|Year : 2015 | Volume
| Issue : 3 | Page : 748-756
The effect of royal jelly versus sulfasalazine on acetic acid-induced colitis in adult albino rats
Bothina L Mahmoud, Abeer M Shady, Maisa A Kifafy, Ghada H El-Seify, Ramadan A Eldamhogy, Rania S Omara MSc
Department of Histology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||21-Nov-2014|
|Date of Acceptance||09-Dec-2014|
|Date of Web Publication||22-Oct-2015|
Rania S Omara
Sheibin El Kom, Menoufia
Source of Support: None, Conflict of Interest: None
The present study was designed to investigate the therapeutic effect of royal jelly (RJ) as a new treatment for inflammatory bowel disease (IBD), compared with sulfasalazine, in experimental colitis induced with acetic acid.
Ulcerative colitis is an inflammatory disease in which patients experience cyclic bouts of symptoms including diarrhea and rectal bleeding. Sulfasalazine is mainly used for the treatment of IBD. RJ is a natural product commonly used to supplement the treatment of various diseases.
Materials and methods
Sixty adult male albino rats were used in the present work and were divided into two groups: group I (the control group) and group II. In group II experimental colitis was induced with 4% acetic acid enema and the rats were divided into four subgroups: subgroups IIa, IIb, IIc, and IId. The rats in subgroup IIa were killed after 24 h; those in subgroup IIb did not receive treatment; subgroup IIc received sulfasalazine orally at 100 mg/kg daily; and subgroup IId received RJ orally at 150 mg/kg daily. After 1 month the animals were killed and the distal colon was excised and stained with hematoxylin and eosin, alcian blue, and periodic acid-Schiff; immunohistochemical analysis was carried out for inducible nitric oxide synthase and nuclear factor kB. Total Colitis Index score was calculated, and morphometric and statistical studies were carried out.
Rectal instillation of acetic acid resulted in marked disturbed histological structure of the mucosa and submucosa of the colon. The mucosal crypts showed wide separation and sloughing of the surface epithelium, as well as cellular infiltration. Significant decrease in alcian blue and increase in inducible nitric oxide synthase and nuclear factor kB were observed in the colitis and recovery subgroups. Treatment with RJ or sulfasalazine caused restoration of the mucosal architecture with histological variations more superior in the RJ subgroup than in the sulfasalazine subgroup as proved by histochemical analysis and Total Colitis Index score.
RJ had greater therapeutic effect against experimental colitis compared with sulfasalazine.
Keywords: Alcian blue, inflammatory bowel disease, induced colitis, inducible nitric oxide synthase, nuclear factor kB, royal jelly, sulfasalazine
|How to cite this article:|
Mahmoud BL, Shady AM, Kifafy MA, El-Seify GH, Eldamhogy RA, Omara RS. The effect of royal jelly versus sulfasalazine on acetic acid-induced colitis in adult albino rats. Menoufia Med J 2015;28:748-56
|How to cite this URL:|
Mahmoud BL, Shady AM, Kifafy MA, El-Seify GH, Eldamhogy RA, Omara RS. The effect of royal jelly versus sulfasalazine on acetic acid-induced colitis in adult albino rats. Menoufia Med J [serial online] 2015 [cited 2020 Feb 23];28:748-56. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/748/167897
| Introduction|| |
Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa with intervals of remissions and exacerbations that can markedly increase the risk of colon cancers by 10-fold . Individuals with active disease have been found to have high levels of cytokines and reactive oxygen species (ROS) within their colonic mucosa .
The etiology of inflammatory bowel disease (IBD) is still largely unexplained, although it is believed to be multifactorial. Hereditary (genetic) and environmental (microbial and behavioral) factors have been claimed to interact to result in the disease .
The diagnosis and classification of IBD is usually established by a combination of tests (laboratory, endoscopic, and/or radiologic) in the presence of clinical symptoms .
