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Anti-inflammatory and immunomodulatory effects of Spirulina platensis in

comparison to Dunaliella salina in acetic acid-induced rat experimental colitis

Article  in  Immunopharmacology and Immunotoxicology · January 2015

DOI: 10.3109/08923973.2014.998368 · Source: PubMed

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RESEARCH ARTICLE

Anti-inflammatory and immunomodulatory effects of Spirulina platensisin comparison to Dunaliella salina in acetic acid-induced ratexperimental colitis

Mohamed M. Abdel-Daim1, Sameh M. Farouk2, Fedekar F. Madkour3, and Samar S. Azab4

1Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt, 2Cytology & Histology Department, Faculty of

Veterinary Medicine, Suez Canal University, Ismailia, Egypt, 3Marine Science Department, Faculty of Science, Port Said Univeristy, Port Said, Egypt,

and 4Pharmacology & Toxicology Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

Abstract

Context: Spirulina platensis (SP) is used as a source of protein and vitamin supplement inhumans without any significant side-effects. Dunaliella salina (DS) is also regarded as one of therichest natural producers of carotenoid, thus used as a source of antioxidants to protect cellsfrom oxidative damage.Objective: The aim of the present study is to compare the ameliorative effect of Spirulina andDunaliella in experimental colitis.Materials and methods: Spirulina and Dunaliella were investigated at the same dose of 500 mg/kgbody weight for their modulatory effect against acetic-acid induced ulcerative colitis (UC) in rats.The colonic lesion was analyzed by examining macroscopic damage, bloody diarrheascores, colon weight/length and change in body weight of tested rats. Colon lipid peroxidationand oxidative stress markers were examined by evaluating malondialdehyde (MDA), proteincarbonyl (PCO), catalase (CAT), reduced glutathione (GSH) and superoxide dismutase (SOD).Colon inflammatory markers; myeloperoxidase (MPO) and prostaglandin (PGE2) as well asproinflammatory cytokines; tumor necrosis factor (TNF-a) and interleukins (IL-1b, IL-6) were alsostudied.Results: The colonic mucosal injury, biochemical and histopathologic results suggest that bothSP and DS exhibit significant modulatory effect on acetic acid-induced colitis in rats, which maybe due to a significant increase of antioxidant enzymes activity and significant inhibition of lipidperoxidation and inflammation markers.Discussion: Results showed that in comparison to Sulfasalazine, SP exhibited better therapeuticand safety profile than DS against acetic acid-induced UC.Conclusion: This study suggests potential benefits of SP and DS in an experimental modelof colitis.

Keywords

Lipid peroxidation, myeloperoxidase,oxidative stress, protein carbonyl,tumor necrosis factor

History

Received 10 July 2014Revised 27 October 2014Accepted 5 December 2014Published online 8 January 2015

Introduction

Ulcerative colitis (UC) and Crohn’s disease (CD) are immuno-

logically mediated disorders that are collectively referred to as

inflammatory bowel diseases (IBD). Ulcerative colitis affects

primarily the mucosal lining of the colon and rectum, whereas

CD may involve any segment of the gastrointestinal tract1.

Etiology of IBD remains obscure, although environmental

factors, in combination with genetic factors2,3 and altered

immune response driven by microbial factors in the enteric

environment4 are proposed to be involved in its pathological

events. For instance, the mucosal immune system is reported as

the main mediator of intestinal inflammation and injury, with

cytokines playing a central role in initiating inflammation5,6.

The activation of the intestinal immune system results in the

production of proinflammatory cytokines, such as tumor

necrosis factor (TNF-a) and interleukin-1b (IL-1b), prosta-

glandins (PG) and leukotrienes (LT)7.

Importantly, infiltration of inflammatory cells, such as

neutrophils, in addition to the overproduction of proinflam-

matory cytokines8 ultimately gives rise to mucosal disruption

and ulceration2. Neutrophils’ infiltration, assessed by myelo-

peroxidase (MPO), is a key source of production of the

cytotoxic reactive oxygen species (ROS)9. In addition to free

radicals, both the nitric oxide (NO) system and cycloxygenase

(COX-2) have been shown to modulate many events in the

gastrointestinal tract. Several studies are reporting that both

inducible nitric oxide synthase (iNOS) and COX-2 are

evidently upregulated after the stimulation of host cells with

bacteria or inflammatory cytokines, such as TNF-a and IL-1,

Address for correspondence: Samar S. Azab, Pharmacology &Toxicology Department, Faculty of Pharmacy, Ain Shams University,Cairo 11566, Egypt. Tel: +2-01003814389 (Mobile). Fax: +202-24051107. E-mail: samar_saad_azab@pharma.asu.edu.eg

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indicating their role in exacerbation of the underlying

ulcerative pathogenesis10–12.

Currently available therapies for IBD are only effective in

ameliorating the disease symptoms, while having many

concomitant disadvantages13. In this context, a number of

recent studies have renewed interest in the antioxidant potential

of Spirulina platensis (SP) and Dunaliella salina (DS) for

the management of inflammatory conditions and oxidative

damages14–16. Spirulina (SP) refers to the dried biomass of

Arthrospira platensis, an oxygenic photosynthetic microscopic

cyanobacterium found worldwide in fresh and marine waters.

