activation can exert neuroprotective effects, potentially mitigating the neural abnormalities observed in IBD[1]. On the other hand, CNR2 is highly expressed in the immune system, and its activation can reduce inflammation and attenuate excessive immune responses. Therefore, these receptors can be used as potential therapeutic targets for IBD treatment[14].

There are over 800 G protein-coupled receptors (GPCRs), with GPR18 and GPR55 being suggested to be part of the ECS[15]. The expression of GPR18 was observed to be abundant in the brain, including the hypothalamus, in cells that belong to the immune system, and in the colon when referring to humans, while in mice, GPR18 is also located in the gastrointestinal tract[16]. GPR55 is expressed in various locations, including the brain (with a notable presence in the cerebellum) and in the jejunum and ileum of the gastrointestinal tract[17]. Additionally, its expression levels exhibit variations in mucosal biopsies from individuals with IBD, particularly in cases of CD[18].

Among the numerous receptors within the gastrointestinal tract that are suggested to contribute to the pathophysiology of IBD, PPAR gamma (PPARG) and TRP cation channel, subfamily V, member 1 (TRPV1) have been identified and are under consideration for their potential effectiveness. Lately, there has been a growing recognition of PPARG’s involvement in intestinal disorders, particularly in the context of colon cancer and intestinal inflammation, since studies are reporting its role in maintaining the balance of the immune system in the intestine, and may also exert anti-inflammatory effects[19,20]. TRPV1 has been reported to play a role in triggering pain and inflammation, particularly in IBD, and its increased expression was linked to the exacerbation of clinical symptoms and histopathological alterations[21]. A deeper investigation could offer a new perspective for drug development in IBD. The activity of endocannabinoid-degrading enzymes (FAAH and MAGL) can impact central and peripheral levels of endocannabinoids. Dual inhibition of FAAH and cyclooxygenase enzymes induces protection against intestinal inflammation[22]. Thus, utilizing inhibitors targeting both FAAH and MAGL could offer an alternative therapeutic approach for managing IBD[23]. While research is still in its early stages, the potential of cannabinoids in managing IBD symptoms is a promising area of study.

In the present work, we investigate the expression levels of ten genes belonging to ECS in the colonic mucosa from IBD patients and non-IBD controls in order to identify: (1) An ECS gene expression signature for IBD; (2) An ECS gene expression signature specific for UC or CD; and (3) A possible correlation between this signature and the most important clinical features.

MATERIALS AND METHODS

Patients and sample collection

Thirty adult IBD patients with a diagnosis of UC (n = 17) or CD (n = 13) were enrolled in the study at the Fundeni Clinical Institute, Bucharest, Romania, where the paired biopsies of endoscopically inflamed (IM) and noninflamed (NIM) colonic mucosa were obtained. Mucosal biopsies were taken with a minimum of 10 cm between the IM and NIM areas. Patients had been prescribed a standardized colonoscopy bowel preparation of 3-4 L of polyethylene glycol. The diagnosis was made according to European Crohn’s and Colitis Organization Guidelines. Clinical status was classified according to the CD activity index and Mayo Clinic score. Patients were considered in remission if they had a CD activity index score < 150 or a Mayo score of 2 or fewer points, along with not having more than a point in any individual subscore. The extension of UC was classified as the longest extension from disease diagnosis.

The colonic mucosa from 17 non-IBD controls, collected during a colonoscopy screening. The inclusion and exclusion criteria have been previously described. The exclusion criteria for non-IBD controls were as follows: (1) Presence of digestive symptoms; (2) Current or previous nonsteroidal anti-inflammatory treatments (within the past 3 months); and (3) Current or previous anticoagulant/antiplatelet treatments (within the past 3 months). The study has been approved by the local ethical committees of the of the Fundeni Clinical Institute (No. 50290/October 11, 2019) and Victor Babes National Institute of Pathology (No. 103/July 23, 2022). All the participants signed the written informed consent before being enrolled in the study.

