Benecial role of Pistacia lentiscus aqueous extract in experimental colitis: Anti-inammatory and potential therapeutic effects

Background: Pistacia lentiscus L. (PL) is a owering plant traditionally used in the treatment of gastrointestinal disorders. The extracts of this plant are endowed with strong pharmacological activities. The aim of our current study was to investigate the anti-inammatory and potential therapeutic effects of PL leaves aqueous extract (PLAE) against Dextran Sulfate Sodium (DSS)-induced acute colitis. Materials and methods: The therapeutic effect of PLAE was evaluated after orally administration of 3% DSS alone or concomitantly with PLAE (50, 100 or 200 mg/Kg). Mucosal lesions were assessed by macroscopic and histopathological examination. In this context, hemorrhage, diarrhea, weight loss, and disease activity index (DAI) were determined daily throughout the experiment. In the same way, hematoxylin-eosin and Alcian blue staining of colonic mucosal were used to evaluate respectively mucosal damages and mucus production. Furthermore, the levels of nitric oxide (NO), and pro-inammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] were measured in plasma, as well as in colonic explants and peritoneal macrophages cultures supernatants. Results: Administration of DSS+PLAE indicated a signicant reduction in clinical score of acute colitis DAI compared to DSS alone administration. Interestingly, histological analysis of the mucosa showed that DSS+PLAE-treated groups exhibited almost normal histology evidenced by an intact epithelium structure and less inammatory cell inltration in the mucosa. Alcian bleu staining revealed that DSS+PLAE-treated groups displayed almost normal mucus production. Importantly, a signicant decrease in pro-inammatory mediators (NO, IL-6 and TNF-α) levels in dose dependent manner was reported in plasma, and culture supernatants of colonic explants and peritoneal macrophages from DSS+ PLAE-treated mice compared to the DSS group. Conclusion: Our results showed that the systemic and local anti-inammatory


Introduction
In ammatory bowel disease (IBD) constitute a group of chronic and relapsing idiopathic in ammatory disorder of the intestine that include Crohn's disease (CD) and ulcerative colitis (UC) (Caruso et al. 2020, Geboes et al. 2018. The exact factors that trigger the chronic and relapsing intestinal in ammation remain unknown. Multiple lines of evidence suggest that the disease is caused by a con uence of genetic and environmental factors that alter gut homeostasis. It is also thought that IBD results from an aberrant and continuing immune response against endogenous ora and luminal antigens (Zhang and Li, 2014, Sou i et al. 2016, Toumi et al. 2021. Although the pathogenesis of both Crohn's disease and ulcerative colitis involves intestinal in ammation, these two disorders differ in several features, including the association with speci c susceptibility loci and the type of immune response and pathology associated with disease (McGovern et al. 2015). CD has long been considered to be driven by Th1 and Th17 responses, whereas UC has been associated with a non-conventional Th2 response (Duché, 1992;Dray and Marteau, 2007). Immunological studies have recently focused on the mucosal innate immune responses, such as epithelial barrier integrity, innate microbial sensing, autophagy and the unfolded protein response. During the innate response, the intestine of patients with IBD is massively in ltrated by in ammatory cells, mostly macrophages and neutrophils that release large amounts of pro-in ammatory cytokines, such as TNF-α, IL-6, IL-17, IL-12, and interferon-γ (Abraham and Medzhitov 2011, Sou i et al. 2016, Toumi et al. 2021). The pro-in ammatory cytokines are involved in the up-regulation of inducible nitric oxide synthase (iNOS) expression as well as nitric oxide (NO) production. In fact, during IBD, the balance between pro-in ammatory cytokines, anti-in ammatory cytokines , and immunoregulatory cytokines (IL-10 and transforming growth factors-β) is disturbed .
The most commonly used treatments for IBD are 5-aminosalicylates (5-ASA), corticosteroids, immunesuppressors and immunomodulatory therapies that involve the use of humanized monoclonal antibodies mainly against TNF-α. However, treatments targeting the immune system have only an immunosuppressive effect (Taylor and Irving, 2011), and most of them have limitations in side effectsand safety, such as drug dependence, adverse reaction, decline of immune function, and risk of cancer (Danese et al. 2012).
In this sense, it appears important to research and develop an affordable, cost-effective and side-effectfree therapeutic strategy. Therefore, highlighting a new therapeutic approach that targets the deregulated immune response is of great interest. There is growing evidence and focus that herbal remedies can help cure gastrointestinal illness and prevent relapse (Ukwe et al. 2010;Bozorgi et al. 2013;Zahouani et al. 2020). Among those medicines gures Pistacia lentiscus L. (PL), a owering plant growing in the Mediterranean region. In fact, most members of the Pistacia have chemical and therapeutic similarities. Results from several studies have revealed potent pharmacological activities of various parts of this genus plants (Paterniti et al. 2017). The fruits, nuts, resin and leaves of Pistacia lentiscus are used for the treatment of eczema, asthma, kidney stones, throat infections, stomach and diarrhea ache, with astringent, antipyretic, antibacterial, anti-in ammatory and antiviral activities (Bozorgi et al. 2013;Paterniti et al. 2017).
The aim of our present study was to evaluate the anti-in ammatory and potential therapeutic effects of aqueous extract of Pistacia lentiscus leaves (PLAE) in the experimental model of Dextran Sulfate Sodium (DSS)-induced acute colitis.