This alga has been used as a source of protein and vitamin

supplement in humans without any significant side-effects.

Apart from its high (up to 70%) content of protein, it

also contains vitamins, especially B12 and provitamin A

(b-carotenes), and minerals, especially iron. It is also rich

in phenolic acids, tocopherols and g-linolenic acid17.

C-phycocyanin (C-PC), one of the major biliproteins of SP,

is reported to exhibit an antioxidant, radical scavenging

properties, as well as selective cyclooxygenase-2 inhibition,

anti-inflammatory and anticancer effects18.

On the other hand, DS is a unicellular marine phytoplankton

that belongs to the phylum Chlorophyta. Dunaliella is regarded

as food supplement as it is one of the richest natural producers

of carotenoid, producing up to 15% of its dry weight under

suitable conditions. Therefore, DS could be used as hepato-

protective, antioxidant, free radical scavenger, protect cells

from oxidative damage16,19. Until today, b-carotene remains

the major natural product harvested from DS. b-carotene

prevents cancer of various organs, including ovary, prostate,

cervix, breast, pancreas, lungs, stomach, rectum and colon by

antioxidant activity20. It boosts immune response21, inhibits

neoplastic transformation and controls of growth22.

To date, however, the possible modulatory role of either SP

or DS in colon inflammation has not been yet verified; hence,

we aimed in the current investigation to evaluate and compare

the possible modulating effect(s) of SP and DS on acetic acid-

induced ulcerative colitis model in rats.

Materials and methods

Chemicals

Pure premium SP powder was purchased from (HerbaForce,

Berkshire, UK). Dunaliella salina was collected from a highly

saline concentrating pond at the solar saltern of Port Fouad,

Port Said, Egypt, during summer 2010. The collected DS was

examined, microscopically identified, cultivated and har-

vested by centrifugation at 3500 rpm for 15 min. The dried

material was mixed with methanol and sonicated, then placed

on the shaker platform for 24 h for cold extraction. The filtrate

was evaporated by rotary evaporator at 30–35� and the mass

obtained was dissolved in distilled water as a vehicle and

employed for further experiments16. Sulfasalazine (SSZ) was

obtained from El-Kahira Pharmaceutical Company, Cairo,

Egypt. MDA, GSH and SOD kits were purchased from

Biodiagnostic Co., Giza, Egypt. Acetic acid was obtained

from El-Nasr Chemical Co. (Cairo, Egypt). IL-1, IL-6, PGE2

and TNF-a ELISA kits were purchased from R&D Systems

GmbH, Wiesbaden, Germany. All other chemicals used

were of analytical grade.

Animals

Forty male Wistar albino rats, with a mean weight of

150–170 g were obtained from the animal house of the

National Central Institute; Dokki, Cairo, Egypt, and allowed

to acclimatize to their environment for 1 week before the

experiment. The rats were housed in stainless-steel cages (eight

animals per cage) and kept on an equal light and dark cycle and

constant environmental conditions. The rats were allowed to

free access to water and food (fed on standard pellet). All

efforts were made to minimize animal pain or suffering during

experimentation. The research was conducted in accordance

with the internationally accepted principles for laboratory

animal use and care as found in the European Community

Guidelines (EEC Directive of 1986; 86/609/EEC) and was

approved by the Animal Care and Use Committee of Faculty of

Veterinary Medicine, Suez Canal University, Cairo, Egypt

(Approval no; 20146).

Study groups

Animals were divided into five groups (eight rats each). The

first group received vehicle (oral saline) for 15 days followed

by single rectal instillation of saline on day 16 and served as

normal control. The second group (acetic acid; AA group) was

given acetic acid intrarectally at day 16 as single dose and

served as positive control (ulcerated, non-treated). The third

group was given sulfasalazine 500 mg/kg orally for three

consecutive days (13th, 14th and 15th) then subjected to rectal

instillation of acetic acid, as in group 2 on day 16. Groups 4 and

5 were given orally SP and DS at 500 mg/kg, respectively, for

15 consecutive days, then also subjected to rectal instillation of

acetic acid, as in group 2 on day 16. On the 17th day, 24 h after

induction of colon ulceration, rats were sacrificed under

anesthesia and laparotomy was performed. Sulfasalazine, SP

and DS doses were determined from reports of previous

work16,23,24. Colonic segments were excised, freed of adherent

adipose tissue, washed with saline, and were used for

macroscopic scoring, histopathological examination and bio-

chemical studies. Colonic samples were stored immediately at

�20 �C till analysis of oxidative stress, inflammatory and

immunomodulatory markers.

