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ORIGINAL ARTICLE

Marjoram oil attenuates oxidative stress and improves colonic epithelial barrier function in dextran sulfate sodium-induced ulcerative colitis in Balb/c mice

Bouayad DI1*, Hadria Grar1,2, Sadia Berzou3, Omar Kheroua1, Djamel Saidi1,4, Hanane Kaddouri1

1Laboratory of Physiology of Nutrition and Food Safety, Department of Biology, Faculty of Natural and Life Sciences, University Oran 1 Ahmed Ben Bella, Oran, Algeria;

2Department of Biology, Faculty of Natural and Life Sciences, University of Mostaganem, Algeria;

3Laboratory of Clinical and Metabolic Nutrition, Department of Biology, University Oran 1 Ahmed Ben Bella, Oran, Algeria;

4Higher School of Biological Sciences of Oran (ESSBO), Oran, Algeria

Abstract

In this study, we explored the effects of marjoram oil (MO) on dextran sulfate sodium (DSS)-induced colitis in Balb/c mice. Marjoram oil was found to significantly reduce the severity of DSS-induced colonic inflammation, as reflected by improved disease activity index, prevented colon length shortening, lower histopathological score, decreased myeloperoxidase activity, and reduced interlukin 6 (IL-6) levels. Moreover, marjoram oil pretreatment enhanced the colonic epithelial integrity by decreasing paracellular permeability. Marjoram oil was found to clearly reduce the colonic levels of thiobarbituric acid reactive substances assay and enhance the activity of superoxide dismutase, catalase, and glutathione sulfhydryl content. Marjoram oil could exert a protective effect on ulcerative colitis through its anti-inflammatory and antioxidant properties.

Key words: dextran sulfate sodium, experimental colitis, Origanum majorana, oxidative stress, inflammation, Ussing chamber

*Corresponding Author: Ismahan Bouayad Debbagh, Laboratory of Physiology of Nutrition and Food Safety, Department of Biology, Faculty of Natural and Life Sciences, University Oran 1 Ahmed Ben Bella, BP 1524 EL M’Naouer, Oran, Algeria. Email: [email protected]

Received: 26 December 2022; Accepted: 7 February 2023; Published: 16 March 2023

DOI: 10.15586/ijfs.v35i1.2320

© 2023 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

Inflammatory bowel disease (IBD) is a long-term gastrointestinal disorder marked by recurrent inflammation and extensive mucosal destruction in the intestine (Mijan and Lim, 2018). Histologically, extensive ulcerated regions with significant neutrophil infiltration and epithelial cell necrosis characterize ulcerative colitis (UC) (Wang et al., 2019). Although the cause of IBD is unknown, various factors are known to have a role in its development, including breakdown of the epithelial barrier, a decrease in commensal bacteria tolerance, immunologic alterations, oxidative stress, and an increase in inflammatory mediators (Liu et al., 2017; Goyal et al., 2014). Immunological cell infiltration into the gut’s lamina propria, as well as uncontrolled synthesis of pro-inflammatory mediators and reactive oxygen species (ROS) in colonic tissues, contributes to the breakdown of intestinal barrier integrity and an increased inflammation of the mucosa (Maloy and Powrie, 2011).

An overproduction of ROS causes lipid peroxidation which reduces cellular antioxidant capacity and leads to severe colonic inflammation (Tahan et al., 2011). Endogenous antioxidants can ordinarily protect the intestinal mucosa from oxidative damage (Mandalari et al., 2011). Nevertheless, inflammation increases the demand for these antioxidants, resulting in a pro-oxidant–antioxidant imbalance, resulting in mucosal damage (Mandalari et al., 2011; Zhu and Li, 2012).

Myeloperoxidase (MPO) activity is another indicator of the severity of ulcerative colitis. In colitis, MPO is widely used as a marker of oxidative stress and gastrointestinal inflammation (Klebanoff et al., 2005). A previous investigation demonstrated that inflammatory cytokine-caused apoptosis was found to have a deleterious impact on the structure of adherent junction and epithelial barrier functions (Su et al., 2013).

