The possible ameliorative effect of simvastatin versus sulfasalazine on acetic acid induced ulcerative colitis in adult rats

Accepted Manuscript The possible ameliorative effect of simvastatin versus sulfasalazine on acetic acid induced ulcerative colitis in adult rats Nema A. Soliman, Walaa A. Keshk, Fatma H. Rizk, Marwa A.A. Ibrahim PII:

S0009-2797(18)31193-1

DOI:

https://doi.org/10.1016/j.cbi.2018.11.002

Reference:

CBI 8453

To appear in:

Chemico-Biological Interactions

Received Date: 8 September 2018 Revised Date:

20 October 2018

Accepted Date: 3 November 2018

Please cite this article as: N.A. Soliman, W.A. Keshk, F.H. Rizk, M.A.A. Ibrahim, The possible ameliorative effect of simvastatin versus sulfasalazine on acetic acid induced ulcerative colitis in adult rats, Chemico-Biological Interactions (2018), doi: https://doi.org/10.1016/j.cbi.2018.11.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT The possible ameliorative effect of simvastatin versus sulfasalazine on acetic acid induced ulcerative colitis in adult rats Nema A. Solimana,*, Walaa A. Keshka, Fatma H. Rizkb, Marwa A.A. Ibrahimc

RI PT

Departments of aMedical Biochemistry, bPhysiology and cHistology.Faculty of Medicine, Tanta University, Egypt. *

Corresponding author: Nema Ali Soliman, Department of Medical Biochemistry,

M AN U

+2-01147347680; E-mail: [email protected]

SC

Faculty of Medicine, Tanta University, Al-Geish Street, Postal No: 31527, Egypt, Tel:

Abstract:

Objective: Inflammatory bowel diseases (IBD) are chronic and recurrent disorders of the gastrointestinal tract with unknown etiology and have two major forms, ulcerative colitis (UC) and Crohn diseases. In view of the adverse effects and incomplete

TE D

efficacy of currently administered drugs, it is essential to investigate new and harmless drugs with more desirable beneficial effects. Statins have many additional

EP

pleiotropic effects other than their lipid-lowering effect. This study aims to investigate the role of simvastatin (SIM) at different doses against induced UC in rats. Methods:

AC C

SIM (10, 20 mg/kg), and sulfasalazine as a standard therapy (100 mg/kg) were given from five days before and seven days after induction of UC by acetic acid (AA). Colonic mucosal inflammation was evaluated macroscopically and microscopically. Furthermore, the colonic tissue tumor necrosis factor-α (TNF-α), interleukin 1beta (IL 1B),

nod-like

receptor

family

pyrin

domain-1

containing

3

(NLRP3),

malondialdehyde (MDA), reduced glutathione (GSH) and super oxide dismutase (SOD) were assayed in addition to immunohistochemistry of caspase-1 and cyclooxygenase-2 (COX2). Results: SIM in a dose dependant manner significantly

ACCEPTED MANUSCRIPT improved macroscopic and histological scores, diminished colonic levels of IL 1B, TNF-α, NLRP3, MDA, caspase-1 and COX2 and elevated GSH and SOD. Conclusion: SIM has anti-inflammatory, cytoprotective and antioxidants effects that

this makes it a new therapeutic target for UC.

RI PT

are not directly related to its cholesterol lowering activity against AA induced colitis

Key words: Ulcerative colitis, Simvastatin, NLRP3 inflammasome, oxidative stress, COX2.

SC

Abbreviations

IBD; inflammatory bowel diseases, UC; ulcerative colitis, SIM; simvastatin, AA;

M AN U

acetic acid, TNF-α; tumor necrosis factor-alpha, IL 1B; interleukin 1beta, NLRP3; nod-like receptor family pyrin domain-1 containing 3,MDA;malondialdehyde, GSH; reduced glutathione, SOD; super oxide dismutase, COX2; cyclooxygenase-2; DAI; disease activity index, ROS; reactive oxygen species, NF-κB; nuclear factor-kappa B.

TE D

1. Introduction:

Inflammatory bowel diseases (IBD) have two major forms, ulcerative colitis (UC) and Crohn diseases are characterized by epithelial ulceration. It can impair patients'

EP

quality of life and raise the risk of cancer colon (1). Many factors as genetic,

AC C

environmental and immunological may predispose to UC (2). Plentiful chemicalinduced animal models of colonic inflammation have been extensively used to clarify its underlying pathophysiologic mechanisms and evaluate the potency of therapeutic agents as acetic acid (AA) induced UC (3).Pro-inflammatory cytokines are very important for the pathogenesis of inflammatory disease, one of which is interleukin 1beta (IL 1B), which is expressed as an inactive pro-IL 1Band its activation is under the control of caspase-1 (4).Caspase-1 activation is achieved by cytosolic multiprotein complexes called inflammasomes. A number of inflammasomes have been described.

ACCEPTED MANUSCRIPT The Nod-like receptor family pyrin domain-1 containing 3 (NLRP3) inflammasome plays an important role in several inflammatory disorders (5). Data regarding the role of NLRP3 activation in IBD are limited. Therefore, a better understanding of the mutual regulation of inflammasomes will be crucial for discovering new therapeutics

RI PT

for IBD (6). At present, the most widespread drugs used for the management of colitis include 5-aminosalicylates, sulfasalazine, corticosteroidsand immunosuppressants, which diminish the intestinal inflammatory burden and improve the disease-related

SC

symptoms. However, most drugs cause multiple adverse effects (7). As a result, searching for safer and more helpful agents of management for colitis remains an

M AN U

important issue. Statins include a class of cholesterol-lowering drugs used in the treatment of hypercholesterolemia and coronary artery diseases acting as 3-hydroxy3-methylglutaryl-coenzyme A reductase inhibitors. In addition, it was suggested that statins have anti-inflammatory and endothelial cell defending actions, independent of

TE D

their anti-hyperlipidemic effects, they also reduced highly sensitive C-reactive protein levels and vascular proinflammatory cytokines associated hyperlipidemia (8). Simvastatin (SIM) is a commonly given statin with anti-inflammatory and antioxidant

EP

effects. However, the mechanisms by which statins regulate NLRP3 inflammasome

AC C

stay largely unknown. In the present study, SIM was used as a promising antiinflammatory, cytoprotective and antioxidant agent to investigate its possible protective effects in experimental model of colitis induced by AA in rats. 2. Material and methods: 2.1. Drugs and chemicals SIM (ZOCOR 20 mg) and sulfasalazine (500 mg) tablets was purchased from Pfizer Company for Pharmaceutical & Chemical industries (New York, USA). AA, other chemicals and solvents used except otherwise described were purchased from Sigma

ACCEPTED MANUSCRIPT (Sigma Chemicals Co., St. Louis, MO, USA.). All other chemicals and solvents were of high analytical grade. 2.2. Animals Twelve weeks adult male Wister albino rats weighting180-210 g were received from

RI PT

Animal house of Faculty of Medicine, Tanta University. Animals were randomly divided into five groups (number = 10 per group). The animals were housed in sanitary well-ventilated cages, under standard laboratory conditions (12: 12 light -

SC

dark cycle, environmental temperature 25 ± 2°C and free access to water and fed standard pellet chow) and allowed 2 weeks for getting used to laboratory conditions

M AN U

before the beginning of the experiment. This study was conducted in accordance to the guiding principles of the National Institutes of Health for the care and use of laboratory animals (NIH Publications No. 8023, revised 1996) to decrease animal distress and in accordance to the guiding principles of the Ethical Committee of

32525/08/18).