Induction of colonic inflammation in rats

The animals were fasted for 24 h with access to water ad

libitum before induction of colitis. Induction of colitis was

performed using a modification of the method described by

Millar et al.25. Each rat was sedated by an intraperitoneal

injection of pentobarbitone (35 mg kg�1). A solution of 1 ml

(4%, v/v) acetic acid (Merck, Germany) in saline was

infused for 30 s using a polyethylene tube (2 mm in

diameter), which was inserted through the rectum into the

colon to a distance of 8 cm. The rats were then maintained

in a supine Trendelenburg position for 30 s to prevent early

leakage of the intracolonic instillate. The acetic acid was

retained in the colon for 30 s after which the fluid was

withdrawn26.

Colitis-macroscopic scoring

Mucosal damage was assessed macroscopically at post-

mortem laparatomy by the scoring system developed by

2 M. M. Abdel-Daim et al. Immunopharmacol Immunotoxicol, Early Online: 1–14

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Millar et al.25, where 6 cm of colon extending proximally for

2 cm above the anal orifice were cut, weighed then split

longitudinally. In each rat, the macroscopic injury of each

ulcer was scored by an independent observer according to a

scoring system described by Cao et al.27 ranging from 0 to 5

as follows: (0) no macroscopic changes or observed damage;

(1) localized hyperemia with no ulcers; (2) liner ulcers with

no significant inflammation; (3) liner ulcers with inflamma-

tion at one site; (4) more ulcerative and inflammatory sites,

the size of ulcers51 cm and (5) multiple inflammations and

ulcers, the size of ulcers �1 cm. Intermediate values reflected

intermediate appearances. Furthermore, the condition of stool

of the rats was evaluated one day before and after acetic acid

treatment. The score for stool occult blood for each rat was

determined as follows: (0: negative, 100: positive).

Assessment of oxidative stress and lipid peroxidationmarkers

The colon lipid peroxidation was evaluated in colon tissue

homogenate by measurement of colonic MDA content

according to Mihara and Uchiyama28, and colonic protein

carbonyl (PCO) was assessed according to Levine et al.29. The

carbonyl content was calculated in terms of nmol/mg protein.

The non-enzymatic antioxidant biomarker; reduced glutathi-

one (GSH) was assessed according to Beutler et al.30. The

enzymatic antioxidant biomarker: superoxide dismutase

(SOD) was evaluated according to the study of Nishikimi

et al.31 and catalase (CAT) according to Aebi32.

Evaluation of the inflammatory andimmuno-modulatory markers

Myeloperoxidase (MPO) activity was used as an index of

leukocyte adhesion and accumulation in several tissues,

including the intestine. Estimation of MPO activity was

carried out according to Krawisz et al.33. The proinflammatory

cytokines: IL-1b, IL-6, TNF-a and PGE2 were assessed

using commercially available kits from R&D Systems

GmbH, Wiesbaden, Germany and followed Reinecker

et al.’s34 method; and were quantified by enzyme-linked

immunoabsorbent assay [ELISA] (Amersham Pharmacia

Biotech, Little Chalfont, UK) and the results were expressed

as pg/g wet tissue. PGE2 was also measured by ELISA kits

(R&D Systems, Minneapolis, MN)35, and expressed as pg/g

wet tissue.

Colitis-histopathological evaluation

The prepared colonic sample sections were stained using the

following stains36: Harris hematoxylin and eosin (H&E),

periodic-acid Schiff (PAS) technique, Alcian blue technique

(pH 2.5) and Masson’s trichrome. The stained sections were

examined for the histopathological findings of colonic

architectural changes. Representative photomicrographs

were taken using Olympus BX41 research optical photo-

microscope fitted with Olympus DP25 digital camera (Tokyo,

Japan) in Cytology and Histology Department, Faculty of

Veterinary Medicine, Suez Canal University.

Colitis-immunohistochemical examination

Immunohistochemical examination was carried out as previ-

ously described37,38 using specific antibodies targeting

COX-2 (Novus Biologicals NBP100-689, Littleton, CO) and

iNOS (Novus Biologicals NBP1-50606, USA). Images were

taken using Leica DM2500 microscope (Wetzlar, Germany)

and analyzed using an image analyzer Leica Q win V.3

program in the Histology Department, Faculty of Medicine,

Ain Shams University. Area percentage of the immunohisto-

chemical stains was assessed for five fields per slide in all the

treatment groups.

Statistical analysis

All data are expressed as mean ± standard error of the mean

(S.E.M.) of eight rats per experimental group. Statistical

analysis was performed using Instat 3.06 statistical software

package (San Diego, CA). Parametric one-way analysis of

variance (ANOVA) followed by the Tukey–Kramer multiple

comparisons test was used to compare the mean values of

quantitative variables among the groups. The minimal level

of significance was identified at p50.05. Correlation coef-

ficient was determined by linear regression analysis23.

Significance of non-parametric data used for analyzing the

macroscopical tests (score of bloody diarrhea and lesion

Table 1. Effects of same doses of Sulfasalazine (SSZ), Dunaliella (DS) and Spirulina (SP) on colon lesion parameters from rats with acetic-acid-induced ulcerative colitis.