Animal models of IBD are suitable for studying the pathogenesis of the disease and developing new drugs. Dextran sulfate sodium (DSS)-provoked acute colitis is among the most regularly adopted methods (Bang and Lichtenberger, 2016) because of its great homogeneity, lesions’ reproducibility predominantly in the distal colon, and similarity to clinical IBD (Valatas et al., 2013).

Currently, several therapies, such as nonsteroidal anti-inflammatory medications, corticosteroids, immune-suppressing medications, and biological agents are being investigated (Baakhtari et al., 2018; Yang et al., 2015). Nevertheless, homeostasis and maintenance of intestinal barrier are rarely emphasized in conventional pharmacotherapy, and interest in integrative and complementary medicines is growing (Larussa et al., 2017). Patients with IBD frequently use complementary and alternative medicines (CAMs) (Cheifetz et al., 2017; Lin and Cheifetz, 2018), which may include plants or herbal items, probiotics, and prebiotics (Cheifetz et al., 2017). Thus, in IBD patients, use of CAM, particularly herbal medicines, is increasing as a safe anti-oxidant/anti-inflammatory therapeutic option (Wan et al., 2014).

Owing to their high phenolic content, plants belonging to family Lamiaceae are studied extensively as natural antioxidant sources (Trouillas et al., 2003). Marjoram (Origanum majorana) is the most important member of family Lamiaceae, which includes a wide variety of aromatic herbs and shrubs and volatile oil plants (Paterson et al., 2006).

Polyphenols, such as flavonoids, having several therapeutic properties are found in abundance in Origanum majorana (Goel and Vasudeva, 2015). Leaves of Origanum majorana are found to exhibit antioxidant (Duletic et al., 2018; Erenler et al., 2016), antimicrobial (Della Pepa et al., 2019; Guerra-Boone et al., 2015), anti-neurodegenerative (Della Pepa et al., 2019; Duletic et al., 2018; Erenler et al., 2016; Guerra-Boone et al., 2015), hepatoprotective (Mossa et al., 2013), cardioprotective (Ramadan et al., 2013), antiulcer (Al-Howiriny et al., 2009), anti-inflammatory (Arranz et al., 2015), and anticancer properties (Al Benhalilou et al., 2019 Dhaheri et al., 2013). Origanum majorana has also demonstrated positive effects in acute infectious diarrhoea (Makrane et al., 2018). Recent studies have demonstrated that essential oil of Origanum majorana suppressed the development of colon cancer cells through p38 mitogen-activated protein kinase (MAPK)-provoked apoptosis in vitro and in vivo (Athamneh et al., 2020).

To date, no scientific data exist on the effect of Origanum majorana on intestinal inflammation. Therefore, the purpose of our study was to evaluate the possible protective effect of Origanum majorana on DSS-provoked colitis in Balb/c mice.

Materials and Methods

Chemicals

Marjoram oil was obtained from commercial CAP Pharm (EL Captain Company, Egypt). Dextran sulfate sodium (40 kDa), fluorescein isothiocyanate conjugated dextran (FITC-Dextran; catalog number: FD4 [4 kDa]), and mouse interlukin 6 (IL-6) enzyme-linked-immunosorbent serologic assay (ELISA) kit were purchased from Sigma Aldrich (France). The remaining solvents and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Marjoram oil composition was analyzed using Perkin Elmer Clarus 500 gas chromatograph capillary column HP-5 (30 m × 0.25 mm ID, 0.25-µm film thickness), coupled to Clarus 500 mass spectrometer (GC-MS). More details about the chemical composition of marjoram oil were reported earlier by Bouayad Debbagh et al. (2021).

Experimental animals

Male Balb/c mice, 8-week old, were kept at the University of Oran’s animal facility with a 12-h light–dark cycle at 23 ± 2°C. Animals had free access to commercial standard diet and water. All experimental procedures involving animals were approved by the current Algerian legislation covering the protection of animals.

Colitis induction and design of treatment

Animals were divided into the following four groups, with eight mice in each group: control group (Ctrl) received tap water; DSS group (DSS) received DSS 4% (40 kDa; Sigma-Aldrich) in their drinking water ad libitum for 7 days; marjoram oil group (M) received 0.5-mL/kg body weight per os (by gavage) of marjoram oil for 14 day; and marjoram oil + DSS group (M+DSS), in which mice received MO (0.5 mL/kg body weight) orally for 14 day and were exposed to DSS 4% for 7 days (day 15–21) (Figure 1).