The therapeutic plan for IBD includes several drug modalities - namely, aminosalicylates (sulfasalazine, mesalamine), corticosteroids, azathioprine, cyclosporine, and infliximab. The choice among these drugs depends on the site, extent, and severity of the disease .
Sulfasalazine is mainly used for treatment of IBD, including UC and Crohn's disease. It is also effective in several types of arthritis, particularly rheumatoid arthritis .
Royal jelly (RJ) is a traditional product commonly used to supplement the medical treatment of various diseases as it contains many important compounds such as free amino acids, proteins, sugars, fatty acids, minerals, and vitamins such as thiamine, niacin, and riboflavin ,. The therapeutic effect of on UC has not been well studied. Hence, the aim of this study was to evaluate the therapeutic effect of RJ, versus sulfasalazine, on an experimental model of UC.
| Materials and methods|| |
Sixty adult male albino rats weighting 200-250 g were used in this study. They were kept under the same environmental condition with free access to food and water.
- Acetic acid (Merck Laboratories, Darmstadt, Germany): acetic acid was the chemical agent used to induce colitis in experimental groups. It was diluted with distilled water to obtain a concentration of 4% acetic acid .
- RJ: RJ was obtained from an apiary in El-Menoufia Governorate, Egypt. It was diluted with distilled water and given orally at a dose of 150 mg/kg .
- Sulfasalazine (Salofalk 500 mg tablets; Minapharm, Helipolice, Cairo, Egypt): sulfasalazine tablets were ground into powder and given orally at a dose of 100 mg/kg .
The rats were divided into two main groups as follows:
Group I (the control group): this group was composed of 20 adult rats that were divided into four subgroups. Subgroup Ia received no medication; subgroup Ib received normal saline enema once; subgroup Ic received oral sulfasalazine; and subgroup Id received RJ orally. All rats were killed at the end of the experiment.
Group II (the acetic acid-induced colitis group): this group consisted of 40 rats that received 4% acetic acid dissolved in 0.9% NaCl enema once for induction of colitis , and then divided into four equal subgroups as follows:
Subgroup IIa (the acetic acid-induced colitis group) was killed 24 h after enema.
Subgroup IIb (the recovery group) was left without further treatment until the end of the experiment.
Subgroup IIc (the sulfasalazine-treated group) received oral sulfasalazine treatment at a dose of 100 mg/kg daily for 4 weeks .
Subgroup IId (the RJ-treated group) received oral RJ treatment daily at a dose of 150 mg/kg for 4 weeks .
At the end of the experiment the animals were killed. The distal 8 cm of the colon was excised, fixed in 10% formol saline, and processed for paraffin blocks. Sections of 5-6-μm thickness were cut and stained with hematoxylin and eosin for routine histological examination , with alcian blue and periodic acid-Schiff's stain for histochemical study , and with inducible nitric oxide synthase (iNOS) and nuclear factor kB (NF-kB) for immunohistochemical study . Total Colitis Index was calculated by summation of the grades of inflammation severity, inflammation extent, and crypt damage using a previously validated scoring system, as shown in [Table 1] .
Quantitative morphometric study
The optical densities of iNOS and NF-kB reactions were measured in the colon crypts in 10 fields per specimen at a magnification of 100. The measurements were obtained using a Leica Qwin 500 Image Analyzer (Leica UK, Department of Anatomy, Faculty of Medicine, Menuophya University) computer system.
The data obtained for optical densities of iNOS and NF-kB reactions and also for the Total Colitis Index [Table 1] for all groups were expressed as mean ± SD and subjected to statistical analysis using one-way analysis of variance (ANOVA) for comparison between the different groups. The post-hoc test was used to find the statistical significance between groups when ANOVA was statistically significant. Results were tabulated and considered significant when P values were less than or equal to 0.05.
| Results|| |
The sections of all control subgroups showed similar results. The colon showed a normal histological architecture with characteristic mucosal folds, and parallel, tall and regularly arranged crypts. Intact cells of two types were seen lining the intestinal crypts: the principal (absorptive) tall columnar cells with basal oval vesicular nuclei, and numerous mucous-secreting goblet cells. The submucosa was thin and contained connective tissue with numerous small blood vessels [Figure 1]a and b.