ECS gene expression analysis

After the colonoscopy, the biopsies were preserved in RNA protect solution (Qiagen, Germany) and later stored at -80 °C until RNA isolation. The RNA isolation from the frozen biopsies was conducted using the RNeasy mini kit (Qiagen, Germany). The quality and the concentration of the RNA were determined using the Nanodrop 2000 (Thermo Scientific, MA, United States) by measuring the absorbance at 260 nm, 230 nm, and 280 nm. Both 260/280 nm and 260/230 nm parameters were > 1.9. RNA was reverse-transcribed to cDNA using the RT2 First Strand Kit (Qiagen, Germany), and quantitative polymerase chain reaction was performed on the ABI-7500 fast instrument (Applied Biosystems, CA, United States) using SYBR Green chemistry. The primers were purchased by the Qiagen company and are identified by the following codes: CNR1 (No. PPH01504A), CNR2 (No. PPH02723A), diacylglycerol lipase alpha (DAGLA) (No. PPH14872A), diacylglycerol lipase, beta (DAGLB) (No. PPH12132A), FAAH (No. PPH23936C), GPR18 (No. PPH10393A), GPR55 (No. PPH11293B), MGLL (No. PPH19478A), PPARG (No. PPH02291G), and TRPV1 (No. PPH08086F). The gene expression levels of the investigated genes, expressed in Ct values, were normalized according to the ΔCt method, against the geometric mean of two housekeeping genes, hypoxanthine phosphoribosyltransferase 1 (HPRT1) (No. PPH01018C) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (No. PPH21138F), whose stability was previously assessed[24,25].

Statistical analysis

The Shapiro-Wilk test was applied to test the normal distribution of the gene expression levels. Since the normal distribution was lacking, non-parametric tests were used. Given that the non-IBD controls (CTRL) compared to both the IBD group and the UC group were not homogeneous in terms of sex distribution, the differences in gene expression between CTRL vs IM and CTRL vs NIM were evaluated using the nonparametric one-way covariance analysis (ANCOVA) (Quade’s) test, followed by Bonferroni correction. The differences in gene expression between CTRL and the CD groups were analyzed using the Mann-Whitney U test. The statistical significance between IM vs NIM was evaluated using the Related Samples Wilcoxon Signed Rank test. The statistical analysis was performed using Statistical Package for the Social Sciences (SPSS version 20.0, IBM, NY, United States), and the graphs were generated with GraphPad Prism 8.4.3 (GraphPad Software, San Diego, CA, United States). Using the G*Power 3.1.9.7 tool, we performed a power analysis to compute the required sample size for the paired analysis (IM vs NIM). We found that for the alpha error of 0.05 and a power (1-beta err prob) of 0.95, the required sample size was 47. Thus, a sample size of 60 mucosa (30 IM vs 30 NIM) included in this study should be considered sufficient.

RESULTS

The 30 IBD patients and the 17 non-IBD controls did not differ in terms of age (P = 0.756), but were not homogeneous in terms of sex distribution (P = 0.023). Stratifying IBD patients by UC and CD, we found that there was no difference in sex distribution for the CD group (P = 0.283) compared to the CTRL group, whereas it differed in the UC group compared to the CTRL group (P = 0.026). The socio-demographic and clinical features of the enrolled IBD patients and controls are reported in Table 1.

Table 1 Sociodemographic and clinical data of the individuals involved in the study.

Feature	CTRL (n = 17)	IBD (n = 30)	UC (n = 17)	CD (n = 13)	P value
Age	48.12 ± 17.46	49.60 ± 14.53	48.82 ± 11.89	49.31 ± 17.94	P = 0.756 (CTRL vs IBD); P = 0.741 (CTRL vs UC); P = 0.829 (CTRL vs CD)
Sex M, F	52.9% F; 47.1% M	20% F; 80% M	11.76% F; 88.24% M	30.77% F; 69.23% M	P = 0.020; χ2 = 5.419 (CTRL vs IBD); P = 0.026; χ2 = 6.585 (CTRL vs UC); P = 0.283; χ2 = 1.475 (CTRL vs CD)
Disease status		23.3% remission; 76.7% active	23.5% remission; 76.5% active	23.1% remission; 76.9% active	P = 0.999; χ2 = 0.977 (UC vs CD)
Age at onset (mean ± SD)		44.3 ± 14.55	48.82 ± 11.72	42.31 ± 17.93	P = 0.522 (UC vs CD)
Therapeutic regimen		23.3% drug free; 56.7% 5-ASA; 6.7% AZA; 13.3% biological treatment	29.4% drug free; 64.7% 5-ASA; 5.9% AZA	15.4% drug free; 46.2% 5-ASA; 7.7% AZA; 30.7% biological treatment	P = 0.096; χ2 = 6.336 (UC vs CD)
Extension (UC) and Location (CD)- Montreal			5.9% E1; 35.3% E2; 8.8% E3	15.4% L1; 23.1% L2; 61.5% L3
Behaviour- montreal				46.2% B1; 38.4% B2; 15.4% B3