Figure 1. Experimental protocol of the study.

Clinical colitis severity assessment

The disease activity index (DAI) was measured daily on the basis of clinical manifestations of colitis (Cooper et al., 1993) comprising body weight, faecal bleeding, stool consistency, and diarrhoea (Table 1).

Table 1. Disease activity index (DAI) score (Cooper et al., 1993) (n = 8).

Score Weight loss Stool consistency Bleeding
0 None Normal No bleeding
1 1–5%
2 5–10% Loose stools Slight bleeding
3 10–15%
4 More than 15% Watery diarrhoea Gross bleeding

Analysis of colon inflammation

The animals were sacrificed after 21 days of treatment. Blood was collected and the plasma was kept at −80°C for assays. The colon was excised and washed with saline solution. The length and weight of colon was measured. A 5-mm section of the distal colon was put in freshly prepared 10% (w/v) formaldehyde (pH 7.0), processed, and paraffin-embedded for histopathological evaluation. The mid-distal portion (30–60% region) was used for electrophysiological studies. The remaining colon tissue was kept at −80°C for additional biochemical analysis.

Ex vivo experiments: Ussing chamber assay

After sacrifice, distal colon was removed and excised through mesenteric border. Colonic tissue samples were immediately placed in Ussing chambers as sheets (Physiologic Instrument, San Diego, CA) with an exposed area of 0.2 cm2. Both sides were filled with Ringer solution (5 mL), oxygenated continuously (95% O2 and 5% CO2), and maintained at 37°C. The mucosal-to-serosal flux of FITC-Dextran with a 4-kDa mean molecular mass (FD4) was measured across colonic samples to determine paracellular permeability.

Colonic paracellular permeability was determined by measuring mucosal-to-serosal flux of FITC-Dextran across colonic samples. After a stabilization period of 15–25 min, the mucosal reservoir was injected with FD4 at a concentration of 2.2 mg/mL. For measuring FD4, basolateral samples (500 μL replaced by appropriate 500-μL buffer solution) were taken for 90 min at 30-min intervals for recording electrophysiological parameters. The concentration of FD-4 was determined using a fluorescence microplate reader with a 520-nm emission wavelength and a 490-nm excitation wavelength (Perkin-Elmer, Waltham, MA, USA). The apparent permeability coefficient (Papp) was calculated for this period as follows:

Papp (cm/s × 10−6) = dc/dt × (V/(A × C0)),

where dc/dt is the change in serosal concentration during 60–90 min (mol/L/s) period, C0 is the initial marker concentration in mucosal reservoir (mol/L), “V” is the volume of serosal reservoir (cm3), and “A” is the exposed intestinal area in the chamber (cm2).

Histological assessment of colitis

Distal colon sections were taken and fixed in phosphate-buffered formalin (10%), and then dried and paraffin-embedded. For light microscopic examination, these samples were stained with hematoxylin and eosin (H&E). Histological damage was determined using the scoring system of Stillie and Stadnyk (2009).

Measurement of IL-6 level

Level of IL-6 in plasma samples was measured in duplicate using commercially mouse IL-6 ELISA kit (Sigma-Aldrich) according to manufacturer’s instructions.

Myeloperoxidase activity assay

The MPO activity is defined as the quantity of enzyme that degraded 1 μmol of H2O2 per minute. It was measured in units per milligram of protein. In this study, the MPO activity was measured in proximal colon tissue according to Lenoir et al. (2011). In a 50-mM phosphate buffer with hexadecyl trimethyl ammonium bromide (0.5%), a section of colon was homogenized in ice with a T25 Ultra-Turrax system (HTAB; IKA, Staufen, Germany). The homogenate was sonicated in ice for 30 s prior to centrifugation at 3,200 g for 10 min at 4°C. The supernatants were frozen and thawed thrice prior to being centrifuged for 10 min at 3,200×g at 4°C.

