Therapeutic effect of gold nanoparticles on DSS-induced ulcerative colitis in mice with reference to interleukin-17 expression

Ulcerative colitis (UC) is among the most challenging human diseases. Nanotechnology has incontestable promising outcomes in inflammatory bowel diseases. This study aimed to investigate the therapeutic effect of naked gold nanoparticles (AuNPs) on dextran sodium sulphate (DSS) induced ulcerative colitis in mice. We also examined the expression of interleukin-17 (IL-17) following AuNPs treatment. Mice were randomly divided into control, DSS and DSS+ AuNPs groups. Severity of colitis was assessed by disease activity index (DAI) measurement. At the end of the experiment, the final body weights were recorded. The colon was dissected and processed for histopathological examinations by light and electron microscopes. Colon homogenates were prepared for assay of tissue malondialdehyde (MDA) and real-time PCR analysis of IL-17A. Immunohistochemical localization of IL-17A was carried out. Scanning electron microscopy (SEM) and Energy Dispersive X-ray (EDX) detector were used to detect the presence of AuNPs in the colonic tissue of DSS+ AuNPs groups. Our results showed that AuNPs effectively targeted the colonic tissue, and reduced changes induced by DSS. The underlying mechanisms could be related to anti-oxidant effect (as evident by decreasing tissue MDA) and anti-inflammatory potential of AuNPs. Our study draws attention to as a novel therapeutic strategy for treating UC.

. Body weights, colon length and tissue malondialdehyde (MDA) level in different groups. Values are expressed as mean ± standard error of means (SEM) of n = 8 animals. a P compared with control group. b P compared with DSS group. * Significant difference (P < 0.05). * * Highly significant difference (P < 0.001).
non-immunogenicity, antimicrobial properties and facile surface chemistry are some of valuable features of AuNPs 15 . AuNPs were applied as a therapeutic modality in different diseases such as rheumatoid arthritis, diabetes mellitus and chronic myeloid leukemia 16 .
Based on this background, this study aimed to investigate the therapeutic effect of naked AuNPs on DSS induced ulcerative colitis in mice. We also examined the expression of IL-17 following AuNPs treatment.

Results
AuNPs characterization. The AuNPs used in this study had an average diameter of 5 nm, as revealed by transmission electron microscopy ( Fig. 1).
The control subgroups Ia and Ib showed nearly similar results Consequently, only results of the control subgroup Ia were presented in tables and figures. Results of the control subgroup Ib were listed at the end of this section.
Body weight measurements. Statistical analysis of the final body weight measurements revealed a significant decrease in the DSS group (0.8 fold) compared to the control (p < 0.05). There was a non-statistical significant difference in the DSS+ AuNPs group compared to the control (Table 1).
Colon length measurements. The length of the colon was significantly shorter in the DSS group compared with the control group (p < 0.001), whereas the DSS+ AuNPs group was comparable to the control group (Table 1).
Disease activity index (DAI) measurement. DAI showed a highly significant increase in the DSS group compared to the control (p < 0.001). On the other hand, the DSS+ AuNPs group revealed a highly significant decrease compared to DSS group (Fig. 2).

Biochemical results.
• Tissue malondialdehyde (MDA) levels Measurements of MDA levels revealed a highly significant increase in the DSS group (4.4 folds) compared to control group as (p < 0.001). There was a non-statistical significant difference in the DSS+ AuNPs group treated group compared to the control group (Table 1) • IL-17A gene expression results There was a highly significant increase (5.8 folds) in IL-17A gene expression in the DSS group (p < 0.001) www.nature.com/scientificreports www.nature.com/scientificreports/ when compared to normal controls. On contrary, There was a significant decrease in the DSS+ AuNPs group when compared to the DSS group (p < 0.05) (Fig. 2B).