Experimental groups

Parameters Control Acetic acid SSZ DS SP

Initial body weight 226.25 ± 4.905 225.63 ± 4.06 226.25 ± 3.26 226.14 ± 3.02 226.57 ± 4.36Final body weight 258.5 ± 3.67 238 ± 4.14a 252.125 ± 3.85 263.43 ± 2.91b 271.29 ± 4.51b,c

Body weight change 32.25 ± 1.95 12.375 ± 0.595a 25.88 ± 2.07b 37.29 ± 1.6b,c 44.71 ± 1.67a,b,c,d

Bloody diarrhea 0 ± 0 100 ± 0a 12.5 ± 12.5b 57.14 ± 18.89a 14.29 ± 13.36b

Colon weight 1.57 ± 0.024 2.42 ± 0.028a 1.56 ± 0.03b 1.76 ± 0.02a,b,c 1.59 ± 0.03b,d

Colon length 17.34 ± 0.54 12.88 ± 0.305a 15.85 ± 0.35b 14.92 ± 0.47a,b 16.54 ± 0.29b

Colon weight/length 90.83 ± 2.44 188.61 ± 3.86a 98.91 ± 3.24b 119.04 ± 5.37a.b,c 96.49 ± 2.71b,d

Lesion score 0 ± 0 4.88 ± 0.125a 1.38 ± 0.18a,b 1.71 ± 0.17a,b 0.57 ± 0.19b,c,d

aStatistical significance as compared to the control.bStatistical significance as compared to the acetic acid-treated group.cStatistical significance as compared to the sulfasalazine-treated group.dStatistical significance as compared to the Dunaliella-treated group.

DOI: 10.3109/08923973.2014.998368 Spirulina and Dunaliella modulate ulcerative colitis 3

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score) was achieved using GraphPad Prism software version 5

(Graph Pad Software Inc., San Diego, CA) and was evaluated

by the Kruskal–Wallis test [non-parametric ANOVA] fol-

lowed by Dunn’s multiple comparisons test, p50.0539.

Results

Macroscopic scoring and colon lesion parameters

The body weight and macroscopic lesion parameters are

presented in Table 1. Acetic acid caused severe macroscopic

edematous inflammation in the colon, as assessed by the

high score of colonic lesion and bloody diarrhea, in addition

to the increased colon wet weight and colon weight/length.

Spirulina platensis and Dunaliella salina significantly

ameliorated all these lesions, and both were comparable to

the standard drug; SSZ effects except DS which failed to

affect the bloody diarrhea score.

Oxidative stress markers

The overwhelmed defense systems resulted in a significant

increase in concentration of MDA and PCO by 476 and 223%

in acetic acid ulcerated group compared to the control

group (Table 2). Regarding the redox state, acetic acid group

caused a significant decrease in colonic non-enzymatic;

GSH by 49% and enzymatic; CAT, SOD defense systems

by 47 and 44% respectively; however, these effects were

significantly reversed in all treated animals of groups 3, 4 and

5, approaching the normal values with SP treatment.

Treatment with SSZ, DS or SP produced a marked decrease

in MDA by 58, 62 and 47% and in PCO levels by 50, 72 and

Table 4. Scoring the severity of the histopathological alterations in colon of different experimental groups.

Experimental groups

Parameters Control Acetic acid SSZ DS SP

Mucosal ulceration �� +++ �� + ��Mucosal necrosis �� +++ �� + ��Mucosal hemorrhage �� +++ + ++ ��Submucosal edema �� +++ ++ +++ ++Submucosal hemorrhage �� +++ �� + ��Submucosal inflammatory cells infiltration �� +++ + ++ +Submucosal congestion �� + + �� ��

+++: Severe histopathological alteration.++: Moderate histopathological alteration.+: Mild histopathological alteration.��: Nil histopathological alteration.

Table 2. Effects of same doses of Sulfasalazine (SSZ), Dunaliella (DS) and Spirulina (SP) on colon oxidative stress marker and antioxidant parametersfrom rats with acetic-acid-induced ulcerative colitis.

Experimental groups

Parameters Control Acetic acid SSZ DS SP

MDA (nmol/g) 11.89 ± 0.78 56.55 ± 1.87a 32.76 ± 1.84a,b 34.94 ± 2.23a,b 26.55 ± 1.58a,b,d

PCO (nmol/mg protein) 3.79 ± 0.24 8.44 ± 0.28a 4.24 ± 0.14b 6.04 ± 0.24a,b,c 4.09 ± 0.17b,d

GSH (mg/g) 1330.25 ± 60.15 645.88 ± 36.4a 859.63 ± 45.87a,b 797.43 ± 40.79a 874.57 ± 37.13a,b

CAT (U/g) 24.44 ± 0.73 11.44 ± 0.52a 20.07 ± 1.09a,b 15.11 ± 0.95a,c 21.13 ± 1.13b,d

SOD (U/g) 6.485 ± 0.34 2.845 ± 0.24a 5.2 ± 0.38b 5.71 ± 0.31b 7.67 ± 0.51b,c,d

aStatistical significance as compared to the control.bStatistical significance as compared to the acetic acid-treated group.cStatistical significance as compared to the Sulfasalazine-treated group.dStatistical significance as compared to the Dunaliella-treated group.