Pistacia lentiscus
Pistacia lentiscus L. leaves were collected in East Algeria in December 2017. A specimen of Pisactia lentiscus L. has been identi ed as previously described (Boutemine et al. 2018).

Preparation of aqueous extract of PL (PLAE)
PL leaves were rst washed with water, dried in the shade, and then powdered with a blender. The powder obtained was then dissolved in bi-distilled water. The mixture was subjected to continuous stirring for a period of 24 hours and then ltered and centrifuged. The supernatant obtained was lyophilised and the extract has been stored.
2.3 Acute cell toxicity of PLAEon hepa1c1c7 cell line a-hepa1c1c7cells line culture Mouse hepatoma hepa1c1c7 (ATCC ® CRL-2026) cell line was cultured in DMEM (Dulbecco's Modi ed Eagle's Medium, Hyclone Laboratories, Logan, Utah, USA) supplemented with 10% of fetal bovine serum (FBS), 100 unit/mL of penicillin and 100 g/mL of streptomycin (Gibco®, Life Technologies, Carlsbad, CA, USA). Brie y, cells at exponential phase were seeded into 96-well plates at 10 5 cells/mL per well. Then, different concentrations of PLAE were added (0.304, 0.914, 2.74, 8.13, 24, 74, 220 and 666µg/mL). Each treatment was tested in triplicate and the control group was given only culture medium without PLAE. The above plate were placed in a 5% CO 2 humidi ed-atmosphere incubator at 37 °C for 24h.

b-MTT assay
The cytotoxic effect of PLAE on hepa1c1c7cell line was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5difenyltetrazolium (MTT) assay. In this sense, at the end of culture time, 20 μL MTT (5 g/L) was added to each well and the plate was incubated at 37 °C for 4 h. The plate was then shaken for 15 min. Finally, the absorbance of each well was measured by microplate reader at 492 nm. The proliferation rate was calculated as A (absorbance) 492 of treated/A492 of untreated (control) cells × 100% (Tankiewicz-Kwedlo et al. 2010).

Induction of acute colitis by sodium dextran sulfate (DSS)
Female BALB/c mice (18-22g) were allowed free access to food and water and were kept under normal conditions with a 12 h dark/light cycle. All our experiments were approved by Ethics Committee of the Thematic Research Agency in Health Sciences (N°58/DFPR/ATRSS).
The mice were randomly divided into 6 groups as follows ( Fig.1): 1) DSS group: mice received DSS dissolved in drinking water , plus 200 μl of distilled water by gavage each day (n = 8).
2) DSS+PLAE 1 group: mice received DSS dissolved in drinking water, plus 200 μl of aqueous extract dissolved in distilled water at 50 mg/Kg by gavage each day (n = 8).
3) DSS+PLAE 2 group: mice received DSS dissolved in drinking water, plus 200 μl of aqueous extract dissolved in distilled water at 100 mg/Kg by gavage each day (n = 8). 4) DSS+PLAE 3 group: mice received DSS dissolved in drinking water, plus 200 μl of aqueous extract dissolved in distilled water at 200 mg/Kg by gavage each day (n = 8). 5) PLAE group: mice received drinking water, plus 200 μl of aqueous extract dissolved in distilled water at 200 mg/Kg by gavage each day (n = 4). 6) Control (Ctrl) Group: mice received drinking water, plus 200 μl of distilled water by gavage each day (n = 8).