In microcuves, 50 μL of supernatants were combined with 1 mL of phosphate buffer (10 mM), 500 μL of 0.22% guaiacol, and 10 μL of H2O2 (0.3 %). After 3 min, absorbance was measured at every minute for 20 min at 470 nm. The study was performed in triplicate.

In vivo antioxidant activity

Homogenization of tissues and quantification of proteins

In ice cold water, frozen tissue specimens were weighed (range 100 mg) and homogenized with 0.9-mL potassium phosphate buffer (0.12 mmol/L, pH = 7.4).

The samples were centrifuged twice at 3,000×g for 20 min at 4ºC. Superoxide dismutase, catalase, glutathione peroxidase (Gpx) activities, and reduced glutathione level were determined in all supernatants. Protein content was measured according to the method described by Lowry et al. (1951).

Thiobarbituric acid reactive substances assay (TBARS)

Lipid peroxidation was measured by the method of Salih et al. (1987), In which 2 g of colon was homogenized in 30 mL of trichloroacetic acid (10%) and 200 µL of butyl hydroxyl toluene. Samples were centrifuged for 10 min at 3,500 rpm. Thiobarbituric acid (0.02 M), 5 mL, was then added to 5 mL of supernatant and the mixture was heated for 30 min at 100°C. Absorbance of the supernatant was read against blank specimen at 532 nm. TBARS was expressed in nanomole (nmol) of MDA (malondialdehyde) per gram of tissue.

Catalase (CAT) activity was determined according to the method of Aebi (1984), in which 20-µL of supernatant was mixed with 1.8 mL of 100 mmol/L phosphate buffer (pH 7.4) and 200-µL H2O2 (50 mM). Following a decrease in absorbance at 240 nm, CAT activity was measured spectrophotometrically in units/milligram of protein, compared to a standard curve.

Superoxide dismutase (SOD) was determined according to the method described by Marklund and Marklund (1974). The mixture included 970 µL of Tris-HCl (50 mM, pH 8.2) and 15 µL of supernatant. The reaction began by adding 15 µL of pyrogallol (12-mM pyrogallol in 1-mM HCl) to the mixture. A control was prepared from the same mixture by adding 985-µL Tris-HCl instead of 15-µL of pyrogallol. Change in absorbance was measured during a 5-min period at a wavelength of 325 nm, and variation in absorbance per minute was calculated and expressed per milligram of protein, corresponding to the quantity of enzyme that suppressed auto-oxidation of pyrogallol by 50%.

Glutathione peroxidase was determined by Rodrigues et al. (2002) method, in which 200 µL of tissue homogenate was added to a solution of Tris-HCl (0.4 M, pH 7), 0.1-mL sodium azide (10 mM), and 0.2-mL reduced glutathione (1 mM) followed by 0.1 mL of 0.2-mM hydrogen peroxide. After a 10-min incubation period at room temperature, the mixture was centrifuged at 1,200×g for 10 min after adding 0.4 mL of 10% trichloroacetic acid. After this, 0.2 mL of supernatant and 0.1 mL of Ellman’s reagent were combined, and the absorbance was measured at 340 nm.

Reduced glutathione (GSH) levels in colon tissues were assessed according to the method described by Sedlak and Lindsay (1968). GSH reacts with Dithiobis-2-nitrobenzoic acid (DTNB) to produce thio-2-nitrobenzoic acid, which has a wavelength of 412 nm. For measuring it, 1 mL of tissue homogenate (100-mg sample diluted with 0.9 mL of 0.12 mmol/L phosphate buffer, pH 7.2) was combined with 800 μL of ice-cold distilled water and 200 μL of trichloroacetic acid (50%) and incubated for 15 min; 400 μL of supernatant was mixed with 800 μL of Tris buffer (0.4 mol/L, pH 8.9) and 20-μL DTNB reagent (0.01 mol/L) after centrifugation at 1,200×g for 15 min. After 5 min of incubation, the absorbance of the reaction mixture was measured at 412 nm against a blank reagent.

Statistical analysis

Data were presented as mean ± standard error of mean (SEM). Results were examined by one-way ANOVA followed by Fisher’s least significant difference (LSD) test (Statistica Stat Soft, Maisons-Alfort, France); p < 0.05 was considered as statistically significant.