Table 3. Effects of same doses of Sulfasalazine (SSZ), Dunaliella (DS) and Spirulina (SP) on colon inflammation marker and immunomodulatoryparameters from rats with acetic-acid-induced ulcerative colitis.

Experimental groups

Parameters Control Acetic acid SSZ DS SP

MPO (U/g) 15.88 ± 2.52 59.73 ± 3.19a 37.78 ± 1.74a,b 42.64 ± 2.75a,b 38.35 ± 1.85a,b

TNF-a (pg/g) 61.96 ± 4.44 118.875 ± 5.15a 79.39 ± 3.59b 95.82 ± 5.38a,b 68.06 ± 3.32b,d

IL-1b (pg/mg tissue) 1.71 ± 0.14 12.21 ± 0.71a 3.805 ± 0.29a,b 4.25 ± 0.27a,b 3.45 ± 0.305a,b

IL6 (pg/mg tissue) 2.055 ± 0.17 14.65 ± 0.85a 4.57 ± 0.36a,b 5.105 ± 0.32a,b 4.145 ± 0.37a,b

PGE2 (pg/mg tissue) 202.125 ± 14.3 1535.38 ± 63.19a 712.75 ± 30.47a,b 943.29 ± 38.56a,b,c 720.14 ± 32.24a,b,d

aStatistical significance as compared to the control.bStatistical significance as compared to the acetic acid-treated group.cStatistical significance as compared to the Sulfasalazine-treated group.dStatistical significance as compared to the Dunaliella-treated group.

4 M. M. Abdel-Daim et al. Immunopharmacol Immunotoxicol, Early Online: 1–14

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48% respectively, compared to the acetic acid ulcerated group.

Moreover, the levels of GSH were increased by 133, 123.5

and 135% in SSZ-, DS- and SP-treated groups. Likewise, the

levels of CAT and SOD were also elevated by 175 and 483%

in SSZ group, by 132 and 201% in the DS-treated group and

finally by 185 and 269% in the SP-treated group.

Inflammatory and immunomodulatory markers

In acetic acid ulcerated rats, the macroscopic inflammatory

effect was mirrored by a 376% increase in mucosal MPO

activity, compared to the control group. SSZ, DS and SP

produced a significant reduction in MPO activity compared to

the acetic acid ulcerated group by about 63, 71 and 64%,

respectively (Table 3). Ulcerated non-treated group showed

an elevation by 192% in the colonic TNF-a level, which was

reversed upon treatment with SSZ, DS and SP by 67, 81 and

57%, respectively. Likewise, IL-1b level increased by 714% in

the acetic acid group compared to the control group, and

decreased by about 31, 35 and 28% in SSZ, DS and SP

treatment groups. Moreover, IL-6 and PGE2 levels were

increased in the acetic acid non-treated group by 713 and

759% compared to the control group. Although this effect was

lowered by all treatment regimens, yet the effect of SSZ and

SP 500 mg/kg was more pronounced than DS-treated groups.

The reduction levels in IL-6 level were 31, 35 and 28% for

SSZ, DS and SP groups, respectively. Similarly, the PGE2

reductions level for these groups was 46, 61 and 47%,

respectively.

Histopathological studies

The previous macroscopic findings were emphasized by the

histopathological examination, where the severity of alter-

ations in the colon of different experimental groups was

microscopically scored blindly by an independent

Figure 1. Photomicrographs of colonic sections of different treatment groups stained by H & E. (A) Control group (vehicle treated) showing normalhistological structure of the mucosal layer with underlying submucosa and muscular layer then serosa. (B) Acetic acid-treated group (AA) showingfocal ulceration, necrosis and hemorrhage in the mucosal layer with inflammatory cells infiltration in underlying submucosa (C) Sulfasalazine-treatedgroup (SSZ) showing focal hemorrhage in mucosa with edema, dilated blood capillaries and few inflammatory cells’ infiltration in the submucosa. (D)Dunaliella-treated group (DS) showing focal ulceration, necrosis and hemorrhage in mucosa with inflammatory cells infiltration and hemorrhage inunderlying submucosa. (E) Spirulina-treated group (SP) showing massive goblet cells formation in mucosal lining epithelium with edema and fewinflammatory cells infiltration in underlying submucosa.

DOI: 10.3109/08923973.2014.998368 Spirulina and Dunaliella modulate ulcerative colitis 5

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histopathologist, and the scores are presented in Table 4. In

the colon of the negative control group, there was no

histopathological alteration and the normal histological

architecture was recorded (Figures 1A, 2A, 3A, and 4A).