IBD: Inflammatory bowel disease; UC: Ulcerative colitis; CD: Crohn’s disease; CTRL: Non-inflammatory bowel disease controls; M: Male; F: Female; 5-ASA: Aminosalicylates ; AZA: Azathioprine; E1: Proctitis; E2: Left-sided colitis; E3: Pancolitis; L1: Lerminal ileal; L2: Colon; L3: Leocolon; B1: Non-stricturing, non-penetrating; B2: Stricturing; B3: Penetrating.

Gene expression results: IBD vs CTRL

Among the ten genes analyzed, concerning the comparison between the IM mucosa of IBD patients (n = 30) and CTRL (n = 17), FAAH, PPARG, and TRPV1 genes were found to be significantly differentially expressed. When considering NIM in comparison with the mucosa from the CTRL group, a significant change in gene expression levels was found for FAAH and GPR55. The paired analysis IM vs NIM identified six genes differentially expressed: CNR2, FAAH, GPR18, GPR55, PPARG, and TRPV1. In particular, the level of CNR2, GPR18, and GPR55 was upregulated in the IM compared to the paired NIM, whereas the level of FAAH, PPARG, and TRPV1 was downregulated in the IM compared to both the paired NIM and the CTRL. In addition, FAAH level was significantly downregulated in the NIM compared to the CTRL. The results are presented in Figure 1 and Table 2.

Figure 1 Gene expression levels of the significant genes belonging to the endocannabinoid system in inflammatory bowel disease. Bar graphs refer to the mean of the 2^-ΔCt values in each group, and error bars represent the SEM. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. IBD: Inflammatory bowel disease; UC: Ulcerative colitis; CD: Crohn’s disease; CTRL: Non-inflammatory bowel disease controls; NIM: Noninflamed mucosa, IM: Inflamed mucosa; CNR2: Cannabinoid receptor 2; FAAH: Fatty acid amide hydrolase; GPR: G protein-coupled receptor 18; GPR55: G protein-coupled receptor 55; PPARG: Peroxisome proliferator-activated receptor gamma; TRPV1: Transient receptor potential cation channel, subfamily V, member 1.

Table 2 Gene expression levels in inflammatory bowel disease patients (mean ± SD).

Genes	2−∆Ct, CTRL (n = 17)	2−∆Ct, NIM (n = 30)	2−∆Ct, IM (n = 30)	1P value, case control (CTRL vs NIM)	1P value, case control (CTRL vs IM)	2P value, paired (IM vs NIM)
CNR1	0.014 ± 0.009	0.008 ± 0.005	0.018 ± 0.029	0.525	0.999	0.090
CNR2	0.010 ± 0.007	0.008 ± 0.006	0.025 ± 0.035	0.609	0.141	0.005
DAGLA	0.004 ± 0.003	0.005 ± 0.005	0.003 ± 0.003	0.924	0.999	0.153
DAGLB	0.006 ± 0.003	0.005 ± 0.003	0.005 ± 0.002	0.093	0.659	0.329
FAAH	0.032 ± 0.007	0.019 ± 0.014	0.012 ± 0.007	0.003	< 0.001	0.012
GPR18	0.013 ± 0.006	0.010 ± 0.005	0.014 ± 0.007	0.167	0.999	0.037
GPR55	0.008 ± 0.004	0.004 ± 0.002	0.008 ± 0.004	0.003	0.999	0.001
MGLL	0.107 ± 0.039	0.182 ± 0.266	0.127 ± 0.112	0.999	0.999	0.491
PPARG	0.088 ± 0.038	0.092 ± 0.055	0.045 ± 0.032	0.999	< 0.001	0.001
TRPV1	0.030 ± 0.015	0.023 ± 0.017	0.015 ± 0.010	0.248	0.002	0.032

¹Non-parametric one-way covariance analysis (ANCOVA) (Quade’s) followed by Bonferroni correction.