Effect of PLAE on clinical manifestations of acute colitis
In order to assess the severity of colitis, body weight, stool consistency, and blood in the stool were determined daily throughout the experiment, using the following classi cation system (Azuma et al. 2010b): Weight loss was noted as follows: score 0: none; score 1: 1 to 5%; score 2: 5 to 10%; score 3: 10-20%; score 4: > 20% weight loss.
Blood in the stool was scored as follows: score 0: normal, score 2: slight bleeding; score 4: rectal hemorrhage.
The sum of the three scores allowed us to calculate the disease activity index (DAI), to assess the severity of the in ammation throughout the experiment (Azuma et al., 2010b). The total DAI score ranged from 0 to 12. On day 7, the mice were euthanized, the blood was collected by cardiac puncture and the plasma was separated and stored at -20°C for subsequent analysis. Next, an intraperitoneal washing was performed for the recovery of peritoneal macrophages. After dissection, the colon and cecum of each mouse were isolated by separating them from the small intestine at the ileocecal junction and from the anus at the distal end of rectum. Next, colon was washed using cold PBS to remove faeces and measured. Raw pictures from all groups of mice were taken. Then, each colon was dichotomized as follows: A fragment was immersed in 4% formaldehyde for histological analysis.
Small fragments were placed in DMEM supplemented with 10% FCS, 100 unit/mL of penicillin and 100 g/mL of streptomycin for explant culture. Colon mucosa of mice were xed in 4% buffered formaldehyde and embedded in para n. Tissue sections 2 μm thick were depara nized using xylene and rehydrated through a series of increasingly concentrated ethanol baths. Sections were stained with hematoxylin and eosin. Brie y, the tissue sections were rst immersed in hematoxylin for 3-5 min.After rinsing, they were stained with eosin for 2 min. The histological criteria were based on the degree of tissue structure changes, as well as in ammatory cells in ltration.
Colonic in ammation scores were determined in the basis of previously described system for DSSinduced colitis (Azuma et al. 2010b, Sou i et al. 2015. The histological criteria were noted as follows: For the epithelium: score 0: normal morphology; score 1: Goblet cell loss; score 2: loss of crypt and presence of epithelium; score 3: loss of crypt and epithelium; score 4: loss of crypt and epithelium (large area).
For in ltration: score 0: occasional in ammatory cells in the lamina propria; score 1: increased in ltration in the lamina propria mainly at the base of the crypts; score 2: con uence of the in ammatory in ltrate extending to the mucosa; score 3: con uence of the in ammatory in ltrate extending to the submucosa; score 4: transmural extension of the in ltrate (extensive).

b-Histological staining of colonic mucosal with Alcian Blue
Each piece of colon was stained with Alcian Blue to assess mucus production. This staining will color the contents of the cells pink and the mucus blue. For this, after depara nization and rehydration, colon sections of 2 μm thick were placed in the Alcian Blue dye for 15 min and then in nuclear red for 2 to 3 min. The intensity of the blue color in the crypts re ects a signi cant production of mucus.
Interpretation of all results (H&E and Alcian blue staining) was performed double-blind in the presence of a pathologist. The images were captured with a digital camera (Casio) on an optical microscope (Motic).

Culture of mice peritoneal macrophages
Peritoneal macrophages were obtained by washing the peritoneal cavity of mice using 5 mL of cold PBS (pH = 7.4). The cells suspensions were washed thrice with PBS by centrifugation at 2800 rpm for 5 min. Pellets recovered were re-suspended in DMEM, supplemented with 10% FCS. Cells were allowed to adhere at a density of 10 5 macrophages/mL in a 48-well microplate. After 2 hours, the wells were washed with PBS to remove non-adherent cells. Then, all macrophages were incubated in complete DMEM at 37°C and 5% CO 2 . After 24 hours, the culture supernatants were recovered and then stored at -20°C for the subsequent determination of NO and cytokines levels.

Ex vivo Culture of colonic explants
Ex vivo culture of two pieces of the colon will provide key information regarding the severity of in ammation. In this context, the colonic sections (approximately 0.5 cm), cutlongitudinally, were washed thrice serially in mixture of antibiotics (100 U/mL of penicillin and 0.1 mg/mL of streptomycin). The washed colons explants was then placed in a well (24 well plate) containing a drop of complete DMEM with 1% antibiotics (penicillin & streptomycin), supplemented with 10% FCS. After 2 hours, 500 μL of DMEM were added and the explants were incubated at 37°C and 5% CO 2 for 20h. Then, supernatants were collected and centrifuged for 10 min at 4°C and store at −20°C until determination of NO and cytokines levels.