The microscopic pattern of acetic acid-induced colitis

group is characterized by focal ulceration, necrosis and

hemorrhage were noticed in the mucosal layer associated with

hemorrhage, edema and inflammatory cells’ infiltration in the

submucosa (Figure 1B). The intact regenerative crypts were

lined with columnar absorptive and goblet cells with marked

mucin depletion (Figures 2B and 3B). In addition to a

significant increase in the lymphocytic infiltration, extra-

vesated red blood cells and different types of leukocytes were

noticed among the colonic tunics. The cellular infiltration is

more extensive towards the upper mucosal surface between

the crypts and extends diffusely towards the submucosa and

even towards the muscularis externa (Figure 1B). The stromal

elements separating the mucosal crypts were rich in thickened

collagenic layer, and a lesser extent of collagenic fibers were

noticed toward the colonic submucosa (Figure 4B). The

SSZ-treated group was significantly improved as compared to

the experimental colitis group, where the mucosal layer

showed focal hemorrhagic areas while the underlying sub-

mucosa showed edema, few inflammatory cells’ infiltration

and dilated blood capillaries (Figure 1C). The columnar

absorptive cells as well as the goblet cells comprised fewer

amounts of neutral and acidic mucin as compared to the

normal control group. Fine collagen fibers and engorged

blood vessels were noticed among the colonic lamina propria

and submucosa (Figures 2C, 3C and 4C).

Figure 2. Photomicrographs of colonic sections of different treatment groups stained by PAS. (A) Control group (vehicle treated) showing strong PASreaction in the absorptive columnar and goblet cells. (B) Acetic acid-treated group (AA) showing marked neutral mucins depletion. (C) Sulfasalazine-treated group (SSZ) showing strong PAS positive columnar and goblet cells. (D) Dunaliella-treated group (DS) showing strong PAS positive gobletcells. (E) Spirulina-treated group (SP) showing moderate PAS positive reaction in the goblet cells.

6 M. M. Abdel-Daim et al. Immunopharmacol Immunotoxicol, Early Online: 1–14

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Moreover, histological findings in the DS-treated group

showed focal ulceration with necrosis and hemorrhages in the

mucosal layer while the underlying submucosa showed

hemorrhages, edema and inflammatory cells infiltration

(Figure 1D). The columnar absorptive cells as well as the

goblet cells showed more neutral and acidic mucins content as

compared to the SP-treated group (Figures 2D and 3D). Both

lamina propria and submucosa comprised nearly normal

content of collagen fibers as compared to the control group

(Figure 4D). On the other hand, histological examination

revealed that the group treated with SP showed a substantial

reduction of the mucosal epithelial erosions and marked

decreasing of the cellular infiltrate as compared to the

experimental group. Furthermore, a slight variation in crypt

architecture was also marked, as they are often regular in

shape and size (Figure 1E). Marked increase in neutral and

acidic mucin contents of goblet cells were also observed,

particularly alcianophilic goblet cells (Figures 2E and 3E).

Masson’s trichrome stained colonic sections showed fine

collagen fibrous content of the lamina propria as compared to

the control group (Figure 4E).

Immunohistochemical studies

The underlying inflammatory mechanisms were then inves-

tigated by immunohistochemical evaluation of iNOS and the

PG-generating enzyme, COX-2 expression. Immunoreactivity

of the COX-2 protein was generally intense than that of iNOS

in the different treatment groups. However, the area

Figure 3. Photomicrographs of colonic sections of different treatment groups stained by Alcian blue. (A) Control group (vehicle treated) showing manyAlcian blue positive columnar and goblet cells. (B) Acetic acid-treated group (AA) showing marked acidic mucins depletion. (C) Sulfasalazine-treatedgroup (SSZ) showing strong Alcian blue positive columnar and goblet cells. (D) Dunaliella-treated group (DS) showing strong Alcian blue positivecolumnar and goblet cells. (E) Spirulina-treated group (SP) showing strong alcianophilic goblet cells. For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.

DOI: 10.3109/08923973.2014.998368 Spirulina and Dunaliella modulate ulcerative colitis 7

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percentage of immunoreaction in different treatment groups

for COX-2 expression was strongly correlated with that of

iNOS expression (r¼ 0.865, p50.0001). No immunoreaction

was detected in the control group for either COX-2 or iNOS

(Figures 5A and 6A). On the other hand, severe positive

immunohistochemical signal for COX-2 and iNOS was

detected in ulcerated non-treated samples (Figures 5B

and 6B). Positive cells typically exhibited a brown reaction

product without background staining. The immunoreaction

area percentage varied among histologic structures in

different treatment groups, where the immunoreaction in the

SSZ-treated group was mild for COX-2 and moderate for

iNOS (Figures 5C and 6C). Sections from DS-treated rats

showed moderate immunohistochemical signals for both

COX-2, and iNOS (Figures 5D and 6D). Conversely, sections

from SP-treated rats showed no and mild immunohistochem-

ical signal for COX-2 and iNOS, respectively (Figures 5E

and 6E). The mean areas of immunohistochemistry for

the control, acetic acid, SSZ-, DS- and SP-treated samples

are 2.06 + 0.62, 27.25 + 1.74, 8.95 + 1.93, 16.27 + 1.75

and 6.74 + 0.62 for COX-2 (Figure 5F), and 1.1 + 0.28,

33.4 + 2.28, 12.035 + 1.88, 19.01 + 1.4 and 4.18 + 0.85 for

iNOS (Figure 6F), respectively.