TE D

Medical Research, Faculty of Medicine, Tanta University, Egypt (agreement code:

2.3. Acute colitis model induction and treatment protocols

EP

Rats were starved for at least 24 hrs before the induction of colitis with free access to

AC C

tap water. On the day of the experimentation, water was held two hours before the procedure. Colonic inflammation under light ether anesthesia was induced by administering 2 ml of 3% (v/v) AA in 0.9% NaCl intrarectally using an elastic silicone catheter with an outer diameter of 2 mm. The catheter was inserted rectally into the colon to 8 cm then, rats were holed straight for 2 min to prevent outflow of AA. 2 ml of air were injected to spread the AA totally in the colon before removing the catheter. Control animals underwent the same process using the same volume of 0.9% NaCl instead of AA solution (9, 10).

ACCEPTED MANUSCRIPT 2.4. Animal grouping Group 1 (normal control) received 2 ml of 0.9 NaCl intrarectally under light anesthesia and distilled water orally; group II: served as a colitis control and received 2 ml of 3% (v/v) AA in 0.9% NaCl intrarectally under light anesthesia (9,10); group

RI PT

III and IV (SIM treated groups) (11) received 2 ml of 3% (v/v) AA in 0.9% NaCl intrarectally under light anesthesia + SIM (10 and 20 mg/kg) respectively; group V (sulfasalazine treated group) which was used as a standard therapy for UC (12)

SC

(received 2 ml of 3% (v/v) AA in 0.9% NaCl intrarectally + sulfasalazine 100mg/kg). Both SIM and sulfasalazine were dissolved in distilled water and were taken once

administration of AA (12 days). 2.5. Tissue sampling

M AN U

daily using a gastric gavage needle from 5 days before to 7 days after rectal

After the end of experiment, rats were weighted then anaesthetized with light ether

TE D

and sacrificed. The abdomen was opened; distal 8 cm portions of the colon of each rat was removed and placed on an ice-cold plate, longitudinally opened and cleaned from their luminal contents with cold saline and observed for macroscopic evaluation for

EP

colitis then were cut into three small pieces. One piece was further transversely cut,

AC C

immersed in 10% neutral buffered formalin and fixed for histological and immunohistochemical staining. The second piece was homogenized with 50 mmol/L phosphate buffer (pH 7.4) and then centrifuged at 11 000 g for 20 min at 4 °C then the supernatant was frozen at −80°C until analysis. The residual piece was stored at −80 °C for RNA extraction. 2.6. Macroscopic and microscopic assessments of colitis

ACCEPTED MANUSCRIPT Macroscopic assessment scoring: The colonic samples were at once examined by naked eye and by a magnifying lens (×10). A scale ranging from 0 to 5 was recorded according to Morris et al., 1989 (13). Microscopic assessment scoring: A scale ranging from 0 to 4 was recorded according

RI PT

to Noronha-Blob et al., 1993 (14). 2.7. Disease activity index (DAI)

It was calculated depending on three major signs including % weight loss, diarrhea

SC

and rectal bleeding (15).The presence of occult blood in feces was assayed using benzidine test (16). DAI can be calculated from the following equation. body weight loss score + diarrhea score + rectal bleeding score 3

M AN U

DAI =

2.8. Determination of the ulcer area and ulcer index (UI)

The surface area colonic lesions were measured with the aid of a magnifying lens

TE D

(×10) while UI was determined as earlier described (17) using the following equation. UI =

Total area of the ulcer (mm2) Total area of the colon specimen (mm2)

EP

The UI for each group was calculated as the mean lesion score of all rats in that group; and the inhibition ratio was calculated using the equation (18). UI of AA group − UI of SIM or sulfasalazine groups & 100 UI of AA group

AC C % Inhibition =

2.9. Assessment of oxidative stress and inflammatory biomarkers Spectrophotometric assay of colonic tissue malondialdehyde (MDA), reduced glutathione (GSH) levels were assayed using commercial supplied kits (Biodiagnostic Co., Giza, Egypt) while super oxide dismutase (SOD) activity was done according to the earlier described method of Misra et al 1972(19).

ACCEPTED MANUSCRIPT Colonic tissue tumor necrosis factor alpha (TNF-α) and IL 1B were assayed using commercial supplied kits; both were supplied by Sunred Biological Technology Co., Ltd. Shanghai, China. All ELISA techniques were done according to the manufacturer’s procedure Tissue protein content was determined by the manner of

RI PT

Lowry et al., 1951 (20).

2.10. Estimation of colonic relative NLRP3 mRNA expression by real-time PCR Total RNA was extracted from frozen colonic tissues after processing using Qiagen

SC

RNeasy Total RNA isolation kit (Qiagen, Hiden, Germany) according to the procedure provided by the company, followed by the synthesis of the first strand

M AN U

using Super-Script III First-Strand synthesis kit for real-time PCR (Life Technologies, Carlsbad, California, USA) according to manufacturer’s directions. PCR reactions were performed using Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, California, USA). NLRP3 mRNA transcripts were quantified relative to the

TE D

housekeeping gene glyceraldehyde 3-phosphatede dehydrogenase (GAPDH). Definite primers sequences were designed by Primer3 software (http://bioinfo.ut.ee/primer3/) as follows: rat NLRP3 (Gen Bank accession No. NM_001191642.1): up-stream: 5′-

EP

AGCTAAGAAGGACCAGCCAG-3′,

down-stream:

5′-

AC C

CGTGCATGCATCATTCCACTC-3′ rat GAPDH (Gen Bank accession No. NM_017008): up-stream: 5′-GGTGAAGTTCGGAGTCAACGGA-3′ and downstream:

5′-GAGGGATCTCGCTCCTGGAAGA-3′.

The

thermal

cycling

circumstances were pre-denaturation (95°C for 10 min), followed by 40 cycles of denaturation (95°C for 30 s), annealing (64°C for 1 min) and extension (72 for 30 s) followed by a final incubation at 72°C for 7 minutes. Amplification and data analysis were conducted on a Rotor-Gene Q 6 plex and its specific software (Qiagen, Valencia, California, USA). Relative gene expression was automatically calculated

ACCEPTED MANUSCRIPT using the comparative threshold (Ct) method for the values of the target and the reference genes. 2.11. Histological and immunohistochemical study The colonic sections were stained with Hematoxylin & Eosin (H&E) and Periodic

RI PT

Acid Schiff (PAS) for histological assessment (21). For immunohistochemical staining, the colonic sections were deparaffinized, rehydrated and washed with phosphate buffer (pH 7.4) then incubated with 3% hydrogen peroxide in methanol.