Nitrite concentrations measurement
The Griess reaction was used to determine nitrite levels as an indicator of NO production, in plasma and culture supernatants of Ctrl, DSS, DSS+PLAE 2 and DSS+PLAE 3 groups, as described by Touil-Boukoffa et al. (1997). Brie y, samples were mixed with Griess reagent and distilled water. After incubation, the absorbance was determined at 543 nm by a spectrophotometer. The concentration of nitrites was determined using a standard curve with sodium nitrite.

Measurement of Il-6 and TNF-a levels
The levels of IL-6 and TNF-α were measured in the plasma and the culture supernatants of colonic explants, and peritoneal macrophages of Ctrl, DSS, DSS+PLAE 2 and DSS+PLAE 3 groups, using speci c enzyme immunoassay kits (ELISA), according to the manufacturer's speci cations (Invitrogen ThermoFisher scienti c). Brie y, capture antibodies were xed on 96-well plates. After 24h, the samples were added followed by detection antibodies conjugated with biotin. Then Avidin-HRP followed by tetramethylbenzidine (TMB) was added to reveal the reaction. Finally, the absorbance was measured at 450 nm with an ELISA reader. A standard curve was performed for each assay using recombinant cytokines (Analytical sensitivity: 3.7 pg/mL for TNF-α and < 3 pg/mL for IL-6).

Statistical analysis
Statistical analysis was performed using GraphPad Prism. All data were expressed as mean ± SEM. The statistical signi cance was assessed using Student's t-test, with p < 0.05 being considered signi cant.

Effect of PLAE on cell proliferation and viability
The Acute cell toxicity of PLAE on hepa1c1c7cell line ( Fig. 2) was determined by 3-(4, 5-dimethylthiazol-2yl)-2,5-difenyltetrazolium (MTT) assay. We observed that exposure of cells to up 220µg/mL PLAE (equivalent to raw medicinal material) for 24 h did not reduce the cellular proliferation and viability (Fig. 2) compared to the control group (0µg/mL of PLAE). In addition, our results indicated that exposure of cells to 666µg/mL PLAE (three times more concentrated than raw medicinal material) for 24 h did not alter the cellular proliferation and viability. Our results clearly showed that PLAE has no cytotoxic activity on hepa1c1c7cell line at concentrations used in our experiments.

PLAE decreases the clinical manifestations of DSS-induced acute colitis
We determined daily throughout the experiment hemorrhage, diarrhea and weight loss. These parameters were used to determine the clinical score of acute disease activity index (DAI) (Fig. 3). Our results suggest that concomitant administration of PLAE at 200 mg/Kg with DSS signi cantly reduced the clinical manifestations and clinical score of acute colitis DAI in comparison to DSS administration alone (Fig. 3).
These symptoms and signs of morbidity were more noticeable from day 5 to the end of the experimental period. Administration of PLAE at 100 mg/Kg highly reduced the clinical manifestations and clinical scores of DAI but did not improve mice weight loss induced by DSS (Fig. 3). However, we observed that the concentration of 50 mg/Kg did not improve the DSS-induced clinical manifestations (Fig. 3). Interestingly, the administration of the aqueous extract alone in absence of DSS had no effect on animals and the results were comparable to those obtained for the control group (Fig. 3).

Effect of PLAE on colon histological lesions induced by DSS
The observed blooding and changes in colon size in DSS group suggest signi cant tissue alterations. In order to con rm these observations, we performed histological analysis. Colon tissue sections of control mice revealed a well-differentiated, normal-polarity epithelium and well-preserved crypts, as well as a complete absence of in ammatory in ltrate (score 0) (Fig. 5a). In contrast, DSS-treated mice colon displayed signi cant tissue damage as compared to control group (Fig. 5b). These damages are characterized by crypt deletion, complete goblet cell depletion, ulceration, mucosal erosion and replacement of epithelial cells by granulous tissue (score 4). Importantly, an intense in ltration of in ammatory cells into the mucosa and submucosa with a strongly increased size of mucosa-associated lymphoid tissue was observed, as well as the occasional presence of pseudo-polyps (score 4) (Fig. 5b).
Interestingly, we observed that treatment with DSS+PLAE 3 signi cantly reduced these tissue alterations (Fig. 5e). In fact, normal crypts were observed, with almost an intact epithelium structure, evidenced by a focal decrease in the height of the epithelium and less cellular in ltration in the mucosa. We noted in some cases the presence of some regenerative ulceration corresponding to remissions (score 1) (Fig. 5e). In DSS+ PLAE 2 group, a locally altered epithelium was observed accompanied by a focal deletion of crypts, and less in ammatory in ltrate compared to DSS group (score 2) (Fig. 5d). However, treatment with PLAE at 50 mg/Kg concomitantly with DSS has no effect on DSS-induced histopathological lesions (score 4) (Fig. 5c). In addition, PLAE group ( Fig. 5f) presented histological structure comparable to that of control group (score 0).