Correlation studies

Estimation of colonic damage by mucosal injury scoring was

found to be strongly correlated with the mucosal content of

MDA, PCO, GSH, CAT and SOD (r¼ 0.89, 0.88, �0.7,

�0.79 and �0.77, respectively, p50.0001) as shown in

Figure 4. Photomicrographs of colonic sections of different treatment groups stained by Masson’s trichome. (A) Control group (vehicle treated)showing fine collagenic bundles in the lamina propria and submucosa. (B) Acetic acid-treated group (AA) showing fibrotic lamina propria.(C) Sulfasalazine-treated group (SSZ) showing fine collagen fibers and engorge blood vessels are noticed among the colonic lamina propria andsubmucosa. (D) Dunaliella-treated group (DS) showing fine collagen fibers among the lamina propria and submucosa. (E) Spirulina-treated group (SP)showing fine collagen fibrous content of the lamina propria.

8 M. M. Abdel-Daim et al. Immunopharmacol Immunotoxicol, Early Online: 1–14

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Figure 7(A–E). Estimation of colonic damage by mucosal

injury scoring was found to be strongly correlated with the

mucosal content of MPO, TNF-a, IL-1b, IL-6 and PGE2

(r¼ 0.8, 0.81, 0.935, 0.935 and 0.89 respectively, p50.0001)

as shown in Figure 8(A–E).

Discussion

Ulcerative colitis is an IBD characterized by inflammation of

the colorectal mucosa. Dysregulation in the immune response,

with infiltration of leukocytes into the mucosal interstitium

play an important role in its pathogenesis together with

excessive production of ROS2. The effects of acetic acid on

the colon in our UC experimental model were explained by

the fact that acetic acid could trigger inflammation by many

biological pathways, including direct cytotoxic effects in

addition to apoptotic damage of colonic epithelial cells25.

In addition, there is an increase in colonic weight/length

confirms intensification of intestinal infiltrations, inflamma-

tion and consequent intestinal edema40. The present investi-

gation outlines the anti-inflammatory and antioxidant

activities of DS and SP against acetic acid-induced ulcerative

colitis. Treatment of rats with DS or SP significantly reduced

the wet weight of distal colon segments, the gross lesion

scores, the incidence of diarrhea and occult blood and

significantly inhibited colonic shortening. However, regarding

the final body weight change, SP and DS offered better safety

profile, compared to the acetic acid-treated groups.

Oxidative stress plays a fundamental role in disease

initiation and progression of IBD41. Furthermore, it is well

known that the infiltration of inflammatory cells is considered

as a trigger of free radical production, which then attack

cellular macromolecules, disrupt epithelial cell integrity,

perturbate membrane lipids and delay mucosal recovery

through the impairment of endogenous defense systems42. In

this study, acetic acid-induced ROS formation, as indicated by

Figure 5. Immunohistochemical staining by COX II antibody in colonic sections of different treatment groups. (A) Control group (vehicle treated)showing no immunoreaction in mucosa and submucosa. (B) Acetic acid-treated group (AA) showing severe immunoreaction in mucosa, submucosaand muscularis. (C) Sulfasalazine-treated group (SSZ) showing mild immunoreaction in mucosa and submucosa. (D) Dunaliella-treated group (DS)showing severe immunoreaction in mucosa and submucosa. (E) Spirulina-treated group (SP) showing no immunoreaction. (F) The mean areapercentage of COX II immunohistocehmical staining in the different treatmnet groups.

DOI: 10.3109/08923973.2014.998368 Spirulina and Dunaliella modulate ulcerative colitis 9

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elevation of PCO levels, and reduction of SOD and

CAT activities. Moreover, acetic acid-induced oxidative

stress and lipid peroxidation was indicated by the increase

in MDA, and decrease in GSH levels. Similar findings have

been previously reported by Mustafa et al.26. On the contrary,

PCO and MDA levels were reduced while SOD, CAT

activities and GSH levels were elevated in all treated

groups, especially SP group, which even showed better

improvements than SSZ.

In addition, measurement of MPO activity has been

previously regarded as an indicator of the neutrophil influx

into inflamed gastrointestinal tissue43. A previous study

reported that acetic acid increased the levels of colonic MPO,

indicating infiltration of neutrophils and perturbation of the

inflammatory system33. This result is observed in mice

models44, rat models45, as well as patients with IBD46. In

agreement with previous reports, the present study showed a

significant increase in MPO activity in the acetic acid group.

On the other hand, both SP and DS 500 mg/kg ameliorated

neutrophil infiltration as evidenced by suppression of

colon MPO.

Furthermore, macrophages produce TNF-a and IL-1b, the

levels of which are often elevated in both animal models and

patients with UC44–46. IL-1b and TNF-a, are often regarded

as key immunoregulatory cytokines that amplify the inflam-

matory reaction by triggering a cascade of immune cells,

hence stimulating production of cytokines, arachidonic acid

metabolites, and proteases by intestinal macrophages, neu-

trophils, smooth muscle cells, fibroblast and epithelial cells47.