|Figure 1: (a) A photomicrograph of a section of a control group colon showing intact muscularis mucosa (→) and normal submucosa (thick arrow), and numerous small blood vessels (B). Inset: intact mucosa with many mucosal folds. (b) A photomicrograph of a section of a control group colon showing intact epithelium lining the mucosa (→). Tall, regular, and closely related crypts lined by columnar cells (thick arrow) with vesicular nuclei () and many goblet cells (G). Hematoxylin and eosin, (a– b) ×40.|
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Sections of subgroups IIa and IIb (the induced colitis and recovery subgroups) showed marked disturbed histological structure of the mucosa and submucosa. The characteristic mucosal folds were lost with flattening of the mucosal surface as compared with the control group. The mucosal crypts showed shortening and wide separation. There was sloughing and loss of surface epithelium (ulcers). A disturbed submucosa with wide separation and edema was detected [Figure 2]a-c.
|Figure 2: (a) A photomicrograph of a section of a colon of the induced colitis subgroup (IIa) showing absent crypts (ulcer) (U), sloughing of surface epithelium (→); short, irregular, and widely separated crypts. Inset: Dilated lumen and lost mucosal folds. (b) A photomicrograph of a section of a colon of the colitis subgroup (IIa) showing hemorrhage in the mucosa hemorrhage (H). Infl ammatory infiltrations consist of neutrophils and lymphocytes (→). The columnar cells show pyknotic nuclei (). (c) A photomicrograph of a section of a colon of the recovery subgroup IIb showing wide separation of the submucosa () and widely separated crypts (¡ú). Inset: Dilated lumen and lost characteristic mucosal folds. (d) A photomicrograph of a section of a colon of the recovery subgroup (IIb) showing widely separated crypts (→) and congested blood vessels (B), with inflammatory cell infiltration |
in the mucosa () and loss of the covering epithelium (thick arrow). Hematoxylin and eosin, (a– d) ×40.
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Congested blood vessels with thickened walls and extravasation of blood were observed in the submucosa and between the crypts. The mucosa and submucosa showed inflammatory cellular infiltration consisting of mononuclear cells such as lymphocytes, neutrophils, and eosinophils. The cells lining the crypts showed darkly stained pyknotic nuclei [Figure 2]b and d.
Sections of the sulfasalazine-treated subgroup (subgroup IIc) revealed restored mucosal folds. The crypts were slightly separated and exhibited a preserved surface epithelial lining, formed of simple columnar epithelium with normal vesicular nuclei and normal goblet cells. There was infiltration of the mucosa and submucosa with a few inflammatory cells consisting mainly of the mononuclear type. The submucosa was normal with a few congested blood vessels having thickened walls [Figure 3]a and b.
|Figure 3: (a) A photomicrograph of a section of a colon of the sulfasalazinetreated subgroup (IIc) showing crowded crypts with normal height and cell lining (), congested blood vessels (B), and normal submucosa. Inset: Increased mucosal folds; normal submucosa. (b) A photomicrograph of a section of the colon of the sulfasalazinetreated|
subgroup (IIc) showing infl ammatory cell infiltration (→); the columnar cells lining the crypts show vesicular nuclei (). (c) A photomicrograph of a section of the colon of the royal jelly-treated subgroup (IId) showing tall, crowded, and regular crypts (→). The submucosa is normal (). Inset: Increased mucosal folds. The submucosa is normal. (d) A photomicrograph of a section of the colon of the royal jelly-treated subgroup (IId) showing normal crypts (A) with minimal infl ammatory infi ltration (thick arrow). Intact surface epithelium (¡ú). The columnar cells show vesicular nuclei (). Hematoxylin and eosin, (a– d) ×40.