PLAE inhibits mucus production by Goblet cells in acute colitis
In order to test the effect of PLAE on mucus production, we stained the colonic tissue sections with the Alcian blue stain. Mucosa of control mice had a normal structure and crypts composed of several welldifferentiated Goblet cells stained blue, re ecting normal mucus production (Fig. 6a). On the other hand, the colonic mucosa of the DSS group was characterized by a hypocrine effect due to the disappearance of Goblet cells and their secretory vesicles. A considerable reduction in blue coloration was observed re ecting a decrease of mucus production (Fig. 6b). Interestingly, colonic mucosa in DSS+PLAE 2 and 3treated mice (Fig 6 d, e) exhibited almost normal mucus production with some degree of focal loss of crypts in the DSS+ PLAE 2 group mucosa (Fig 6d). In addition, the PLAE group showed similar mucus production to the control group (Fig. 6f). Nevertheless, the DSS + PLAE 1 group was characterized by a hypocrine effect and disappearance of Goblet cells comparable to that observed in the DSS group (Fig.  6c).
Our macroscopic and histopathological evaluation revealed clearly that the dose 50 mg/Kg is too low and have no effect on DSS-induced colitis. Hence, the doses 100 mg/Kg and 200 mg/Kg were chosen for additional investigations.

Effect of PLAE on systemic, local and peritoneal macrophages productions of NO in acute colitis
It is well known that DSS-induced colitis is associated with prolonged activation of iNOS and increased NO production at systemic and local levels (Sou i et al. , Sou i et al. 2016. In order to evaluate the effect of PLAE on systemic and local production of NO, we measured nitrites in plasma and culture supernatants of colon explants. Our results showed that the plasma levels of nitrites in DSS group were signi cantly higher compared to control group (22.04 ± 3.5 vs 4.70 ± 1.95 μM, P <0.05) (Fig. 7a). In contrast, mice treated with DSS+PLAE showed a dose-dependent decrease in systemic levels of nitrites (DSS+PLAE 2: 11.65 ± 6.76 µM, DSS+PLAE 3: 5.38 ± 1.99 μM). In addition, DSS+PLAE 3 group indicated a signi cantly reduced NO level compared to DSS group (5.38 ± 1.99 μM vs 22.04 ± 3.5, p <0.05).

Effect of PLAE on pro-in ammatory cytokines production by peritoneal macrophages in acute colitis
We then measured the rate of pro-in ammatory cytokines IL-6 and TNF-α secreted by peritoneal macrophages cultured without any form of stimulation. The results showed that IL-6 and TNF-αlevels in culture of peritoneal macrophages obtained from DSS group were higher than those from control group (IL-6: 15.60 ± 6.08 vs 70.43 ± 23.98 pg/mL/10 5 macrophages, p<0.05 ; TNF-α: 58.11 ± 38.2 vs 32.7 ± 2.8 pg/mL/10 5 macrophages). In contrast, the production of IL-6 by peritoneal macrophages from DSS+PLAE 2 and 3-treated mice was signi cantly reduced as compared to DSS group (Fig. 9 a). In addition, we observed that TNF-α levels in DSS+PLAE 3 treated group was highly reduced compared to DSS group (58.11 ± 38.2 vs 39.28 ± 12.42 pg/mL/10 5 macrophages) (Fig.9 b).