SC

Antigen retrieval was performed by microwaving in 2 rounds, each for 20 minutes. Sections were then incubated overnight at 4ºC with rabbit polyclonal anti-caspase-1

M AN U

(ab1872; Abcam, Massachusetts, USA), rabbit polyclonal anti-COX2 (ab15191; Abcam, Massachusetts, USA). After washing with phosphate buffer, sections were incubated with the biotinylated secondary antibody for 30 minutes and then with streptavidin peroxidase conjugate for 30 minutes followed by brief incubation with

TE D

diaminobenzidine (DAB) chromogen. Counterstaining with Mayer's hematoxylin was done. Negative controls were done by excluding the primary antibodies (22). 2.12. Morphometrical analysis

EP

A Leica digital camera (ICC50, Swizterland) coupled to a Leica light microscope

AC C

(DM500, Swizterland) was used for image acquisition. The software “ImageJ” (version 1.48v National Institute of Health, Bethesda, Maryland, USA) was used for image analysis. Ten different non-overlapping randomly selected fields from each slide were examined at a magnification of 400 to quantitatively evaluate the following parameters: 1- Mean number, size and color intensity of PAS positive goblet cells. 2-Mean area percentage and color intensity of caspase-1 and COX2 positive immunohistochemical reaction in DAB stained sections.

ACCEPTED MANUSCRIPT 2.13. Statistical analysis The data were expressed as the mean ± SD. The differences between the studied groups were tested using one-way analysis of variance (ANOVA) followed by a post hoc test (Tukey). P values <0.05 were considered statistically significant. Statistical

RI PT

tests were performed with SPSS (IBM SPSS Statistics for Windows, IBM Corp, version 23.0. Armonk, NY, USA).

SC

3. Results:

3.1. Effect of SIM on clinical symptoms of AA-induced colitis

M AN U

All rats treated with AA, exhibited features of UC. The maximum incidence of both diarrhea and rectal bleeding was observed in group II while rats treated with SIM 10 mg/kg showed moderate incidence of diarrhea and rectal bleeding Moreover, The lowest occurrence of diarrhea with no rectal bleeding was observed in the rats treated

TE D

in with SIM 20 mg/kg and sulfasalazine 100 mg/kg The DAI as a realistic indicator of colonic inflammation showed a significant increase compared to control group and treated groups (p<0.05), however, SIM 10 mg/kg treated group showed no significant

EP

improvement in DAI (Table 1)

AC C

Table 1: Clinical evaluation and disease activity index in all studied groups Group Diarrhea n, (%) Rectal bleeding n, DAI (%) (mean ±SD) 00±00 Group 1 10, (100%) 8, (80%) 6.3±0.44a Group II 8, (80%) 6, (60%) 5.92±0.99a,c Group III 2, (20%) 2.01±0.65a,b Group IV 1, (10%) 1.92±0.23a,b Group V Note: Data represent; n = number, % = percent incidence, mean ± SD. Each group had n = 10 rats.DAI; disease activity index a Significant difference vs group I (P <0.05). b Significant difference vs group II (P <0.05). c Significant difference vs group V (P <0.05).

3.2. Antiulcer properties of SIM

ACCEPTED MANUSCRIPT Rats treated with SIM significantly (P<0.05) and dose dependently (10 and 20 mg/kg) decreased ulcer area and index as compared to AA group (group II) and thus showed significant increase in % of inhibition against ulcer development. There were insignificant differences between rats treated with SIM 20 mg/kg and rats treated with

support cytoprotective and anti-ulcer effects SIM (Table 2)

RI PT

sulfasalazine 100 mg/kg as regard to ulcer area and UI (P>0.05), which powerfully

M AN U

SC

Table 2: Effect of SIM on ulcer area and ulcer index (UI) of rats in all studied groups Group Ulcer area (mm2) UI % of inhibition 00±00 00±00 Group 1 a a 35.08±1.98 52.45±2.12 Group II 33.82±2.12a,b,c 48.12±3.23a,b,c 8.2 Group III 10.23±2.98a,b 8.54±2.13a,b 83.71 Group IV a,b a,b 9.78±2.23 6.23±2.12 88.12 Group V Note: Data represent mean ± SD. Each group had n = 10 rats. UI; ulcer index a Significant difference vs group I (P < 0.05). b Significant difference vs group II (P < 0.05). c Significant difference vs group V (P < 0.05).

3.3. Effect of SIM on microscopic and macroscopic assessment scores

TE D

Animals that received AA without added treatment showed a significant (P<0.05) increase in both microscopic and macroscopic assessment scores as compared to

EP

normal rats. Treatment with SIM (10 and 20 mg/kg) significantly and dose dependently decreased (P<0.05) in both scores as compared with AA group with

AC C

higher significant improvement in the SIM 20 mg/kg treated group (P<0.05). There were insignificant differences between rats treated with SIM 20 mg/kg and rats treated with sulfasalazine 100 mg/kg in both scores (P>0.05), which powerfully support the action of SIM against UC in rats (Table 3). Table 3: Effect of SIM on macroscopic and microscopic scores in all studied groups Group Group 1 Group II Group III Group IV

Macroscopic score 00±00 3.01±0.23a 2.99±0.12a,b,c 1.24±0.54a,b

Microscopic score 00±00 3.81±0.22 a 2.51±0.24a,b,c 1.90±0.76a,b

ACCEPTED MANUSCRIPT 1.01±0.13a,b 1.82±0.15a,b Group V Note: Data represent mean ± SD. Each group had n = 10 rats. a Significant difference vs group I (P < 0.05). b Significant difference vs group II (P < 0.05). c Significant difference vs group V (P < 0.05).

3.4. Effect of SIM on oxidant/antioxidant status

RI PT

In the AA group, GSH level and SOD activity were significantly lower than the normal control group, whereas MDA level was significantly higher in the AA group than in the normal control group (P<0.05). The treatment with SIM 10 mg/kg showed

SC

insignificant reduction of the aforementioned biomarkers while SIM 20 mg/kg showed significant improvement as well as sulfasalazine 100 mg/kg when compared

M AN U

to group II (Table 4).