Histopathological results.
• Light microscope results

Results of Haematoxylin & Eosin (H&E) stain.
Examination of the H&E stained sections of the colon of the control group revealed normal arrangement of the lining layers: mucosa, submucosa and musculosa. The crypts were lined by columnar absorptive cells with acidophilic cytoplasm and basal oval nuclei. Numerous goblet cells appeared with basal nuclei and vacuolated cytoplasm. There was thin lamina propria containing few lymphocytes. Prominent muscularis mucosa, normal submucosa and musculosa were seen ( Fig. 3A-C). Sections of DSS group showed disturbed architecture of colon. There were few crypts, intense inflammatory cell infiltrations, mucosal ulcers and submucosal edema, separated muscle fibers of musculosa. The crypts were lined with few goblet cells, many vacuolated cells and some cells with dark nuclei. Congested blood vessels were also observed ( Fig. 3D-G). Sections from DSS+ AuNPs group showed apparently normal structure of the lining layers of the colon. Preserved crypts with many goblet cells were seen. Some inflammatory cell infiltrations in the lamina propria were still observed ( Fig. 3H-J).
We summarized the histological changes using a scoring system that weighted the severity of inflammatory cell infiltrate, ulceration, crypt damage and edema. The total damage score were represented in (Fig. 4).
Results of toluidine blue stain. Toluidine blue-stained semi-thin sections of the control group showed parts of crypts lined with columnar cells and many goblet cells. Some of them opened into the lumen, while in the basal part muscularis mucosa was seen (Fig. 5A,B). DSS group showed mucosal ulcers. The crypts were lined with few goblet cells. Diffuse inflammatory cell infiltrations were noticed in lamina propria (Fig. 5C,D). Section from DSS+ AuNPs group revealed nearly normal colonic crypts that lined with columnar cells and many goblet cells (Fig. 5E,F).
Results of Mallory's trichrome stain. Examination of Mallory's trichrome-stained sections of the control group showed few collagen fibers in lamina propria and submucosa (Fig. 6A). Sections from DSS group showed numerous collagen fibers in the lamina propria and submucosa (Fig. 6B). DSS+ AuNPs group revealed some collagen fibers in lamina propria and submucosa (Fig. 6C).
Results of alcian blue stain. Alcian blue-stained sections of the control showed mucosal crypts lined by abundant goblet cells (Fig. 6D). DSS group showed crypts lined by few goblet cells (Fig. 6E). DSS+ AuNPs group revealed that the crypts were lined by many goblet cells (Fig. 6F). . a: P compared with control group; b: P compared with DSS group; Asterisks * and ** denote p < 0.05 and p < 0.001 respectively; n = 8. (B) Real-time PCR analysis of IL-17A expression levels in the colons of study groups. Values are estimated as a fold-increase compared to IL-17A expression in the calibrator control which is equal to 1. a: P compared with control group; b: P compared with DSS group; Asterisks * and ** denote p < 0.05 and p < 0.001 respectively; n = 8.

• Electron microscope results
Electron microscope examination of the ultrathin sections of the control colon showed absorptive columnar cells with regular oval euchromatic nuclei, numerous microvilli and plentiful mitochondria. Well-developed cell junctions were seen (Fig. 7A). Numerous goblet cells appeared with basal euchromatic regular nuclei, plentiful rough endoplasmic reticulum and several apical mucous granules that might coalesce (Fig. 7B). A thin core of lamina propria containing fibroblasts with elongated nuclei was noticed (Fig. 7C).
Ultrathin sections of the colon from the DSS group showed columnar cells with few microvilli and abnormal cell junctions (Fig. 7D). Damaged cells appeared with dark small nuclei, rarified cytoplasm and degenerated organelles. Separations between cells could be observed (Fig. 7E). Abnormal goblet cells had few apical mucous granules. Some vacuoles were also seen (Fig. 7F). Atypical goblet cells appeared with electron dense condensed secretions surrounded by a space (Fig. 7G). Parts of lamina propria showed abundant collagen fibers and fibroblasts with secretory granules containing immature collagen (Fig. 7H). Intra epithelial infiltrating plasma cells with characteristic cartwheel nuclei and extensive array of rough endoplasmic reticulum were seen (Fig. 8A). Infiltrating eosinophils appeared with bilobed nuclei and specific granules having an electron dense crystalline core surrounded by a less dense material (Fig. 8B). Infiltrating neutrophils appeared in the lamina propria with their segmented nuclei (Fig. 8C).
Ultrathin sections of DSS+ AuNPs treated group showed luminal surface of columnar cells with numerous microvilli, mitochondria and normal cell junction. Goblet cells with many mucous granules, abundant rough endoplasmic reticulum and basal nuclei were observed ( Fig. 8D-F). AuNPs were demonstrated in the cytoplasm and some lysosomes (Fig. 8D,E). There was relatively thin core of lamina propria containing connective tissue cells (Fig. 8G).

Scanning electron microscopy (SEM) and Energy Dispersive X-ray (EDX) detector results.
Analysis of SEM image and EDX spectrum revealed the presence of significant amounts of AuNPs ( Fig. 9).
Morphometric results. Statistically analyzed results for mucosal and submucosal thickness measurements were represented in (Table 2). The number of goblet cells, the area percentage of collagen fibers and the area percentage of positive anti-IL-17A immune reactions were summarized in (Table 3).