²Wilcoxon signed-rank test.

IBD: Inflammatory bowel disease; UC: Ulcerative colitis; CD: Crohn’s disease; CTRL: Non-inflammatory bowel disease controls; NIM: Noninflamed mucosa; IM: Inflamed mucosa; CNR2: Cannabinoid receptor 2; FAAH: Fatty acid amide hydrolase; GPR: G protein-coupled receptor 18; GPR55: G protein-coupled receptor 55; MGLL: Monoglyceride lipase; PPARG: Peroxisome proliferator-activated receptor gamma; TRPV1: Transient receptor potential cation channel, subfamily V, member 1; DAGLA: Diacylglycerol lipase alpha; DAGLAB: Diacylglycerol lipase beta.

Gene expression results: UC vs CTRL

In the UC subgroup, the analysis of IM vs CTRL identified FAAH, PPARG, and TRPV1 genes as differentially expressed, as also observed in the previous analysis (IBD vs CTRL). The IM vs NIM analysis conducted in the UC patients revealed a significant difference in CNR2, FAAH, GPR55, PPARG, and TRPV1 gene levels. Moreover, the comparison of NIM vs CTRL identified a significant dysregulation in the expression of FAAH and GPR55 genes. The results are presented in Figure 1 and Table 3.

Table 3 Gene expression levels in ulcerative colitis patients (mean ± SD).

Genes	2−∆Ct, CTRL (n = 17)	2−∆Ct, NIM (n = 17)	2−∆Ct, IM (n = 17)	1P value, case control (CTRL vs NIM)	1P value, case control (CTRL vs IM)	2P value, paired (IM vs NIM)
CNR1	0.014 ± 0.009	0.009 ± 0.006	0.014 ± 0.012	0.525	0.999	0.113
CNR2	0.010 ± 0.007	0.008 ± 0.007	0.021 ± 0.016	0.609	0.141	0.009
DAGLA	0.004 ± 0.003	0.005 ± 0.006	0.003 ± 0.003	0.924	0.999	0.113
DAGLB	0.006 ± 0.003	0.004 ± 0.002	0.005 ± 0.002	0.093	0.659	0.210
FAAH	0.032 ± 0.007	0.019 ± 0.011	0.012 ± 0.008	0.003	< 0.001	0.010
GPR18	0.013 ± 0.006	0.011 ± 0.006	0.014 ± 0.006	0.167	0.999	0.062
GPR55	0.008 ± 0.004	0.004 ± 0.002	0.007 ± 0.003	0.003	0.999	0.004
MGLL	0.107 ± 0.039	0.151 ± 0.127	0.089 ± 0.050	0.999	0.999	0.102
PPARG	0.088 ± 0.038	0.084 ± 0.045	0.047 ± 0.0035	0.999	< 0.001	0.019
TRPV1	0.030 ± 0.015	0.021 ± 0.011	0.014 ± 0.009	0.248	0.002	0.028

¹Non-parametric one-way covariance analysis (ANCOVA) (Quade’s) followed by Bonferroni correction.

²Wilcoxon signed-rank test.

IBD: Inflammatory bowel disease; UC: Ulcerative colitis; CD: Crohn’s disease; CTRL: Non-inflammatory bowel disease controls; NIM: Noninflamed mucosa; IM: Inflamed mucosa; CNR2: Cannabinoid receptor 2; FAAH: Fatty acid amide hydrolase; GPR: G protein-coupled receptor 18; GPR55: G protein-coupled receptor 55; MGLL: Monoglyceride lipase; PPARG: Peroxisome proliferator-activated receptor gamma; TRPV1: Transient receptor potential cation channel, subfamily V, member 1; DAGLA: Diacylglycerol lipase alpha; DAGLAB: Diacylglycerol lipase beta.