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Sections of the RJ-treated subgroup (subgroup IId) showed improvement of colonic tissues. The architecture of the mucosa was restored to that of the control group. The characteristic mucosal folds of the colonic mucosa were restored with intact surface epithelium. The crypts were intact, regular, closely related with almost normal height, and lined with simple columnar epithelium with vesicular nuclei. There was less prominent cellular infiltration and vascular congestion. Blood vessels had normal wall and the submucosa was normal [Figure 3]c and d.
Alcian blue and periodic acid-Schiff's stain
0Sections of the control subgroups Ia, Ib, Ic, and Id stained with alcian blue and periodic acid-Schiff's stain showed a strong diffuse cytoplasmic alcian blue in the goblet cells lining the intestinal crypts [Figure 4]a. There was weak reaction in the subgroups IIa and IIb (the induced colitis and recovery subgroups), indicating glandular atrophy and loss of goblet cells [Figure 4]b and c.
|Figure 4: (a) A photomicrograph of a section of the colon of the control group rat showing strong diffuse cytoplasmic periodic acid-Schiff's stain (PAS) and alcian blue staining, especially in the goblet cells lining the crypts (). (b) A photomicrograph of a section of the colon of the colitis subgroup (IIa) showing very weak alcian blue reaction in the mucosa (). (c) A photomicrograph of a section of the colon of the recovery subgroup (IIb) showing mild alcian blue reaction in goblet cells lining the mucosal crypts (). (d) A photomicrograph of a section of the colon of the sulfasalazine-treated subgroup (IIc) showing moderate alcian blue reaction in the mucosa (). (e) A photomicrograph of a section of the colon of the royal jelly-treated subgroup (IId) showing strong alcian blue reaction in the mucosa (). PAS and alcian blue, ( a– e) ×40.|
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Sections of sulfasalazine and RJ subgroups (IIc and IId) showed strong reaction and increase in the number of goblet cells stained with alcian blue, indicating an increased amount of mucus stained with alcian blue in the colonic mucosa [Figure 4]d and e.
Inducible nitric oxide synthase
Histological examination of iNOS immunoreactions in the colon sections of the control group showed a very weak diffuse cytoplasmic immunostaining for iNOS [Figure 5]a.
|Figure 5: (a) A photomicrograph of a section of the colon of the control group showing weak immunoreactivity in the mucosa. (b) A photomicrograph of a section of the colon of the colitis subgroup (IIa) showing strong immunoreactivity in the mucosa. (c) A photomicrograph of a section of the colon of the recovery subgroup (IIb) showing strong immunoreactivity in the upper third of the mucosa. (d) A photomicrograph of a section of the colon of the sulfasalazine-treated subgroup (IIc) showing mild immunoreactivity in the cells lining the crypts, and the surface epithelium. (e) A photomicrograph of a section of the colon of the royal jelly-treated subgroup (IId) showing weak immunoreactivity in the mucosa. Inducible nitric oxide synthase, (a– e) ×40.|
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Subgroups IIa and IIc showed strong positive cytoplasmic iNOS immunostaining in the epithelial cells lining the crypts [Figure 5]b and c.
Subgroup IIc showed mild positive cytoplasmic iNOS immunostaining in the epithelial cells lining the crypts [Figure 5]d.
Subgroup IId (the RJ group) showed a weak positive cytoplasmic iNOS immunostaining in the epithelial cells lining the crypts [Figure 5]e.
Nuclear factor kB
Histological examination of NF-kB immunoreactions in the colon sections of the control group showed a very weak diffuse cytoplasmic immunostaining for NF-kB [Figure 6]a
|Figure 6: (a) A photomicrograph of a section of the colon of the control group (I) showing weak immunoreactivity in the mucosa. (b) A photomicrograph of a section of the colon of the colitis subgroup (IIa) showing strong immunoreactivity in the mucosa. (c) A photomicrograph of a section of the colon of the recovery subgroup (IIb) showing strong immunoreactivity in the mucosa. (d) A photomicrograph of a section of the colon of the sulfasalazine-treated subgroup (IIc) showing mild immunoreactivity in the cells lining the crypts, and surface epithelium. (e) A photomicrograph of a section of the colon of the royal jelly-treated subgroup (IId) showing weak immunoreactivity in the mucosa. Nuclear factor ¦ÊB, (a– e) ×40.|
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Subgroups IIa and IIb showed strong positive cytoplasmic NF-kB immunostaining in the epithelial cells lining the crypts [Figure 6]b and c.