Discussion
Over the previous few decades, the prevalence and incidence of inflammatory bowel disease (IBD) have been developing international 17 . IBD is the fundamental risk factor for the development of gastrointestinal cancers, including colorectal cancer 18 .
In the present work, there was a significant loss in the body weights and a highly significant increase in the disease activity index (DAI) in the DSS group compared to the control group. Weight loss is an indicator of the severity of intestinal inflammation and correlates with the histopathological changes of colitis 19 . Weight loss could be contributed to the inflammatory state in IBD which results in mal-absorption of nutrients; a generalized catabolic state; and alterations in the levels of metabolic hormones affecting satiety 20,21 .
MDA is a marker for oxidative stress. MDA results from lipid peroxidation of polyunsaturated fatty acids and plays an important role in the tissue damage of the UC model 22,23 . In the present work, MDA levels were significantly elevated in the DSS group. DSS directly destructs the epithelial cells and triggers abnormal intrusion of microbial flora into the cell, which is capable of starting innate immune responses and leads to the generation of www.nature.com/scientificreports www.nature.com/scientificreports/ reactive oxygen species (ROS) and reactive nitrogen metabolites 24 . Abundant infiltrations by neutrophils are the main source of those harmful metabolites 25 .
In the current work, microscopic examination of the colon the DSS group revealed disrupted architecture as demonstrated by the high histological damage scores. Our results were in line with other investigators 26,27 . The crypts fail to regenerate as a result of colitis 28 . This might explain the presence of few damaged crypts in our study. Further, widened submucosa might be explained by the increased collagen deposition, edema or inflammatory cell infiltrations.
The augmented formation ROS plays an important role in the pathophysiology of UC. It initiates damage of cellular DNA, proteins, lipids and organelles, with disturbance of cell membrane integrity 29 . Other than, T regulatory (Treg) cells maintain immune cell homeostasis by suppressing the functions of other immune cell types, particularly T helper 17 (Th17) cells 30 . Th17/Treg imbalance may play a crucial role in this inflammatory process of UC 31 . Serum concentrations of Th17-related pro-inflammatory interleukins (IL-6, IL-17A, and IL-17F) will increase and Treg-related anti-inflammatory cytokines (IL-10, IL-2, and TGF-β) will decrease accordingly 32 . In the same context, we revealed that IL-17 gene expressions and IL-17 immune reactions were significantly up regulated in the DSS group compared with the control.
In UC-associated inflammatory processes, electron microscope examination of the DSS group revealed inflammatory cell infiltrations including eosinophils, neutrophils and plasma cells, which might represent an abnormal mucosal immune response to bacterial antigens. Recent data showed that eosinophils have key functions in IBD as well 33 . The inflamed colon contains multiple ligands for CD300f-expressing cells including monocytes, neutrophils and eosinophils which are recruited to the colon following DSS treatment 34 . IL-17 is also involved in the proliferation and chemotaxis of neutrophils 35 . Regarding goblet cells, morphometric analysis of alcian blue-stained sections showed a highly significant reduction in the number of goblet cells compared with the control group. These results were in agreement with others 36 . Impaired differentiation of goblet cells from intestinal stem cells was found in inflammatory bowel diseases 37 . Our ultra-structural examination revealed that www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ goblet cells exhibited few mucous granules. Some cells appeared with abnormal condensed secretion. Secretion of abnormal mucins with decreased glycosylation and sulfation is associated with decreased viscosity and increased susceptibility to erosion and colonic inflammation 38,39 .
Organogold compounds have been used since the 1930s to treat rheumatic diseases. Gold compounds decrease the expression of inflammatory cytokines (IL-1, IL-6 and TNF) in rheumatoid arthritis patients 40 . Gold   Table 2. Mucosal and submucosal thickness of different groups. Values are expressed as mean ± standard error of means (SEM) of n = 8 animals. a P compared with control group. b P compared with DSS group. * Significant difference (P < 0.05). * * Highly significant difference (P < 0.001).  www.nature.com/scientificreports www.nature.com/scientificreports/ also inhibits the expression of nuclear factor kappa beta (NF-kB) which has been associated with chronic inflammatory diseases e.g. IBD 41 .