Gene expression results: CD vs CTRL

In the IM mucosa from CD patients, three genes (FAAH, PPARG, and TRPV1) were found to be differentially expressed compared to the CTRL mucosa, whereas the comparison of IM vs NIM revealed only PPARG as significantly downregulated. In addition, FAAH, GRP18, and GPR55 levels were significantly dysregulated in the NIM compared to the CTRL. The results are presented in Figure 1 and Table 4. When analyzing the gene expression levels in relation to the clinical characteristics of the patients, such as the age at onset, the therapeutic regimen, and the Montreal classification (including extension of UC, and location and behavior for CD), we did not find any significant association.

Table 4 Gene expression levels in Crohn’s disease patients.

Genes	2−∆Ct, CTRL (n = 17)	2−∆Ct, NIM (n = 13)	2−∆Ct, IM (n = 13)	1P value, case control (CTRL vs NIM)	1P value, case control (CTRL vs IM)	2P value, paired (IM vs NIM)
CNR1	0.014 ± 0.009	0.007 ± 0.003	0.024 ± 0.043	0.113	0.457	0.552
CNR2	0.010 ± 0.007	0.007 ± 0.006	0.030 ± 0.052	0.213	0.432	0.173
DAGLA	0.004 ± 0.003	0.004 ± 0.004	0.004 ± 0.002	0.536	0.742	0.701
DAGLB	0.006 ± 0.003	0.005 ± 0.004	0.005 ± 0.003	0.079	0.229	0.861
FAAH	0.032 ± 0.007	0.020 ± 0.017	0.012 ± 0.007	0.003	<0.001	0.279
GPR18	0.013 ± 0.006	0.009 ± 0.004	0.014 ± 0.008	0.048	0.967	0.249
GPR55	0.008 ± 0.004	0.004 ± 0.003	0.008 ± 0.006	0.008	0.869	0.064
MGLL	0.107 ± 0.039	0.222 ± 0.390	0.175 ± 0.152	0.680	0.263	0.507
PPARG	0.088 ± 0.038	0.102 ± 0.069	0.041 ± 0.030	0.805	<0.001	0.023
TRPV1	0.030 ± 0.015	0.026 ± 0.023	0.017 ± 0.011	0.320	0.007	0.279

¹Mann-Whitney U test.

²Wilcoxon signed-rank test.

IBD: Inflammatory bowel disease; UC: Ulcerative colitis; CD: Crohn’s disease; CTRL: Non-inflammatory bowel disease controls; NIM: Noninflamed mucosa; IM: Inflamed mucosa; CNR2: Cannabinoid receptor 2; FAAH: Fatty acid amide hydrolase; GPR: G protein-coupled receptor 18; GPR55: G protein-coupled receptor 55; MGLL: Monoglyceride lipase; PPARG: Peroxisome proliferator-activated receptor gamma; TRPV1: Transient receptor potential cation channel, subfamily V, member 1; DAGLA: Diacylglycerol lipase alpha; DAGLAB: Diacylglycerol lipase beta.

DISCUSSION

In this work, we focused on the gene expression analysis of ten components of the ECS in the paired colonic mucosa (IM vs NIM) of patients with IBD and normal mucosa from controls, and identified an impairment of six genes: CNR2, FAAH, GPR18, GPR55, PPARG, and TRPV1. Stratifying the IBD patients into UC and CD groups, the same impairment trend in terms of gene expression was observed, except for GPR18. The ECS is involved in intestinal homeostasis and plays an important role in IBD by modulating visceral sensation, intestinal permeability, gastrointestinal mobility, and inflammation[4,26]. Despite the substantial knowledge on the beneficial impacts of cannabinoids in IBD, a significant gap remains between the preclinical findings and understanding the role of the ECS in gastrointestinal diseases in humans[4,27-29]. A limited number of studies have investigated the expression profile of the ECS in the human gut, and no study has been conducted on the Romanian population.