Table 4: Effect of SIM on oxidative stress biomarkers in all studied groups Group I

Mean ± SD Improvement (%)

1.91±0.13

Group III

64.41± 2.38

Group IV

Group V

Colonic tissue MDA (pg/mg protein) 3.97±0.43a,d,e 3.84±0.30a,d,e 2.42 ± 0.33a,b,c,e 39.04%

2.01±0.26b,c,d 49.37%

Colonic tissue SOD (µmol/min per mg protein) 33.82±1.87a,d,e 34.79±3.91a,d,e 57.18±3.2a,b,c,e

62.33±2.43b,c,d

TE D

Mean ± SD Improvement (%)

Group II

69.07% Colonic tissue GSH (mg/gm wet tissue) 2.51±0.20a,d,e 2.25±0.25a,d,e 3.32±0.25a,b,c,e

AC C

EP

4.16±0.15 Mean ± SD Improvement 32.27% (%) Note: Data are expressed as mean ± SD.Each group had n = 10 rats.ap<0.05 vs groupI ,bp<0.05 vs groupII, cp<0.05 vs group III, dp<0.05 vsgroup IVand ep<0.05 vsgroupV.MDA, malondialdehyde, GSH, reduced glutathione, SOD, super oxide dismutase

3.5. SIM can attenuate inflammatory biomarkers AA administration showed a significant (P<0.05) increase the inflammatory biomarker as colonic TNF-α and IL 1B levels as well as NLRP3 gene expression as compared with normal control rats. Treatment with SIM (10 and 20 mg/kg) significantly and dose dependently decrease (P<0.05) in those inflammatory biomarkers as compared to AA group with significant improvement in the SIM 20 mg/kg treated group compared with SIM 10 mg/kg treated group (P<0.05).

84.30% 3.84±0.36a,b,c,d 52.99%

ACCEPTED MANUSCRIPT Sulfasalazine 100 mg/kg treated group showed significant decrease of those inflammatory biomarkers when compared to AA and SIM (10 and 20 mg/kg) groups except TNF-α at SIM 20 mg/kg treated group (P>0.05) that reflect the anti-

RI PT

inflammatory effect of SIM in the highest dose in UC treatment (Table 5, Fig. 1) Table 5: Effect of SIM on inflammatory biomarkers in all studied groups Group I

Mean ± SD

0.86 ± 0.06

Group II

Group V 0.83 ± 0.06b,c,d

18.75%

62.95%

SC

41.52%

Colonic tissue IL 1B (pg/mg protein) 126.10±11.64a,c,d,e 108.74±8.31a,b,d,e 63.53±3.69a,b,c,e

64.55± 2.20

13.77% 49.62% Colonic tissue TNF-α (pg/mg protein) 75.72 ±4.52a,b,c 108.63±9.35a,c,d,e 100.24±9.88a,b,d,e

48.04± 4.08b,c,d

M AN U

46.58±4.24

Improvement (%) Mean ± SD

Group IV

Relative colonic tissue NLRP3 gene expression 2.24 ±0 .32a,c,d,e 1.82± 0 .15a,b,d,e 1.31±0 .07a,b,c,e

Improvement (%) Mean ± SD

Group III

TE D

7.72% 30.30% Improvement (%) Note: Data are expressed as mean ± SD. Each group had n = 10 rats.ap<0.05 vs group I ,bp<0.05 vs group II, cp<0.05 vs group III, dp<0.05 vs group IV and ep<0.05 vs group V.TNF-α, tumor necrosis factor-alpha; IL 1B, interleukin 1beta; NLRP3, nod-like receptor family pyrin domain-1 containing 3

3.6. Results of H&E staining

H&E stained sections of the colonic mucosa from the control group showed that it

EP

was formed of closely packed simple tubular glands (crypts of Lieberkuhn) embedded in the lamina propria and extending down to the muscularis mucosa. The crypts were

AC C

lined with surface epithelial columnar absorptive cells with apical brush border and basal oval nuclei, and abundant goblet cells which constituted most of the crypts’lining. Goblet cells showed a vacuolated cytoplasm and basal nuclei. Few intraepithelial lymphocytes could also be observed. The basal parts of the crypts contained undifferentiated cells with basal oval vesicular nuclei (Fig. 2A). Examination of H&E stained sections of group II showed focal areas of extensive mucosal distortion, exfoliation and ulceration with loss of the entire lining of crypts.

63.06% 69.64±1.85b,c 35.90%

ACCEPTED MANUSCRIPT Some crypts showed dilatation with marked decrease or absence of goblet cells. Degenerated crypt cells with cytoplasmic vacuolation and small dark stained nuclei were frequently observed. Areas of separation and hemorrhage were detected. The mucosa was heavily infiltrated with mononuclear cells extending to the underlying

RI PT

muscularis mucosa (Figs. 2B, C, D, E).On the other hand, sections of group III revealed no mucosal ulceration but focal areas of crypt distortion with degeneration and discontinuity were detected. Some degenerated crypt cells showed cytoplasmic

SC

vacuolation and small dark stained nuclei. Some goblet cells were encountered. Mononuclear infiltration was extensive reaching down to the muscularis mucosa (Fig.

M AN U

2F). Sections of group IV showed apparently normal mucosal architecture with minimal signs of crypt degeneration. Goblet cells were apparently near normal in number and size. Mononuclear cells infiltration could be minimally observed (Fig. 2G). Nevertheless, sections from group V revealed near normal histological findings

TE D

with the crypts of Lieberkuhn lined with surface epithelial columnar cells and intact goblet cells. Few intraepithelial lymphocytes were observed (Fig. 2H). 3.7. PAS histochemical staining

EP

PAS stained sections from control group revealed intact PAS positive brush border of

AC C

the surface epithelium and numerous fully distended goblet cells in between them with characteristic strong magenta red coloration (Fig. 3A). However, sections of group II showed a few number of small sized goblet cells with pale staining (Fig. 3B). On the other hand, sections from group III showed a moderate number of partially distended goblet cells of an apparently moderate PAS staining (Fig. 3C). Nevertheless, sections of group IV depicted an abundant number of distended goblet cells with apparently strong PAS staining (Fig. 3D). Sections from group V revealed similar normal findings as control group (Fig. 3E).

ACCEPTED MANUSCRIPT Morphometrical analysis of the mean number, size and color intensity of positive PAS stained goblet cells revealed a highly significant decrease in all parameters in group II compared with the control group, yet group III showed a significant decrease in all parameters compared with the control group. However, a non-significant difference

as between group V and the control group (Table 6). 3.8. Caspase-1 immunohistochemical staining

RI PT

was recorded in all three parameters between group IV and the control group as well

SC

Immunostained sections from control group revealed few cells with a weak positive cytoplasmic caspase-1 immunohistochemical reaction in the form of a brownish

M AN U

coloration (Fig. 4A). However, sections from group II depicted numerous cells with a strong positive mainly nuclear caspase-1 reaction (Fig. 4B). On the other hand, sections from group III showed multiple cells with a moderately positive cytoplasmic caspase-1 reaction (Fig. 4C). Nevertheless, sections from group IV showed few cells

TE D

with a weak positive cytoplasmic caspase-1 reaction (Fig. 4D).Section from group V showed a near normal caspase-1 reaction (Fig. 4E). Morphometrical analysis of mean percentage and color intensity of positive caspase-1

EP

reaction revealed a highly significant increase in both parameters in group II

AC C

compared with the control group. While group III showed a significant increase in both parameters compared to the control. However, a non-significant difference was recorded in both parameters between group IV and the control group as well as between group V and the control group (Table 6). 3.9. COX2 immunohistochemical staining Immunostained sections from control group revealed some cells with a moderately positive cytoplasmic COX2 immunohistochemical reaction in the form of a brownish coloration (Fig. 5A). However, sections from group II depicted numerous cells with a

ACCEPTED MANUSCRIPT strong positive cytoplasmic COX2 reaction (Fig. 5B). On the other hand, sections from group III showed multiple cells with a strong positive cytoplasmic COX2 reaction (Fig. 5C). While sections from group IV showed some cells with a moderately positive cytoplasmic COX2 reaction (Fig. 5D).Section from group V

RI PT

showed a near normal COX2 reaction (Fig. 5E). Morphometrical analysis of mean percentage and color intensity of positive COX2 reaction revealed a highly significant increase in both parameters in group II compared to the control group, yet group III

SC

depicted a significant increase in both parameters compared to the control group. However, a non-significant difference was recorded in both parameters between

(Table 6).