In addition, IL-1b and TNF-a induce epithelial cell necrosis,

edema, neutrophil infiltration, stimulate proliferation of

intestinal smooth muscle cells and fibroblasts and induce

synthesis of IL-6, IL-8 and PGE2 by these cells48. In this

context, it had been documented that blocking of the action of

endogenous IL-1b and TNF-a attenuates acute and chronic

experimental colitis and its further systemic complications49.

Moreover, it is well documented that during the pathologic

course of experimental colitis, TNF-a and IL-1b are released

Figure 6. Immunohistochemical staining by iNOS antibody in colonic sections of different treatment groups. (A) Control group (vehicle treated)showing no immunoreaction. (B) Acetic acid-treated group (AA) showing severe immunoreaction in mucosa, submucosa and muscularis.(C) Sulfasalazine-treated group (SSZ) showing moderate immunoreaction. (D) Dunaliella-treated group (DS) showing severe immunoreaction.(E) Spirulina-treated group (SP) showing mild immunoreaction. (F) The mean area percentage of iNOS immunohistocehmical staining in the differenttreatmnet groups.

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with subsequent activated synthesis of PGE2 and exacerbation

of tissue damage49. In this study, acetic acid caused elevations

in colonic levels of TNF-a and PGE2, which could mediate

epithelial cell necrosis, edema and neutrophil infiltration.

These elevations are in harmony with the previous finding that

TNF-a is widely expressed in the gut of IBD patients6.

Moreover, augmented levels of PGE2, goes in harmony with

previous studies which proved that elevated level of PGE2 is

explained by its enhanced synthesis rather than reduced

catabolism, both of which are mediated by TNF-a50.

Importantly, SP and DS decreased significantly the production

of both TNF-a and PGE2 compared to the acetic acid group.

Finally, the antiulcerogenic morphological effect of DS or

SP was further confirmed by histological preservation of the

colon architecture and correlated to its anti-inflammatory

properties. Treatment with either SP or DS significantly

attenuated the extent and severity of the histological features

of cell damage compared to the acetic acid group, an effect

which was less observed in the DS-treated group. In the

present study, immunohistochemistry showed significantly

increased COX-2 expression in the ulcerative lesions, sug-

gesting that endogenous PGs production may be involved in

exacerbation of the underlying ulcerative damage. This

concept is in agreement with a previous study, as higher

level of PGE2 was detected in the ulcerated group than in the

treated groups39. Furthermore, in the current study, immunor-

eactivity of COX-2 was correlated to that of iNOS. Our

results are in agreement with several previous observations

Figure 7. Correlation analysis. Analysis of the correlation coefficients between gross lesion score and MDA (A), PCO (B), GSH (C), CAT (D) andSOD (E). A significant positive correlation was observed between gross lesion score and colonic MDA, PCO, GSH, CAT and SOD (r¼ 0.89, 0.88,�0.7, �0.79 and 0.77 respectively, p50.0001).

DOI: 10.3109/08923973.2014.998368 Spirulina and Dunaliella modulate ulcerative colitis 11

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that suggest an interaction between COX-2 and iNOS51,52.

Inflammatory cells, such as neutrophils and macrophages,

express iNOS, produce NO, and also produce superoxide53.

Thus, iNOS is considered as a hallmark of inflammation,

which is consistent with previously published data document-

ing elevated mRNA levels of iNOS in the colon of ulcerated

rats54. The iNOS upregulation during inflammation is not

surprising since iNOS has been reported to be localized in

infiltrated neutrophils and macrophages in the colonic mucosa

and submucosa in animal models of IBD55.

To our knowledge, the antioxidant and anti-inflammatory

effects of SP or DS have not been examined previously in

the models of UC. In accordance with our results, it was

proven that the antioxidant properties of SP24 and DS16

contributed to its beneficial effect in treating various patho-

logical conditions.

Conclusion

This study suggests potential benefits of SP and DS in an

experimental model of colitis at the tested dose (500 mg/kg).

These effects, which are comparable or even better than SSZ

in case of SP, and are possibly attributed to their anti-

inflammatory and antioxidant properties. Hence, a strategy

that addresses the possible therapeutic efficacy of SP and DS

in the treatment of UC, following the appearance of its

symptoms, should merit further investigations.

Declaration of interest

All authors declare that they have no competing financial or

personal interest or any kind of conflict of interest relevant to

this study.

Figure 8. Correlation analysis. Analysis of the correlation coefficients between gross lesion score and MPO (A), TNF-a (B), IL-1 b (C), IL 6 (D) andPGE2 (E). A significant positive correlation was observed between gross lesion score and colon MPO, TNF-a, IL-1 b, IL 6 and PGE2 (r¼ 0.8, 0.81,0.935, 0.935 and 0.89, respectively, p50.0001).

12 M. M. Abdel-Daim et al. Immunopharmacol Immunotoxicol, Early Online: 1–14

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