Subgroup IIc showed mild positive cytoplasmic NF-kB immunostaining in the epithelial cells lining the crypts [Figure 6]d.
Subgroup IId showed weak positive cytoplasmic NF-kB immunostaining in the epithelial cells lining the crypts [Figure 6]e.
Quantitative and statistical results
Using ANOVA and the T-test the following statistical results were found.
Total Colitis Index
The Total Colitis Index was significantly increased in experimental colitis (subgroup IIa) compared with the control group (Ia). The Total Colitis Index was also significantly increased in the recovery subgroup (subgroup IIb), and was nonsignificantly decreased by administration of sulfasalazine (subgroup IIc), but was significantly decreased by administration of RJ (subgroup IId) when compared with the control group [Table 2].
|Table 2: Comparison between different studied groups as regards total colitis index|
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Inducible nitric oxide synthase
There was significant difference in the percentage of area stained with iNOS between subgroups (IIa, IIb, and IIc) and between them and the control group (P < 0.05), and a nonsignificant difference between the RJ-treated subgroup (IId) and the control group.
There was significant difference between the sulfasalazine-treated subgroup (IIc) and the RJ subgroup (IId) (P < 0.05) [Table 3].
|Table 3: Comparison between different studied groups as regards percentage of stained area of inducible nitric oxide synthase|
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Nuclear factor kB
There was significant difference in the percentage of area stained with NF-kB between subgroups (IIa, IIb, IIc) and between them and the control group (P < 0.05), and a nonsignificant difference between the RJ-treated subgroup (IId) and the control group.
There was significant difference between the sulfasalazine-treated subgroup (IIc) and the RJ-treated subgroup (IId) (P < 0.05) [Table 4].
|Table 4: Comparison between different studied groups as regards percentage of stained area of nuclear factor κB|
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| Discussion|| |
UC is an inflammatory disease in which patients experience cyclic bouts of clinical symptoms including diarrhea, rectal bleeding, and anemia resulting from intestinal inflammation, edema, and ulceration of the colonic mucosa .
Sulfasalazine is mainly used for treatment of IBD, including UC and Crohn's disease . Natural compounds, such as components of green tea and RJ, can play a significant role against inflammation, cancer, cell proliferation, and tissue damage .
Experimental studies have contributed significantly to advancing the understanding of the etiology of IBD. One of the well-established models of induced colitis - the acetic acid model - was chosen in the present study to detect the morphological and pathological changes associated with colitis .
Wada-Hiraike et al.  stated that estrogen has a protective effect on the colonic mucosa and reported that relative absence of estrogen in postmenopausal women as well as in men was associated with increased levels of ROS as well as with the risk of several diseases such as UC. We used male albino rats in the present study to avoid the protective effect of estrogen.
In the present study the induced colitis subgroup IIa revealed the microscopic criteria for colitis, such as marked disturbed histological structure of the mucosa with loss of characteristic mucosal folds in both the colitis and recovery subgroups. These finding could be due to increased nitric oxide (NO) release, which induces smooth muscle relaxation, colonic dilatation, and vasodilatation. This explanation was in agreement with previous research ,.