M AN U

group IV and the control group as well as between group V and the control group

Table 6: Morphometrical and statistical analysis of all studied groups

Mean number of goblet cells

Group I

Mean color intensity

10.57±0.87a,c,d,e

EP

26.65±1.29

AC C

Improvement (%)

Group III Colonic goblet cells 16.88±1.21a,c,d,e 38.66±2.74a,b,d,e

49.71±4.82

Improvement (%) Mean size of goblet cells (µm2)

Group II

TE D

Parameters

35.73±1.62

Mean area percentage %

5.23±0.19

Improvement (%)

56.33%

62.47%

64.396%

19.84±1.88a,b,d,e

24.61±3.09b,c

25.49±4.19b,c

27.69±2.76a,b,d,e

36.47±4.95

12.23±1.58a,b,d,e 66.47%

16.29±1.03

57.05% 33.38±3.79b,c 45.51%

58.53% 34.21±5.44b,c 46.83%

Colonic caspase-1 a,c,d,e

Improvement % Mean color intensity

48.17±3.11b,c

34.31%

Improvement (%)

Group V

47.98±6.34b,c

46.72%

18.19±1.83a,c,d,e

Group IV

32.08±3.84a,c,d,e

21.87±1.44a,b,d,e 31.83%

6.61±2.91b,c,e 81.88% 17.02±2.42b,c 46.95%

5.74±1.88b,c,d 84.26% 16.59±3.76b,c 48.29%

ACCEPTED MANUSCRIPT Colonic COX2 Mean area percentage %

10.24±1.65

41.91±5.44

a,c,d,e

12.08±2.46b,c

62.61%

Improvement (%) Mean color intensity

15.67±2.55a,b,d,e

53.33±5.8a,c,d,e

27.94±1.59

71.18%

46.53±3.99a,b,d,e

29.05±2.86b,c

11.01±2.59b,c 73.73% 28.31±2.98b,c

RI PT

12.75% 45.53% 46.92% Improvement (%) Note: Data are expressed as mean ± SD. Each group had n = 10 rats.ap<0.05 vs group I ,bp<0.05 vs group II, cp<0.05 vs group III, dp<0.05 vs group IV and ep<0.05 vs group V.COX2; Cyclooxygenase2.

SC

4. Discussion

The management policy of IBD should be focused on alleviating inflammatory

M AN U

mediator and ROS (23). Protonation and migration of AA molecule into colonic microflora after intrarectal administration of AA to produce protons lead to epithelial destruction increase the influx of bacterial into the lamina properia and stimulate the activated enterocytes to produce a variety of cytokines. Moreover, uncontrolled release of pro-inflammatory

TE D

cytokines is a fundamental characteristic in IBD development (24).

Present study discovered the special effects of SIM on AA induced UC in rat model. UC was assessed in this study by the assessment of DAI as an indicator of the severity

EP

of intestinal inflammation, ulcer area, UI and estimation of microscopic and macroscopic scores. Administration of SIM caused a significant decrease in AA

AC C

induced ulcer area, UI with improvement of % of ulceration inhibition and clinical symptoms of UC in the form of diarrhea and rectal bleeding which were reflected on declining of DAI in a dose dependant manner. Better values were observed with a dose of 20 mg/kg to become insignificant with the sulfasalazine 100 mg/kg treated group as a standard therapy (Tables 1, 2). These results run hand in hand with the results of Choi et al., 2018 (25). Goblet cells are mucin-secreting intestinal cells, produce mucins to replenish and preserve the mucus barrier. UC has been connected to a defective colonic mucus layer

ACCEPTED MANUSCRIPT and a reduced number of goblet cells (26). Current work confirmed; loss of intestinal crypt architecture, sloughing of intestinal cells and reduced goblet cell number, size and color intensity as well as presence of inflammatory cell infiltration in AA colitis group (Figs. 2 B, C, D, E, 3B and Table 6) when compared to control group (Figs. 2A,

RI PT

3A. and Table 6). SIM in a dose dependant manner (Figs. 2F, G, 3C, D respectively and Table 6) and sulfasalazine 100 mg/kg (Fig. 2 H, 3E and Table 6) treatment increased goblet cells number, size and color intensity. Superior values were observed

SC

with a dose of 20 mg/kg SIM to be not significant compared to normal control group. The raise in goblet cells was concurrently associated with intestinal epithelial

M AN U

restoration and regeneration of the distorted intestinal crypts, as previously reported (27). These results can reflect cytoprotective and anti-ulcer effects of SIM. Colonic protection effects of SIM may be due to its ability to scavenge free radicals, embarrassment of neutrophil activity, diminution of oxidative stress, preservation of

TE D

vascular integrity and attenuation of pro-inflammatory cytokines (28). IL1B appears early in intestinal inflammation and their pro-forms are activated via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. NLRP3 is a

EP

component of the inflammasome involved in the activation of caspase-1 and the

AC C

subsequent maturation of IL 1B (29). Hirota et al., 2011 (30) reported that, defect in NLRP3 inflammasome protected mice from induced UC, making it a probable target for the development of new therapeutics for patients with IBD. Existing work established a significant increased IL 1B level and NLRP3 mRNA gene expression as well as caspase-1 expression in colonic tissue of AA group (Tables 4, 6 and Figs. 1, 4B) when compared to control group (Tables 4, 6 and Figs. 1, 4A). Of notes, Lazaridis et al., 2017 (31) reported that, expression of the NLRP3 protein, caspase-1 and their downward pathway product IL 1B was obviously enhanced in the

ACCEPTED MANUSCRIPT inflamed colonic mucosa and this definitely correlated with disease severity. SIM in a dose dependant manner (Tables 4, 6 and Figs. 4C, D respectively) inhibited the elevated levels of aforementioned biomarkers. Superior values were observed with a dose of 20 mg/kg of SIM.

RI PT

Since statins are well tolerated and have an outstanding protection evidence, existing study explored the statins effect against NLRP3 inflammasome in AA induced colitis in rats to be aware of the molecular mechanisms contributing to their effects and

SC

elucidate its capacity to treat a wide diversity of diseases like UC.