Control group DSS group DSS+ AuNPs group
The role of nano-medicine in the gastrointestinal tract is regarded a promising therapeutic tool. Different types of nanoparticles have prospective uses in gastroenterology as they overcome the traditional treatment in several disorders 42 . AuNPs have begun to be actively used for diagnostic and therapeutic applications in several fields of nano-medicine 43 . AuNPs are attractive for biological applications due to their low toxicity, excellent biocompatibility, and shape-related optoelectronic properties 44 .
The studies of oxidative stress and inflammatory pathways have improved the understanding of the pathogenesis of UC. Hence, these pathways are the therapeutic targets in the new drug studies of UC 22,45 . In the current work, we used AuNPs of 5 nm size. The size of NPs is a critical parameter in the mechanism by which AuNPs inflammation. Sumbayev et al. proved that 5 nm AuNPs completely block the inflammatory process while 15 nm and 35 nm are less effective 46 . AuNPs (coated with tiopronin) smaller than 10 nm have shown interesting preferences over NPs larger than 10 nm in the terms of their capacity to interact with cells, they highly accumulated in cancer cells and distributed throughout the cytoplasm and nucleus 47 . Further, the smaller AuNPs are more quickly absorbed by the intestinal epithelial cells and more rapidly spread through the interior of the cells. Conversely, the larger AuNPs (100 nm) accumulate inside the cells because of the slow rate excretion; hence increase the mitochondrial toxicity 48 . Concerning the tissue distribution and nano-toxicity, Chen et al. reported that intra peritoneal injection of 5 nm AuNPs didn't induce sickness or lethal effects in mice, pathological examination of different organs (liver, kidney lung, brain, heart, and spleen) didn't show any abnormalities 49 . The authors added that 5 nm AuNPs were mainly excreted in urine.
Our findings indicated an overall improvement of DSS-induced adverse effects upon AuNPs treatment in DSS+ AuNPs-treated group. Body weight measurements significantly increased compared to the DSS group. DAI measurements were down regulated. Biochemical analyses revealed decreased MDA levels in colon homogenates, confirming the antioxidant effects of AuNPs. Similarly, AuNPs are able to inhibit lipids from peroxidation and prevent ROS formation, thus restoring the oxidative imbalances in acute inflammation model induced by carrageenan 50 . AuNPs are anti-oxidative agents that inhibit the generation of ROS and scavenge free radicals to improve antioxidant defence enzymes in diabetic mice 51 .
In the same group, the histopathological examination showed that the colon retained about normal structure except for some inflammatory cells. The low histological damage scores and morphometric analysis proved the previous findings. This might be attributed to the anti-oxidant and the anti-inflammatory effects of AuNPs. In agreement with the previous findings, other investigators reported that AuNPs reduced leukocyte migration following carrageenan injection 52 and also macrophage infiltration in a model of arthritis 53 .
AuNPs interfere with transmission of inflammatory signaling. Sumbayev et al. debated that the anti-inflammatory activity of AuNPs was mostly attributed to extracellular interactions with IL-1β which aggregates around AuNPs, thus inhibiting IL-1β binding to cellular receptors 46 . AuNPs interact strongly with the cell membrane 54 as well as also being internalized and trapped in endosomes 55 . Moreover, AuNPs ameliorated the inflammatory response by decreasing the mRNA expression of IL-1β, IL-6, TNF-α and inducible nitric oxide synthase 56 . Gold Nano-constructs are readily taken up by the reticulo-endothelial system 57 .
Interestingly, our study showed that AuNPs could ameliorate the progression of inflammation as evidenced by decreasing the pro-inflammatory cytokine IL-17 mRNA expression and immune-histochemical reactions in the colon compared to the DSS group. IL-17 coordinates tissue inflammation by inducing the expression of pro-inflammatory cytokines (such as IL-6 and TNFα and matrix metalloproteases, which mediate tissue infiltration and tissue destruction 58 . IL-17 is also a potent inducer for nitric oxide (NO) synthase and cyclooxygenase expression and this process is synergized by TNFα and IL-1β 59,60 . Consequently, IL-17 down regulation might partially contribute to reduce inflammation and counteract oxidative stress.
Analysis of Mallory's trichrome-stained sections of the same group showed a highly significant decrease in collagen fibers when compared to DSS group. One of the anti-inflammatory mechanisms of action of gold compounds is inhibition of the synthesis of collagen in rheumatoid arthritis 61 . IL-17 exerts pro-fibrotic effects in the lung, liver, and heart. IL-17 deficiency reduces fibrosis in models of skin inflammation 62 , and treatment with IL-17 enhances cardiac fibroblast proliferation and migration in pulmonary fibrosis models 63 .
Accordingly, we postulated that IL-17 reduction might be partially implicated in the decrease of collagen fibers expression.