There was shortening and wide separation of mucosal crypts together with sloughing of the surface epithelium (ulcer). Extravasated blood and hemorrhage was observed between the crypts. The mucosa showed inflammatory cellular infiltrations consisting of mononuclear cells and leukocytes, mainly lymphocytes, neutrophils, and eosinophils. The cells lining the crypts showed darkly stained pyknotic nuclei. This could be due to immunological processes and ROS, as had been previously reported by Osama et al.  who stated that immunological processes and ROS, such as peroxide anion, hydrogen peroxide, and hypochloric acid, contribute considerably to the development of tissue injury. Oxidative stress and its consequent lipid peroxidation is able to aggravate free radical chain reactions, disrupt the integrity of the intestinal mucosal barrier, and activate inflammatory mediators, resulting in tissue damage, as shown in both human and experimental animal studies. Also, Monique et al.  stated that neutrophils and macrophages contributed to the initiation and progression of epithelial injury. They attributed this effect to the production of ROS such as hydrogen peroxide, hydroxyl radicals, and NO.
Mangerich et al.  stated that the molecular and chemical mechanisms linking colonic inflammation with cancer remain unclear, with potential involvement of tumor-initiating DNA damage and interference with tumor-suppressive mechanisms. This may explain the pyknotic nuclei in the colitis and recovery subgroups detected in the present study, which could be considered as precancerous lesions.
In the present study the submucosa in the untreated colitis and recovery subgroups showed wide separation mostly due to edema. Congested blood vessels with thickened blood vessel walls and extravasation of blood were observed within the submucosa. Vacuolar degenerative changes were seen inside the muscularis mucosa. This result is in agreement with some previous reports in animal models of colitis, which demonstrated similar macroscopic and microscopic pictures and tissue damage after induction of colitis using acetic acid ,.
Histological examination of sulfasalazine-treated rats revealed restoration of the characteristic mucosal folds. The crypts were slightly separated and exhibited preserved surface epithelial lining, and the intestinal crypt appeared lined with simple columnar epithelium with normal vesicular nuclei. The submucosa appeared normal, together with cellular infiltration in both the mucosa and submucosa consisting mainly of mononuclear cells. Some congested blood vessels with thickened walls were detected in the submucosa.
This effect of sulfasalazine was explained by many previous studies and was attributed to the inhibitory effect on a number of proinflammatory mediators released by the mucosa, including ROS, leukutrins, interleukin (IL) 1, and tumor necrosis factor-a (TNF-a), and decreased immunoglobulin production by plasma cells. Hence, most of the inflammatory cascades in IBD are in some manner influenced by this drug .
Histological examination of the colon of rats treated with RJ showed restoration of normal histological architecture, with less vascular congestion and cellular infiltration. The reduction of inflammation caused by RJ in the present study is in agreement with a previous study that suggested that improvement may be due to the anti-inflammatory, cell regeneration, and immunomodulatory effects of RJ . Also, RJ had an ability to scavenge oxidants such as ROS and free radicals, and inhibit proinflammatory cytokine production by activated macrophages and leukocytes, which participate in tissue damage in UC ,.
The present study revealed that RJ was superior to sulfasalazine treatment in reducing tissue inflammation, as proven by the Total Colitis Index.
Previous studies stated that long-term administration of sulfasalazine is accompanied by considerable side effects (headache, nausea, and fatigue), which are dose related and respond to a reduction in the dose. An allergic reaction to sulfa includes rash, fever, hepatitis, pneumonitis, hemolytic anemia, and bone marrow suppression. Sulfasalazine has been reported to cause infertility in men due to abnormalities in sperm morphology and impaired motility .
These side effects can be avoided by using RJ in the treatment of UC. RJ is used as a supplement in the treatment of liver damage, hepatitis, and anemia and increases both male and female fertility ,,,.
The colonic mucosa of the colitis subgroup showed glandular atrophy and loss of goblet cells, indicated by the reduction in the number of alcian blue-positive cells in the mucosa of the colon. Reduction in the number of alcian blue-positive cells had been previously related to alterations in the normal pattern of maturation of mucin in goblet cells that decreased the protective effect of mucus as an active barrier leading to accumulation of ROS and development of the inflammation process causing tissue damage .
In the present study glandular atrophy and loss of goblet cells were observed in the recovery subgroup as well, whereas the number of active alcian blue-positive cells was increased in the sulfasalazine and RJ subgroups, which could indicate increased amounts of mucus in the colonic mucosa.