Earlier studies of Yu et al., 2017 (32) and Palmer et al., 2004 (33) discovered the anti-

M AN U

NLRP3 of statins in patients with cardiovascular disease and in a collagen induced arthritis model in rats respectively, both revealed that statins clearly diminished NLRP3 inflammasome level. Meanwhile, Kim et al., 2013 (34) concluded that excessive activation of inflammasomes might lead to tissue harm and could implicate

TE D

in pro-inflammatory diseases, however, the mechanisms by which statins regulate NLRP3 inflammasome stay largely unidentified. Homeostasis of the intestinal epithelium is extremely sensitive to the expression

EP

levels of the inflammasome components; NLRP3, caspase-1 and IL 1B, dys-regulated

AC C

expression of one of them might severely affect the vulnerability to IBD (31). As a result, targeting the NLRP3 inflammasome and its downstream signaling may potentially be a key strategy for developing new therapies for IBD. A diversity of dissimilar stimuli have been known to provoke activation of NLRP3 inflammasomes system. Such hazard stimuli include; microbial products, cholesterol crystal and reactive oxygen species (ROS) (35). Oxidative

stress

is

defined

as

the

shift

of

balance

between

the

pro-oxidant/anti-oxidant systems in favor of the pro-oxidant one due to extreme

ACCEPTED MANUSCRIPT production of ROS; oxidative injure has been established to have significant serious effects on IBD pathogenesis (36). AA treatment was established to stimulate an increase in MDA level which is a marker for lipid peroxidation and a decline in the GSH level and SOD activity in the

RI PT

colonic tissue. The findings of present study are consistent with Sepehrimanesha et al., 2018 (36) who reported that, AA deteriorated the anti-oxidant system and induced colitis through excessive ROS generation.

SC

SIM 20 mg/kg treated group showed enhancement of anti-oxidant/pro-oxidant system when compared to AA colitis group (Table 5). An earlier study of Shin et al., 2017

M AN U

(37) reported that, SIM protective effect is created by scavenging free radicals. Release of ROS during episodes of intestinal inflammation is associated with increased amounts of cytokines such as TNF-α which can generate the pathological responses of IBD (38)

TE D

Current work established a significant increased level of TNF-α in colonic tissue (Table 5) when compared to control group. In particular Collins et al., 2014 (39) reported that, TNF-α is considered as the one of the first-line cytokines participating

EP

in the inflammatory course of UC. SIM in a dose dependant manner (Table 5)

AC C

attenuated the elevated level of TNF-α. Better values were observed with a dose of 20 mg/kg. Of notes, TNF-α production in reply to ROS; ROS in turn trigger nuclear factor-kappa B (NF-κB) which is a redox-sensitive factor that controls the transcription of inflammatory genes so enhances more TNF-α and IL 1B production and a vicious series propagation (8). Statins can hinder translocation of NF-κB into the nucleus and keep it in an inactive cytosolic form, thus the expression of proinflammatory mediators including TNF-α and IL 1B will be repressed (25).

ACCEPTED MANUSCRIPT COX2 is the most thoroughly studied and best-understood mammalian dioxygenase that catalyzes the production of prostaglandin E2 (40). The increase of ROS is usually accompanied by increased COX2 as its expression can be induced by inflammatory mediators such as IL 1B and TNF-α. In addition, COX2 acts an important role in

RI PT

NLRP3 inflammasome related diseases through NF-κB and caspase-1 activation as well as augmentation of mitochondrial injure with consequent liberation of mitochondrial ROS (40).

SC

AA treatment notably increased COX2 expression (Table 6 and Fig. 5B) in the colonic tissue when compared to control group (Table 6 and Fig. 5A). SIM in a dose

M AN U

dependant manner treated groups showed a significant decrease of elevated COX2 expression (Table 6 and Figs. 5C, D respectively) when compared to AA colitis group. Moreover, SIM 20 mg/kg treated group showed near normal COX2 expression when compared to control group. In consistent with these results, Massaro et al.,

TE D

2010 (41) reported that statins reduced COX2 expression and activity. Collectively, ROS increase COX2 and trigger NF-κB activation with consequent TNF-α liberation which stimulates NLRP3 inflammasome.

EP

4. Conclusion:

AC C

Present treatment options for UC are limited. New safer and effective therapeutics for it are of concern. Current study clearly demonstrated the protective effects of SIM against chemically induced colitis when compared to sulfasalzine as a standard therapy through its cytoprotective, anti-inflammatory and antioxidant effects along with inhibition of inflammosome complex synthesis in the colonic tissue. Finally, an ever-greater require for more clinical studies designed to prove our experimental results. 5. Authors’ contributions:

ACCEPTED MANUSCRIPT All the named authors participated sufficiently in this research work according to the specialty and expert of each one as follows: Nema A. Soliman designed the research protocol, assay of the biochemical parameters, conducted the statistical analysis, presented the results as tables or figures and wrote and revised the manuscript. Walaa

RI PT

A. Keshk conducted the handling of animals and treatment protocol of the research, recorded the ulcer indices and score, collected samples participated in the assay of the biochemical parameters, conducted the statistical analysis, participated in writing and

SC

revised the manuscript.Fatma H. Rizk conducted the statistical analysis, participated in writing and revised the manuscript. Marwa A.A. Ibrahim conducted the

M AN U

histopathological and immunohistochemical examination. 6. Acknowledgment

The authors would like to acknowledge the technicians in Medical Biochemistry and Histopathology departments and the central laboratory Faculty of Medicine, Tanta

TE D

University, Egypt for the technical assistance. 7. Competing of interests

AC C

8. References

EP

Authors have declared that no competing interests exist.

1- Abraham, B.P.; Ahmed, T.; Ali, T. Inflammatory Bowel Disease: Pathophysiology and Current Therapeutic Approaches. Curr. Pharm. 2017, 239, 115–146. 2- Jiao YF, Lu M, Zhao YP, Liu N, Niu YT, Niu Y, Zhou R, Yu JQ. N Methylcytisine Ameliorates Dextran-Sulfate-Sodium induced Colitis in Mice by Inhibiting the Inflammaory Response. Molecules. 2018 25;23(3). pii: E510. doi: 10.3390/molecules23030510 3- Dothel, G.; Vasina, V.; Barbara, G.; De Ponti, F. Animal models of chemically induced intestinal inflammation: Predictivity and ethical issues. Pharmacol.Ther. 2013, 139, 71–86. 4- Opipari A, Franchi L. Role of inflammasomes in intestinal inflammation and Crohn’s disease. Inflamm Bowel Dis. 2015; 21:173–181.