Conclusion
Our study demonstrated a convenient, therapeutic effect of AuNPs that effectively targeted the colonic tissue, and reduced changes induced by DSS. The underlying mechanisms could be related to anti-oxidant and anti-inflammatory potential of AuNPs.
So, we recommend AuNPs as a novel therapeutic strategy in the treatment of UC. However, their administration to humans should be fully explored. Clinical trials with different doses and durations are needed to guarantee that therapeutic benefits will be balanced against any hazardous risks.
Experimental animals. Forty eight adult mice (BABL/c; 25-28 g weight; 8-12 weeks old) were obtained from the Breading Animal House, Faculty of Medicine, Zagazig University. They were housed in plastic cages with stainless steel wire-bar lid (2 mice per cage separated by partition) in a controlled room (temperature 18-23 °C, light cycle 14-h light/10-h dark, relative humidity of 40-60%) with food and water ad-libitum. Mice were acclimatized to this environment for two weeks before starting the experiment. All experimental procedures were performed in accordance with the guidelines of Institutional Animal Care and Use Committee and approved by Faculty of Medicine; Zagazig University (the protocol approval number: 3653). All data generated or analyzed during this study are included in this article.
Experimental design. The mice were divided randomly into three main groups: • Group I (control group): (24 mice) were equally subdivided into two subgroups (12 mice each); subgroup (Ia) was kept without treatment, subgroup (Ib) received AuNPs (2.5 mg/kg body weight/day) through intra-peritoneal injection starting on the 14 th day for 2 weeks 51 . • Group II (DSS group): (12 mice) was administered 3% (w/v) DSS dissolved in drinking water. DSS was given in a cyclic manner to induce chronic colitis. Mice were placed on 3 cycles; each cycle consisted of 4 days of DSS followed by six days of drinking blank water 64 . • Group III (DSS+ AuNPs group): (12 mice) was given DSS as DSS group and AuNPs (2.5 mg/kg body weight/ day) through intra-peritoneal injection starting on the 14 th day for 2 weeks 51 .
To evaluate the optimal dose of AuNPs, we tried different concentrations. By real-time PCR analysis, we selected the dose of 2.5 mg/kg as the minimal therapeutic dose for accomplishing effective gene down-regulation of IL-17A (Supplementary Fig. 1) and to decrease the risk of adverse effects. Mass of AuNPs (g)/ml was 6.32 × 10 −5 as provided by the supplier.
The selected dose was about 1/1000 of the LD50 of AuNPs (LD50: 2000 mg/kg body weight /day). At the end of the experiment (after 1 month), mice were sacrificed by intra-peritoneal injection of sodium phenobarbital (50 mg/kg body weight) 65 . An incision through the abdominal wall was done to gain access to the colon which was carefully dissected (from cecum to rectum) and promptly washed with saline. The length of the colon of each animal was measured then the colon was divided into three parts, (i) the proximal third was stored at −80 °C to prepare tissue homogenates for biochemical analysis; (ii) the middle third was fixed in glutraldehyde for electron microscope examination; (iii) the distal third was fixed in 10% buffered formalin and processed for light microscope examination. Disease activity index (DAI) measurement. Severity of colitis was assessed by DAI based on the scoring system reported by Zong et al. 66 . During one month of treatment, the changes in body weight, visible stool consistency and rectal bleeding were recorded every 3 days. DAI is the summation of the weight loss index (0-4); stool consistency index (0-3); and faecal bleeding index (0-3). DAI = (combined score of weight loss, stool consistency and bleeding)/3. Scores were assessed as follows: for weight loss, a score of 0 for body weight within the 1% of baseline; 1 for a 1-5% loss; 2 for a 6-10% loss; 3 for an 11-15% loss and 4 for weight losses over 15%. For stool consistency, a value of 0 was assigned for well-formed pellets, 1 for pasty stools, and 3 for liquid stools. Rectal bleeding was graded 0 for none, 1 for occult bleeding, and 3 for gross bleeding. Occult blood in faeces was detected by using benzidine method (The principle of the test is based on that haemoglobin and its derivatives react in a similar way to peroxidase enzymes-by catalysing the oxidation of benzidine with production of a blue colour) 67 .