Discussion
Due to the side effects of the drugs currently available, the use of medicinal plants for treatment of acute and chronic intestinal in ammatory diseases is attracting increasing interest. In our current study, we demonstrated for the rst time the anti-in ammatory and therapeutic activities of the aqueous extract of Pistacia lentiscus leaves (PLAE) on DSS-induced acute colitis.
Crohn's disease and ulcerative colitis are the major forms of intestinal bowel diseases (IBD) characterized by bowel in ammation and abdominal pain. In recent decades, many animal models have been developed to characterize the complexity of IBD pathogenesis, de ning the underlying molecular mechanisms and evaluating potential therapeutics for humans (Chassaing et al. 2015). In the present study, we chose to use experimental colitis model based on administration of dextran sodium sulphate (DSS) in mice. This model has been recognized for its speed, simplicity and reproducibility.
Our results showed that after DSS administration, all animals developed the symptoms of acute colitis characterized by progressive weight loss, diarrhea and rectal bleeding. In fact, we observed rst clinical signs of colitis on day 3 after starting DSS administration, whereas on day 5 most animals started to present heavy bleeding. At macroscopic scale, we observed hemorrhagic erosion zones and ulcerations scattered throughout the colon, the cecum, and the rectum accompanied by colon size shortening. From histological point of view, we observed that DSS causes erosions and thinning of the epithelium surface, edematous zones, crypts dilation, as well as the in ltration of immune cells, mainly polynuclear neutrophils (PNN) and macrophages. The deterioration of the lower intestinal tract was accompanied by a depletion of Goblet cells and a decrease in mucus secretion. These results are consistent with those reported byseveral authorswho speculated that DSS is a chemical colitogen with anti-coagulant properties capable of causing severe murine colitis which closely resembles human ulcerative colitis (Okayasu et al. 1990, Abdelouhab et al. 2012, Chassaing et al. 2015. The mechanism by which DSS induces intestinal in ammation has not yet been fully described. However, some authors suggest that DSS carries a highly negative charge provided by sulfate groups, toxic to the colon epithelia monolayer. It thus induces erosions that compromise the barrier integrity leading to increased colonic epithelial permeability and allow to the dissemination of the pro-in ammatory intestinal contents in the underlying tissues. Poritz et al. (2007) observed that the administration of DSS induces changes in tight junction protein expression as early as one day after treatment and an in ammatory response characterized by leukocyte recruitment and increased production of pro-in ammatory cytokines . These initial effects are followed by increasingly severe symptoms with severe diarrhea, bleeding and acute anemia. In addition, DSS anticoagulant property aggravates intestinal bleeding (Chassaing et al. 2014).
All of these reactions cause narrowing of the colon size, progressive weight loss and death of the animals.
Importantly, our study showed that the oral administration of PLAE concomitantly with DSS signi cantly reduced the clinical, macroscopic and histopathological colitis scores in comparison to the administration of DSS alone. Our results are in line with those of several studies. Zahouani et al. (2020) revealed that pre-treatment with PLAE signi cantly reduced the lesion areas in acetic acid-induced acute colitis. In addition, Dellai et al. (2013) suggested that pre-treatment with PLAE at 200mg/Kg decreased the intensity of gastric mucosal damages at 74.9% compared with ethanol-HCl group in an experimental model of gastric ulcer. Furthermore, several other studies showed the bene t of Pistacia lentiscus (PL) extracts on ulcerative colitis. Recently, Ostovan et al. (2020) demonstrated that Mastic Oil of PL signi cantly reduced the total colitis index, including in ammation severity, in ammation extent, and crypt damage scores. Razi et al. (2000) suggested that supplementation with oleogum resin from PL delayed the onset and progression of the disease and helped prevent weight loss in the DSS model of colitis. In addition, Kim et al. (2009) showed that PL mastic decreased the macroscopic and histological damage in TNBS-induced colitis in rats. Our results are also in concordance with our previous study establishing that PL fatty oil pre-treatment or treatment signi cantly reduced ulcerated and hemorrhagic areas in ethanol-induced gastric ulcer (Boutemine et al. 2018).