Results

Marjoram oil improved DSS-induced colitis

Mice exposed to DSS-induced colitis, as evidenced by severe anorexia, weight loss (Figure 2A), reduction in DAI score from day 5 until the last experimental day (Figure 2B), decreased colon weight (Figure 3B), and drab hair color. The length of colon in DSS-treated mice was significantly shorter by 43.69% (p < 0.001) than that in mice of both control and marjoram groups (Figure 3A), indicating the successful establishment of mice colitis.

Figure 2. Marjoram oil attenuated clinical signs of dextran sulfate sodium (DSS)-induced colitis in mice. (A) Body weight changes *p < 0.05, **p < 0.01 J21 vs J14. (B) Scores of disease activity index (DAI) in marjoram pre-treated mice and colitic (DSS) mice monitored every day. Data are expressed as the mean ± SD (n = 6 mice/groups). *p < 0.05 compared with DSS.

Figure 3. (A) Colon length. (B) Colon weight. (C) Colon macroscopic analysis. Data are expressed as mean ± SD (n = 6 mice/groups). ##p < 0.01, ###p < 0.001, compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the DSS group.

Pre-treatment of colitic mice with marjoram oil showed a reduction in the severity of colitis as evidenced by a decreased weight loss, prevented colon shortening, and reduced DAI score on day 8 (p < 0.05). The length of colon was significantly restored (27%) in the (M+DSS) group, compared to the DSS group (p < 0.001).

Marjoram oil alleviated DSS-induced colonic cellular permeability

After DSS treatment, FITC-dextran permeation was significantly higher, compared to both control and marjoram groups (p < 0.05). However, mice from the (M+DSS) group possessed a lower colonic permeability to FITC-dextran (p < 0.05; Figure 4A). To determine the functional viability and integrity of colonic epithelium under ex vivo conditions, transepithelial ion conductance measurements were done for 120 min. As illustrated in Figure 4B, transepithelial ion conductance in colonic tissues from DSS group was considerably (p < 0.05) higher than that of the control group, indicating that colonic permeability to ions was relatively increased in colitic group. In contrast, the (M+DSS) group showed a significant recovery (p < 0.01) in transepithelial ion conductance. These results suggested that marjoram oil could decrease paracellular permeability by maintaining colonic epithelial integrity.

Figure 4. Analysis of basal gut permeability in male Balb/c mice. (A) Colonic permeability to FITC-dextran (FD4) was measured in Ussing chamber. (B) Transepithelial ion conductance was measured in the colon. Bars indicate mean and SD. n = 6/group. (*p < 0.05, **p < 0.01 compared to DSS group; #p < 0.05, ##p < 0.01 compared to control group).

Marjoram oil ameliorated DSS-induced histopathological damage

Representative results and microscopic scores are illustrated in Figure 5. The DSS-treated mice had severe epithelial damage, including important cellular invasion into the lamina propria and colon mucosa, goblet cell depletion, thickening of the mucosa, and total architecture alteration, leading to a high microscopic damage score (Figure 5B). However, pre-treatment of DSS-treated mice with marjoram oil reduced infiltration of inflammatory cells with a minimum decrease of epithelial cells, resulting in a significantly reduced microscopic damage score when compared to colitic mice. These results suggested the effective protection rendered by marjoram oil to counter DSS-induced colitis.

Figure 5. Effects of marjoram oil on histological damage in DSS-induced mice colitis. (A) Histological appearance of colonic tissue sections (H&E, G×10). (B) Changes in histological score. Data are expressed as the mean ± SD (n = 6). **p < 0.01 is significantly different from the DSS group, and ###p < 0.001 is significantly different from the control group.

Marjoram oil decreased pro-inflammatory cytokine levels

As shown in Figure 6, induction of colitis by DSS produced a marked increase in IL-6 level (p < 0.01). However, pre-treatment with marjoram oil significantly reduced the IL-6 content in plasma (p < 0.05) when compared to the DSS group.