In inflammatory conditions, the ECS is upregulated and acts to restore homeostasis[30]. Our findings suggest that this might be particularly true for CNR2, GPR55, and GPR18 found to be upregulated in the IM of the entire cohort of IBD patients, whereas FAAH, PPARG, and TRPV1 showed a downregulation. In the UC and CD groups, the significant upregulation of CNR2 in the IM was confirmed only in UC patients, while in CD patients, the CNR2 increased levels have not reached statistical significance, probably due to the high standard deviation detected in this IM group. In line with our results, Leinwand et al[31] demonstrated that in the ileum of actively IM 8-12-week-old mice, the expression of CNR2 was significantly increased compared to the control mice. Moreover, treatment with a CNR2 agonist, JWH-133, decreases matrix metalloproteinase-9 and interleukin-8 Levels in IM biopsies from individuals with IBD, suggesting that CNR2 plays a role in promoting mucosal healing in IBD[32]. Furthermore, our study has shown that the expression of GPR55 was upregulated in IM compared to NIM with a statistical significance in the UC group. Together with the available data, our results might indicate that the associations between GPCRs and disease activity, severity, and phenotypes offer the potential to employ GPCR markers for the diagnosis and monitoring of patients with IBD[33]. On the same note, Lin et al[34] observed an upregulation of GPR55 expression at both the gene and protein levels in the IM intestine of rats. This suggests that the activation of GPR55 may play a role in regulating intestinal functions under pathophysiological conditions.

In our study, TRPV1 was downregulated in IM, showing lower expression compared to NIM and CTRL within the IBD and UC groups. This gene is thought to exert a protective role against inflammatory changes by promoting the synthesis and/or release of endovanilloids. Several studies in which TRPV1 agonists were used have demonstrated that TRPV1 receptors could have protective effects by acting as anti-inflammatory and immunomodulatory agents in inflammatory diseases[4,35]. In line with our findings, it has been stated that the expression of TRPV1 was found to be downregulated in patients with active UC and CD compared with controls, and by reducing the expression of TRPV1, the macroscopic epithelial damage in dextran sulfate sodium-induced colitis in mice was reduced[36].

In the context of inflammatory processes, AEA has a protective role in the gut and can be hydrolyzed to arachidonic acid by FAAH[4]. Our results show that the expression of FAAH in IM of both UC and CD patients was downregulated compared to NIM. Given that the levels of AEA were reported to be elevated in the plasma of UC and CD patients, this could explain the low levels of its degrading enzyme, FAAH[30]. Still, we cannot dismiss the possibility that our results could reflect the presence of a compensatory system applicable to the patients included in this study. In the present work, we also found that the expression level of PPARG was significantly decreased in IM than in NIM and mucosa from control, both in patients with UC and CD. Our results are partially in line with the findings of Dou et al[19], reporting that the expression of PPARG was decreased in UC patients and was negatively associated with the disease activity. Additionally, it was shown that in CD patients, mesentery adipocytes overexpress tumor necrosis factor-alpha and PPARG, which could suggest different mechanisms for inflammatory response[37].

The role of ECS in various mechanisms and processes in gastrointestinal physiology, and the exact involvement of this system in IBD, is still under investigation. To accomplish this, assessing the ECS gene expression signature in IBD could serve as a starting point to collect preliminary results. In this light, our findings, showing the impairment of specific ECS genes in the IBD mucosa, albeit preliminary, may be useful for inspiring extensive studies in large cohorts to better understand the potential of cannabinoids in managing IBD. Moreover, the development of targeted and effective cannabinoid-based therapies may offer new hope for individuals struggling with the challenges of this pathology. However, it is crucial to approach these potential treatments with a balanced perspective, considering both the benefits and risks associated with cannabinoid use in medical contexts. The relatively small sample size of this study represents a limitation; thus, extensive research on a larger cohort is needed.

CONCLUSION

The role of ECS in gastrointestinal physiology and the exact involvement of this system in IBD are still under investigation. Given this, our preliminary findings of the impairment of analyzed ECS genes in the IBD mucosa may serve as a basis for more in-depth research in larger cohorts to better understand the potential of cannabinoids in the management of IBD. After the introduction of artificial intelligence in the multi-omics drug delivery pipeline, future therapeutic targets should emerge, allowing for an even more personalized approach to IBD patients