Otherwise, our results revealed that DSS-administration induces a signi cant increase in systemic, local, and peritoneal macrophages production of NO compared to control group. This result suggests that NO is implicated in the intestinal in ammation. Our results are in agreement with previous studies conducted in our laboratory demonstrating that the administration of 3% DSS induced a signi cant decreases in NO production in plasma, colon and macrophages cultures compared to control group (Abdelouhab et al. 2012, Toumi et al. 2014. Other authors demonstrated that the administration of 3% or 5% DSS increases iNOS expression and NO level in rat colon (Arafa et al. 2009, Larrosaet al. 2009). In addition, it was shown that inhibition of iNOS expression blunted DSS colitis in mice (Rahimi et al. 2010) In the second part of our experiments, we aimed to determine the systemic, local, and macrophage production of the pro-in ammatory cytokines IL-6, and TNF-α. In agreement with previous studies, our results suggest that DSS administration signi cantly increase pro-in ammatory cytokines production (Zicong et al. 2020; Toumi et al. 2014). The in ltrated in ammatory cells observed in the mucosa and submucosa after treatment by DSS may secret pro-in ammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-12, IL-6). These cytokines lead with endotoxin (LPS) and many bacterial components present in the colon to activation of the transcription factor NF-κB, and then the expression of iNOS , Sou i et al. 2016. NO produced in large quantities by this enzyme, joins the superoxide anion (O -2 ) to generate peroxynitrite ion (OONO -) known for its deleterious effects on tissues via the nitration of proteins and lipid peroxidation (Korhonen et al. 2005).The latter mechanisms could be involved in the DSS-induced mucosal injuries. In addition, TNF-α can also activate NF-κB and stimulate the production of proin ammatory cytokines (such as IFN-γ) that ampli es its own production (Zhou et al. 2006). IL-6 is another important pro-in ammatory cytokine that plays a central role in acute in ammation (Kishimoto et al. 2005). Thus, IL-6 can activate neutrophils, monocytes/macrophages, and lymphocytes at the site of in ammation. More importantly, IFN-γ, TNF-α, or IL-6 can disrupt the epithelial barrier function by apoptosis-independent mechanisms (Suzuki et al. 2011). Furthermore, these cytokines production upregulates the Cyclooxygenase-2 (COX 2) expression. This enzyme is responsible for producing prostaglandins associated with the mediation of in ammation (Vane et al. 1998). Kim et al. (2015) showed that the COX-2 and prostaglandins-2 levels were increased in the colon tissue concomitantly with TNF-α and IL-6 levels increases in DSS-treated mice as compared to those of control mice.
Interestingly, we observed that systemic, local, and peritoneal macrophages production of NO, IL-6 and TNF-α were signi cantly down-regulated when the DSS is administered concomitantly with PLAE. Our results are in agreement with previous studies. Zahouani et al. (2020) underlined the anti-in ammatory effect of PLAE oral pre-treatment in acetic acid-induced ulcerative colitis, by reducing IL-6 production. Ostovan et al. (2020) showed that PL mastic oil administered intra-rectally signi cantly decreased TNF-α level after 7 days of treatment in acetic acid-induced colitis. Our present nding are also in line with our previous results onethanol-induced gastric ulcer model showing that pre-treatment or treatment with PL fatty oil signi cantly reduced iNOS expression in the gastric mucosa. Systemic and local production of NO was also reduced. Furthermore, a signi cant decrease in IL-6 and TNF-α level was observed in gastric explants culture (Boutemine et al. 2018).
The therapeutic and anti-in ammatory activities of PLAE observed during our study could be due to the presence of many bioactive phyto-chemical compounds. Interestingly, recent phytochemical analyses of PL leavesaqueous extract allowed the identi cation of numerous phenolic compounds such as avonoids (isoquercetin and luterolin), phenolic acids (ellagic and dicaffeoylquinic), avonols (catechin, rutin, and kaempferol) and tannins (Zahouani et al., 2020). Another study conducted on leaves powder, by Arab et al. (2014) revealed the presence of very high alkaloid and polyphenol content, particularly avonoids (isoquercetin and luterolin), leucoanthocyanins and total tannins (mainly gallic tannins). In addition, Bozorgi et al. (2013) showed that PL leaves are rich in monoterpenes (α-pinene, limonene, sabinene and mycene), and in sesquiterpenes (β-caryophyllene). In addition, PL leaves also contain sterols and triterpenes, saponosides as well as reducing compounds (oses, holosides and mucilage).