Figure 6. Effect of marjoram oil on plasma levels of IL-6 in mice treated with 4% DSS. Statistical analysis was performed using one-way Anova. Values are mean ± SD (n = 6 for each group). *Indicates significant differences (*p < 0.05, **p < 0.01) compared with DSS group. ##p < 0.01 compared to control.

Marjoram oil reduced colonic myeloperoxidase activity

In the DSS group, colonic inflammation resulted in an increase in the activity of MPO in comparison to the control group (p < 0.001), reflecting the enhanced neutrophil infiltration that characterized this inflammatory process. However, pre-treatment of DSS-treated group with marjoram oil significantly reduced the colonic activity of MPO, compared to colitic animals (p < 0.001, vs the DSS group; Figure 7).

Figure 7. Colonic myeloperoxidase activity. Data represent mean ± SD (n = 6 per group), ###p < 0.001 compared with control group; ***p < 0.001 compared with DSS group.

Marjoram oil decreased oxidative stress in DSS-induced colitis

In the DSS group, substantial oxidative stress was generated in the colonic mucosa as evidenced by a marked increase in TBARS (Figure 8) levels and a significant decrease in SOD and CAT activity in the colon (Figures 9A and 9B). GSH content in the colon of the DSS-treated mice was relatively lesser than that in the control and marjoram oil groups (p < 0.01; Figure 9D). Interestingly, marjoram oil reversed the loss of SOD and CAT activity (p < 0.01) and increased GSH content, compared to the DSS group. Furthermore, colonic levels of TBARS were reduced in colitic mice pre-treated with marjoram oil. However, no significant changes in glutathione peroxidase activity were observed in the colonic tissues of different groups (Figure 9C).

Figure 8. Effect of marjoram oil on the TBARS levels in colonic tissue of DSS-treated mice (n = 6). Values are expressed as mean ± SD and analyzed using one-way analysis of variance followed by Anova. (##p < 0.01, compared to control; **p < 0.01 compared with DSS group).

Figure 9. Effect of marjoram oil on (A) catalase, (B) superoxide dismutase, (C) glutathione peroxidise, and (D) total glutathione activities in the colonic tissues of DSS-treated mice (n = 6). Values are expressed as mean ± SD and analyzed using one way analysis of variance followed by ANOVA. #p < 0.05, ##p < 0.01, ###p < 0.001, compared to control; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the DSS group).

Discussion

One of the two main types of IBDs is ulcerative colitis, increasing rapidly in prevalence globally (Kokkinidis et al., 2017). Anti-inflammatory medications, including corticosteroids and 5-amino salicylic acid and its derivatives, are frequently used to treat IBD. In contrast, because of the development of additional oxidative stress, these medications are considered as a double-edged sword, as they cause hepatotoxicity and other adverse reactions (Uko et al., 2012). For this reason, natural products have garnered substantial research interest and are being investigated extensively. Our study explored the preventive effects of marjoram oil as a therapeutic strategy for acute ulcerative colitis caused by DSS.

In this study, a colitis model was established with mice being fed with 4% DSS solution for 7 days. DAI score was used as an indicator to evaluate the gravity of gastrointestinal disease in experimental groups. Our results showed that the DSS-treated mice displayed abnormal physiological conditions with an increased clinical score (DAI), indicating the earlier onset of colitis symptoms, such as diarrhoea, bloody stools, and reduced body weight as reported by Cooper et al. (1993). Higher DAI score was related to smaller colon length and elevated histopathological score. DSS is a heparin-like polysaccharide. It is considered to cause mucosal damage and inflammation initially through a direct toxic influence on intestinal epithelial cells by disrupting mucosal barrier to cause colitis (Perse and Cerar, 2012). DSS also causes indirect damage because of modifications in resident bacteria (Okayasu et al., 1990) followed by the mustering and activation of inflammatory cells and the upregulation of inflammatory mediators, resulting in severe colitis (Lewellyn et al., 2017).

The clinical symptoms of IBD induced by DSS were lessened by marjoram oil. Administration of marjoram oil to colitic mice resulted in a considerably reduced symptoms of the disease, such as diarrhoea, bloody stools, and weight loss, which were evidenced by a lower DAI score. In accordance with previous results reported by Al Howiriny et al. (2009), histopathological damage of the gastric mucosa was significantly prevented by administering marjoram extract.