ACCEPTED MANUSCRIPT 5. Shao BZ, Xu ZQ, Han BZ, Su DF, Liu C. NLRP3 inflammasome and its inhibitors: a review. Front Pharmacol. 2015; 6:262. 6-Perera AP, Kunde D, Eri R. NLRP3 Inhibitors as Potential Therapeutic Agents for Treatment of Inflammatory Bowel Disease.Curr Pharm Des. 2017; 23(16):2321-2327. doi: 10.2174/1381612823666170201162414

RI PT

7-Lautenschläger C; Schmidt C; Fischer D; Stallmach A. Drug delivery strategies in the therapy of inflammatory bowel disease.Adv. Drug Deliv. Rev. 2014, 71, 58–76. 8- Khalili H. Editorial: Statins for Inflammatory Bowel Disease: Expanding the Scope of Prevention. Am J Gastroenterol. 2016; 111(10):1424-1426. doi: 10.1038/ajg.2016.338.

M AN U

SC

9- Astha S, Ramica S. Research on anti-inflammatory and anti-oxidant activity of Ocimum sanctum L .ethanolic extract in ulcerative colitis. Wudpecker J Med Plants. 2013; 2(6):66–73. 10- Fatima D, Shaza A, Ahmad A. Ameliorative Effect of OlmesartanMedoxomil on Acetic Acid-Induced Colitis in Rats. International Journal of Pharmaceutical and Clinical Research. 2016; 8(6): 604-609 ISSN- 0975 1556. 11-Maheshwari RA, Balaraman R, Sailor GU, Sen DB. Protective effect of simvastatin and rosuvastatin on trinitrobenzene sulfonic acid-induced colitis in rats. Indian J Pharmacol. 2015; 47(1): 17–21. doi: 10.4103/0253-7613.150311

TE D

12-Manna JM, Abu-Raghif A, Alsaraf KM. Therapeutic Effect of Sildenafil in Experimental Colitis through Anti-Oxidative Stress and Inhibition of Adhesion Molecules.Journal of Pharmaceutical Sciences and Research. 2017; 9(9):1615-1623.

EP

13-Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology. 1989; 96(3):795-803.

AC C

14- Noronha-Blob L., Lowe VC, Muhlahauser RO, Bruch RM. NPC 15669, an Inhibitor of Neutrophil Recruitment Is Efficacious in Acetic Acid-Induced Colitis in Rats. Gastroenterology. 1993, 104, 1021-1029. 15- Meerveld BG, Tyler KR. CPI-1189 protects against DSS induced colitis in mice. Am J PharmacolToxicol. 2006; 1(3):54–59. 16- Everett D. Kiefer M.D. Detection of occult blood in feces. The American Journal of Surgery. 1934; 25(3)530-535. 17-.Rachmilewitz D, Simon PL, Schwartz LW, Griswold DE, Fondacaro JD, Wasserman MA. Inflammatory mediators of experimental colitis in rats.Gastroenterology.1989; 97:326–37. 18- Kumar VS, Rajmane AR, Adil M,Kandhare AD, Ghosh G, Bodhankar SL. Naringin ameliorates acetic acid induced colitis through modulation of endogenous

ACCEPTED MANUSCRIPT oxido-nitrosative balance and DNA damage in rats J Biomed Res. 2014 28(2):132– 145. Published online 2013 Aug 15. doi: 10.7555/JBR.27.20120082. 19- Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972; 247(10):3170-5. PMID: 4623845.

RI PT

20-Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J.. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193(1): 265–275. PMID: 14907713. 21- Bancroft JD, Gamble M. Theory and Practice of Histological Techniques.sixth ed. Philadelphia: Churchill Livingstone: Elsevier . 2008; 126–127.

SC

22- Buchwalow IB, Böcker W Immunohistochemistry: Basics and Methods. Springer, Heidelberg, Dordecht, London, New York. 2010; 31–39.

M AN U

23-Sanei MH, Hadizadeh F, Adibi P, Alavi SA. Inflammatory cells’ role in acetic acid-induced colitis Adv Biomed Res.2014; 3: 193.doi:10.4103/2277-9175.140666 24-Sepehrimanesh M, Poorbaghi SL. Ulcerative colitis: a phytomedical technical note. Online J Vet Res. 2017; 18(2):75-81.

TE D

25-Choi HW, Shin PG, Lee JH, Choi WS, Kang MJ, Kong WS, Oh MJ, Seo YB, Kim GD. Anti-inflammatory effect of lovastatin is mediated via the modulation of NF-κB and inhibition of HDAC1 and the PI3K/Akt/mTOR pathway in RAW264.7 macrophages. Int J Mol Med. 2018; 41(2):1103-1109. doi: 10.3892/ijmm.2017.3309. Epub 2017. 26-Fatani AJ, Al-Hosaini KA, Ahmed MM, Abuohashish HM, Parmar MY, Al-Rejaie SS. Carvedilol attenuates inflammatory biomarkers and oxidative stress in a rat model of ulcerativecolitis. Drug Dev Res.2015; 76(4):204-214. doi: 10.1002/ddr.21256.

AC C

EP

27-Morampudi V, Dalwadi U, Bhinder G, Sham HP, Gill SK, Chan J, Bergstrom KS, Huang T, Ma C, Jacobson K, Gibson DL The gobletcell-derived mediator RELM-β drives spontaneouscolitisin Muc2-deficient mice by promoting commensal microbial dysbiosis. Mucosal Immunol. 2016; 9(5):1218-1233. doi: 10.1038/mi.2015.140. Epub 2016 Jan 27. 28-Statt S, Ruan JW, Hung LY, Chang CY, Huang CT, Lim JH, Li JD, Wu R, Kao CY Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells. Am J Respir Cell Mol Biol. 2015; 53(5):689-702. doi: 10.1165/rcmb.2014-0391OC. 29-Zaki MH, Lamkanfi M, Kanneganti TD. The Nlrp3 inflammasome: contributions to intestinal homeostasis.Trends Immunol. 2011; 32:171–179. 30-Hirota SA, Ng J, Lueng A, Khajah M, Parhar K, Li Y, Lam V, Potentier MS, Ng K, Bawa M, McCafferty DM, Rioux KP, Ghosh S, Xavier RJ, Colgan SP, Tschopp J, Muruve D, MacDonald JA, Beck PL. NLRP3 inflammasome plays a key role in the

ACCEPTED MANUSCRIPT regulation of intestinal homeostasis. Inflamm Bowel Dis. 2011; 17(6):1359-72. doi:10.1002/ibd.21478. 31-Lazaridis L, Pistiki A, Giamarellos-Bourboulis E J, Georgitsi M, Damoraki G, Polymeros D, Dimitriadis G D, Triantafyllou K. Activation of NLRP3 Inflammasome in Inflammatory Bowel Disease: Differences Between Crohn’s Disease and Ulcerative Colitis. Dig Dis Sci. 2017; 62:2348–2356 DOI 10.1007/s10620-017-4609.

RI PT

32-Yu SY, Tang L, Zhao GJ, Zhou SH. Statin protects the heart against ischemiareperfusion injury via inhibition of the NLRP3 inflammasome. Int J Cardiol. 2017, 15;229:23-24. doi: 10.1016/j.ijcard.2016.11.219.