Polyphenols limit the immune response during intestinal in ammation by limiting tissue colonization by PNN. In vitro and in vivo studies have indicated that polyphenols inhibited phospholipase A2, lipoxygenase and COX 2 involved in the production of in ammatory mediators such as prostaglandins (Calder et al. 2006). These enzymes act also on NO production by modulating the activity of iNOS (Sanchez-Fidalgo et al. 2010). In addition, during intestinal diseases, polyphenols have a therapeutic effect similar to that of prebiotics. In fact, most polyphenols pass through the small intestine without being absorbed (Scalbert et al. 2000). Thus, polyphenols are biotransformed to their metabolites by the gut microbiota, resulting in their bioavailability increases. Moreover, polyphenols modulate the composition of the gut microbial community mainly by inhibiting pathogenic bacteria and stimulating bene cial bacteria (Lee et al. 2006, Ozdal et al. 2016. Otherwise, avonoids (such asquercetin) treatment has shown bene cial effects by the amelioration of colonic damage and the reduction of the DAI in the DSS model (Oz et al. 2013). The avonoid antidiarrheal effects have been related to their capacity to inhibit muscle contractility, enhance intestinal motility and reduce uid intraluminal accumulation in the gut lumen (Di Carlo et al. 1993, Vezza et al. 2016. Besides, avonoids signi cantly reduced neutrophil in ltration into the damaged colonic tissue in experimental colitis models (Seibel et al. 2009, Vezza et al. 2016. Flavonoids may inhibit proin ammatory cytokines production by blocking the binding of NF-κB to DNA, as well as inhibiting mitogen-activated protein kinase (MAPK) and STAT activation (Salaritabar et al. 2017). In fact, Camuesco et al. (2004) and Vezza et al. (2016) proposed that the anti-in ammatory effects of quercitrin might be related to a decrease in iNOS expression through down-regulation of NF-κB in colonic tissue. In addition, the administration of quercetin remarkably decreased the levels of different cytokines in the in amed colon induced by DSS (Miller et al, 1999, Westphal et al. 2012, Oz et al. 2013. In another study, Marques et al. (2018) suggest that terpenes (monoterpenes and sesquiterpenes) reduce the pro-in ammatory cytokines TNF-α and IL-1α levels and increase the production of IL-10. In addition, terpenes signi cantly inhibit the production of NO by macrophages in vitro and alter the activity of NF-κB (Marques et al., 2018).
Finally, the study of Andersen and Markham reveal that the total absence of cyanogenic heterosides greatly reduces the toxicological risks associated with the use of Pistacia lentiscus L. These ndings are in agreement with our results suggesting that PLAE has no cytotoxic activity on hepa1c1c7cell line.
In summary, our study and the data on the effects of bioactives components present in PL leaves provides several hypothesis to explain the mechanism of PLAE action.PLAE can act as a therapeutic tool either by i) forming a protective membrane along the colon layers, ii) stimulating secretion of mucus by Goblet cells, iii) interacting directly with the sulfate groups of DSS, thus inhibiting their interaction with the colonic wall, and iv) eliminating pathogenic bacteria (such as Helicobacter pylori) which is involved in the aggravation of DSS-induced colitis. In addition, PLAE may act as an anti-in ammatory agent either at i) cellular level by inhibiting the activation and recruitment of immune cells, ii) membrane level by blocking pro-in ammatory cytokines receptors, or iii) inta-cellular level by reducing the expression of NF-κB, and iNOS, and the production of pro-in ammatory cytokines.
According to our nding we conclude that PLAE is not e cient at 50mg/kKg, this dose was considered too low for therapeutic effects. However, 100mg/Kg PLAE could be prescript to mild colitis patient.
Moreover, 200mg/Kg PLAE could treat the sever forms of colitis.

Conclusion
Collectively, our results suggest that the aqueous extract of the Pistacia lentiscus leaves have a systemic and local anti-in ammatory properties resulting in a therapeutic effect against DSS-induced acute colitis in mice. Thus, the Pistacia lentiscus aqueous extract offers new therapeutic possibilities for the treatment of human IBD.   Effect of PLAE on hepa1c1c7cell line proliferation Acute cell toxicity was determined by 3-(4, 5dimethylthiazol-2-yl)-2, 5-difenyltetrazolium (MTT) assay. Values are means ± SEM of experiments carried out in triplicate. PLAE: Pistacia lentiscus leaves aqueous extract.

Figure 3
Effect of PLAE on clinical manifestations and clinical score of acute colitis DAI (disease activity index).
All parameters were determined in mice (n=8) treated with DSS, in the absence or presence of PLAE after 7 days of treatment as described in material and methods       PLAE inhibits the production of pro-in ammatory cytokines by peritoneal macrophages in acute colitis.
Production levels of (a) IL-6 and (b) TNF-α were determined in culture supernatants of peritoneal macrophages obtained from mice (n=8) treated with DSS, in the absence or presence of PLAE after 07 days of treatment. Legends as in Figure 3. (*p <0.05, ns: not signi cant).

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