Increasing numbers of alcian blue-positive cells in the RJ subgroup could be explained by the cell regeneration effects of RJ, as stated by Niu et al. .
The NO produced by iNOS is the major source of the increased NO production seen in experimental colitis and in ulcerative colitis patient tissues. NO may cause cell apoptosis. Induction of iNOS was also demonstrated in inflammatory cells of the lamina propria but to a less extent .
In the present study the iNOS activity was increased in the epithelium, mucosa, and infiltrating inflammatory cells of the colon in the untreated colitis and recovery subgroups as compared with control groups. This finding is in agreement with other results in both humans and animals, wherein an overproduction of NO and upregulation of iNOS produced by macrophages were noticed in active colitis inflammation .
In the present study there was less iNOS protein expressed in the colon tissue of the RJ-treated subgroup compared with the colitis, recovery, and sulfasalazine-treated subgroups. This was proved by photocytometry and statistical analysis. RJ may act as an iNOS inhibitor and it improved the colon after acetic acid-induced tissue injury and inflammation by reducing nitrosative stress.
Cirillo et al.  stated that the dosage of iNOS inhibitor used in the study, the timing, the route of administration, the animal model used, and most importantly the specificity of nitric oxide synthase inhibitors for different nitric oxide synthase isoform will affect the results.
NF-kB mediates the transcriptional activation of many inflammatory genes. In the normal physiological state, NF-kB exists in the cytoplasma as a heterodimer complex of p65/p50 subunits combined with an inhibitory protein, interleukin kB. Inflammatory stimulation provokes rapid degradation of interleukin kB, and subsequently the free NF-kB molecule translocates into the nucleus, binds to the promoter regions of the target genes, and induces active transcription of inflammatory genes. Thus, NF-kB was considered a causative mediator in the modulation of experimentally induced colitis in the rat .
In the present study increase in NF-kB was detected in the mucosal epithelium of colitis-untreated and recovery subgroups. This is in agreement with the results of a previous study by Monique et al.  that reported increase in NF-kB in trinitrobenzene sulfonic acid-induced colitis tissue leading to release of cytokine gene expression (i.e. TNF-a and IL-1b), resulting in the appearance of inflammation in the colonic tissues and DNA damage.
In the present study expression of NF-kB was reduced in the colonic tissue of RJ-treated subgroups more than in the sulfasalazine-treated subgroup, as shown by photocytometry and statistical analysis. Both IL-1b and TNF-a have been suggested to be important inflammatory mediators involved in the initiation and perpetuation of colonic inflammation in IBD as an important step in the NF-kB pathway .
The precise molecular mechanisms responsible for the anti-inflammatory effects of RJ remain unclear. Mihajlovic et al.  stated that the two most important components of RJ, proteins and fatty acids, are responsible for these different activities, especially the immunomodulatory, antimicrobial, and antioxidative effects.
The major royal jelly protein 3 (MRJP3) inhibits inflammatory cytokines and downregulates T helper 2 cell cytokine production, especially IL-4. Further, the fatty acids in RJ proved to downregulate immune cytokines including IL-1b and TNF-a .
As NF-kB activation is the most critical step for IL-1b and TNF-a gene transcription, activation of NF-kB may be a vital event in proinflammatory signal transduction . Thus, we hypothesized that RJ suppresses inflammatory responses that are possibly associated with the expression of NF-kB through inhibition of IL-1b and TNF-a, which is a vital step in the NF-kB pathway. Thus, the positive anti-inflammatory effect of RJ on induced colitis in the present study could be explained by many mechanisms.
| Conclusion|| |
UC is a multifactorial inflammatory disease causing tissue injury and cellular damage of the colon and may cause cancer. RJ has a positive effect in UC treatment, which is superior to sulfasalazine, and has fewer side effects as a natural product. Thus, it is advised to be used in combination with other drugs in the treatment of UC.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]