IBD is characterized by inflammation that affects the integrity of epithelial barrier (Landy et al., 2016). Tissue conductance or its reciprocal transepithelial electrical resistance is considered as a sensitive biomarker of intestinal barrier integrity and its function (Catanzaro et al., 2015). Changes in transcellular and paracellular ion transport through the epithelium of colonic mucosa, or a disruption in the functional integrity of intercellular tight junctions, could explain an increase in electrical conductance (Mlodzik-Danielewicz and Tyrakowski, 2005).

In the present study, DSS was found to increase the electrical conductivity and cellular permeability to FITC-dextran. Our results were in line with the study conducted by Kitajima et al. (1999), which showed that DSS enhanced colonic mucosal permeability, resulting in the alteration of inflammatory reactions and mucosal barrier function. Indeed, loss of intestinal barrier function in the DSS colitis model was related to changes in tight junction protein distribution (Uko et al., 2012). The impairment of intestinal barrier function induced by DSS in our study was clearly diminished by marjoram oil in colitic mice. This amelioration was related to a decrease in IL-6 level, which plays an integral role in developing IBD (Helieh et al., 2013; Mazur-Bialy et al., 2017). Our data supported the previously reported anti-inflammatory properties of marjoram oil. Arranz et al. (2015) indicated that sabinene hydrate and terpineol in sweet marjoram essential oil inhibited the production of proinflammatory cytokines.

In the current study, the status of inflammation in colitic animals was confirmed by an increase in MPO activity, a specific biomarker of inflammation in colonic mucosa (Malle et al., 2007). However, pre-treatment with marjoram oil significantly reduced the colonic MPO activity. MPO is highly expressed in neutrophils, but less so in monocytes and certain macrophages (Malle et al., 2007). Its activity is linearly related to neutrophil infiltration (Iba et al., 2003). Bhattacharyya et al. (2014) showed that MPO was able to catalyse the generation of ROS.

When ROS formation surpasses the antioxidant capability of cells, cellular macromolecules are destroyed. This oxidative stress is hypothesized to have a role in the development of several human diseases, particularly ulcerative colitis (Mekkioui and Djerdjouri, 2012; Xu et al., 2009). It was reported that DSS-treated mice exhibited a significant increase in lipid peroxidation (Amirshahrokhi et al., 2011; Yan et al., 2015). This was in line with the results of our research, in which TBARS concentrations were higher in colitic mice than in other groups. Interestingly, pre-treatment with marjoram oil resulted in a considerable reduction of TBARS level, demonstrating the antioxidant activity of marjoram oil. This result confirmed the results of our recent study, in which marjoram oil demonstrated a strong in vitro antioxidant power (Bouayad Debbagh et al., 2021).

Glutathione sulfhydryl is thought to be the first line of defense against oxidative stress and inflammatory cascades (Zhu and Li, 2012). GSH is the main reducing agent for colonic epithelial cells; it is frequently reported to diminish during severe DSS-induced colitis (Oz et al., 2005; Wardman et al., 2007). Hence, in agreement with the above findings, we discovered lower GSH levels in the colitic group.

Enzymatic antioxidants are another line of oxidative defense system. In the current study, the activity of CAT and SOD was reduced in the colonic tissues of colitic group. Similar to what was observed in our study and in human IBD patients (Mueller et al., 2013), several studies have revealed that when rodents are exposed to DSS, enzymatic antioxidants are depleted, suggesting enhanced oxidative stress-mediated colon injury (Nishiyama et al., 2012; Pandurangan et al., 2015; Xing et al., 2013).

Conclusions

Our data suggested that marjoram oil lessened DSS-induced experimental colitis as evidenced by reduced severity of ulcerative colitis, preventing oxidative damage, reducing neutrophil infiltration, and inhibiting IL-6 production. Importantly, marjoram oil preserved the intestinal barrier function from damage caused by inflammatory stimuli.

Acknowledgments

This research was supported by the Directorate General for Scientific Research and Technological Development (DGRSDT, MESRS), Algeria.

Conflict of interest

The authors declared no conflict of interest with respect to research, authorship, and/or publication of this article.

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