Biochemical study.
• Tissue MDA MDA was measured in colon homogenates by spectrophotometry according to the method of Ohkawa et al. 68 using diagnostic kits (CAT. No. MD 25 29; Biodiagnostic Company, Dokki, Giza, Egypt). MDA was expressed as nmol/g protein. Total RNA was isolated from homogenates of mucosal extract of the colon using Qiagen RNA isolation kit (RNeasy, Qiagen Ltd, UK) following the manufacturer's protocol. RNA was converted into cDNA by reverse transcriptase (QuantiTect Reverse Transcription Kit, Qiagen, Germany). The extracted RNA was used for the detection of expression of IL-17A genes. 18S rRNA was used as internal control. SYBR Green RT-PCR amplification was carried out using SYBR Green Real-time PCR Master Mix (Roche Diagnostics). The sequences of the primers used were: IL-17A forward, 5′-ATCCCTCAAAGCTCAGCGTGTC-3′ and reverse, 5′-ATCCCTCAAAGCTCAGCGTGTC-3′; 18S rRNA forward, 5′-CGGCTACCACATCCAAGGAA-3′ and reverse, 5′-GCTGGAATTACCGCGGCT-3′ 69 . Amplification was followed by a melting curve analysis to check www.nature.com/scientificreports www.nature.com/scientificreports/ PCR product specificity. Results were normalized to 18S rRNA for stable expression (housekeeping gene). Relative changes of gene expression were calculated from the equation 2 −ΔΔCt .
Light microscope study. Specimens for light microscopy were fixed in 10% buffered formalin and processed to prepare 5-μm-thick paraffin sections for: • H&E stain 70 • Alcian blue stain 70 • Mallory's trichrome stain 70 • Immunohistochemical stains for localization of intrleukin-17A (IL-17A) of by means the avidin-biotin complex (ABC) method (Thermo Scientific ABC Peroxidase Staining Kits, Code No. 32020, Rockford, USA) following the manufacturer's instructions. Paraffin sections (5 μm), mounted on coated slides, were de-waxed and hydrated. To expose target proteins, antigen retrieval was performed using citrate buffer (pH6.0) microwaved for 15 minutes. Tissues were blocked in 3% bovine serum albumin for 30 minutes at room temperature. Then, they were incubated with the specific primary antibody overnight (4 °C SEM and EDX analysis. SEM (JEOL JSM-6510LV electron microscope; Jeol Ltd, Tokyo, Japan) with X-ray analyzer used for EDX (X-Max N 20 SDD system, Oxford Instruments, Oxford, UK) were used to detect the presence of AuNPs in the colonic tissue of DSS+ AuNPs group. In this procedure, the prepared sample was scanned to produce a high-resolution image for analysis. Then, EDX was used for determining elemental compositions. Transmural inflammation (mucosa to muscularis) 5

Crypt damage
None 0 Some crypt damage, spaces between crypts 1 Loss of goblet cells, large spaces between crypts 2 Large areas without crypts, surrounded by normal crypts 3

Presen 1
Histological damage score Sum (max = 13) Table 4. Histological damage scoring system for quantitative evaluation of the colon injury.
www.nature.com/scientificreports www.nature.com/scientificreports/ Morphometric study. Leica QWin 500 image analyzer computer system (Leica Ltd, Cambridge, UK) was used. The data was analyzed by Leica QWin 500 software with the aid of a digital camera connected to an optical microscope (Olympus, Tokyo, Japan) in the Image Analysis Unit at Pathology Department, Faculty of Dentistry, Cairo University. The measurements were performed by an investigator who was unaware of the experiment. The measuring frame of a standard area was equal to 7286, 78 µm 2 . Five non-overlapping fields were randomly chosen from each mouse in each group and examined to measure: • Thickness of mucosa (µm) in H&E stained-sections at a final magnification (100x).
• Thickness of submucosa (µm) in H&E stained-sections at a final magnification (100x). H&E stained sections were scored to evaluate the histological damage using the scoring system described by Stillie and Stadnyk 73 . Slides were scored by a blinded histologist to grade the extent of inflammatory infiltrate (0-5), crypt damage (0-4), ulceration (0-3), and the presence or absence of edema (0 or 1). Histological damage score is the summation of the previous indices (maximum score = 13) ( Table 4).

Statistical analysis.
Data for all groups were expressed as means ± standard error (X ± SE). Analysis was done using Statistical Package for Social Sciences (SPSS) version 22.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance (ANOVA) was used; followed by Tukey's Honestly Significant Difference (Tukey's HSD) test as a post hoc test for pairwise comparisons. The probability values (P) less than 0.05 were considered significant and highly significant when less than 0.001.

Data Availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).