SC

33-Palmer G, Chobaz V, Talabot-Ayer D, Taylor S, So A, Gabay C, Busso N. Assessment of the Efficacy of Different Statins in Murine Collagen-Induced Arthritis ARTHRITIS& RHEUMATISM. 2004; 50(12): 4051–4059. DOI 10.1002/art.20673 © 2004, American College of Rheumatology

M AN U

34-Kim JJ, Jo EK NLRP3 inflammasome and host protection against bacterial infection. Journal of Korean medical science 2013; 28(10):1415-1423. 35-Sutterwala FS, Haasken S, Cassel SL Mechanism of NLRP3 inflammasome activation. Annals of the New York Academy of Sciences 2014; 1319: 82-95.

TE D

36-Sepehrimanesha M, Samimi N, Koohi-Hosseinabadi O, Mokhtari M, AmiriZadehf S, Farjam M. Effects of Cupressus sempervirens extract on the healing of acetic acid-induced ulcerative colitis in rat. Journal of Coloproctology. 2018; doi: 10.1016/j.jcol.2018.07.002 37-Shin SK, Cho JH, Kim EJ, Kim E, Park DK, Kwon KA, Chung JW, Kim KO, Kim YJ. Anti-inflammatory and anti-apoptotic effects of rosuvastatin by regulation of oxidative stress in a dextran sulfate sodium-induced colitis model. World J Gastroenterol. 2017, 7; 23(25): 4559-4568.doi: 10.3748/wjg.v23.i25.4559

AC C

EP

38-Tanideh N, Jamshidzadeh A, Sepehrimanesh M, Hosseinzadeh M, KoohiHosseinabadi O, Najibi A, Raam M, Daneshi S, Asadi-Yousefabad SL. Healing acceleration of acetic acid-induced colitis by marigold (Calendula officinalis) in male rats. Saudi Journal of Gastroenterology 2016 Jan;22(1):50-56. doi: 10.4103/13193767.173759.. 39-Collins CB, Strassheim D, Aherne CM, Yeckes AR, Jedlicka P, de Zoeten EF. Targeted Inhibition of Heat Shock Protein 90 Tumor Necrosis Factor–α and Ameliorates Murine Intestinal Inflammation. Inflamm Bowel Dis. 2014; 20(4): 685694. doi:10.1097/01.MIB.0000442839.28664.75. 40-Hua KF, Chou JC, Ka SM, Tasi YL, Chen A, Wu SH, Chiu HW, Wong WT, Wang YF, Tsai CL, Ho CL, Lin CH. Cyclooxygenase-2 regulates NLRP3 inflammasome-derived IL-1β production. J Cell Physiol. 2015; 230(4):863-874. doi: 10.1002/jcp.24815. 41-Massaro M1, Zampolli A, Scoditti E, Carluccio MA, Storelli C, Distante A, De Caterina R. Statins inhibit cyclooxygenase-2 and matrix metalloproteinase-9 in

ACCEPTED MANUSCRIPT human endothelial cells: antiangiogenic actions possibly contributing to plaque stability. Cardiovasc Res. 2010; 1;86(2):311-20. doi: 10.1093/cvr/cvp375.

Figure legends

RI PT

Fig. 1: Effect of SIM on relative colonic tissue NLRP3 gene expression.

AC C

EP

TE D

M AN U

SC

Fig. 2: H&E staining: A] Group I shows colonic mucosa formed of closely packed crypts of Lieberkuhn (C) extending down to the muscularis mucosa (Ms). The crypts are lined with surface epithelial columnar cells (thin arrows) with apical brush border and basal oval nuclei, and abundant goblet cells (thick arrows) with vacuolated cytoplasm and basal nuclei. Notice few intraepithelial lymphocytes (notched arrow) and basal undifferentiated cells (curved arrows) with basal oval vesicular nuclei. B-E] Group II shows focal areas of extensive mucosal distortion (Ds), exfoliation (Ex) and ulceration (U) with loss of the entire lining of crypts (thin arrows). Some crypts show separation and dilatation (D) with marked decrease or absence of goblet cells (thick arrows). The degenerated crypt cells show cytoplasmic vacuolation (angular arrows) and small dark stained nuclei (arrow heads). Dilated congested blood vessels (V) and areas of hemorrhage (H) are observed. Notice mononuclear cells infiltration (M) extending to the underlying muscularis mucosa. F] Group III shows some areas of crypt distortion with focal degeneration and discontinuity. Some degenerated crypt cells show cytoplasmic vacuolation (angular arrows) and small dark stained nuclei (arrow heads). Some goblet cells (thick arrows) are observed. Mononuclear infiltration (M) is extensive reaching down to the muscularis mucosa G] Group IV shows apparently normal mucosal architecture with intact mucosal surface (thin arrow). Goblet cells (thick arrows) are apparently near normal in number and size. Mononuclear cells infiltration (M) could be minimally observed. H] Group V shows near normal histological findings with the crypts of Lieberkuhn (C) lined with surface epithelial columnar cells (thin arrow) and abundant intact goblet cells (thick arrow). Few intraepithelial lymphocytes are observed [Magnification x400 scale bar=50µm]. Fig. 3: PAS staining: A] Group I shows intact PAS positive brush border of the surface epithelium (thin arrows) and numerous fully distended goblet cells (thick arrows) in between them. B] Group II shows disrupted brush border (thin arrows) and a few numbers of small sized goblet cells with pale staining (thick arrows). C] Group III shows partially intact brush border (thin arrow) and a moderate number of partially distended goblet cells of an apparently moderate PAS staining (thick arrows). D] Group IV depicts an intact brush border (thin arrow) and an abundant number of distended goblet cells with apparently strong PAS staining (thick arrows). E] Group V shows a near control PAS expression [Magnification x400 scale bar=50µm]. Fig. 4: Caspase-1 immunostaining: A] Group I reveals few cells with a weak positive cytoplasmic caspase-1 immunohistochemical reaction (arrows). B]Group II

ACCEPTED MANUSCRIPT depicts numerous cells with a strong positive mainly nuclear caspase-1 reaction. C] Group III shows multiple cells with a moderately positive cytoplasmic caspase-1 reaction. D] Group IV shows few cells with a weak positive cytoplasmic caspase-1 reaction. E] Group V shows a near control caspase-1 expression [Magnification x400 scale bar=50µm].

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 5: COX2 immunostaining: A] Group I reveals some cells with a moderately positive cytoplasmic COX2 immunohistochemical reaction. B] Group II depicts numerous cells with a strong positive cytoplasmic COX2 reaction. C] Group III shows multiple cells with a strong positive cytoplasmic COX2. D] Group IV shows some cells with a moderately positive cytoplasmic COX2 reaction. E] Group V shows a near control COX2 expression [Magnification x400 scale bar=50µm].

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Statins have additional pleiotropic effects beyond their lipid-lowering effect. Both proinflammatory cytokines and oxidative stress play an important role in

RI PT

ulcerative colitis. Simvastain can dowenregulate NLRP3 inflammasome and proinflammatory cytokines and improve oxidant/antioxidant status.

AC C

EP

TE D

M AN U

SC

Simvastain has antioxidants and antinflammatory effects.