Vijayabharathi Rajendiran,Vidhya Natarajan,Sivasithamparam Niranjali Devaraj

Department of Biochemistry,University of Madras,Maraimalai Campus(Guindy),Chennai,600025,India

Keywords:

ABSTRACT

1.Introduction

Chronic inflammation is the major cause of many human cancers,including colorectal cancer(CRC).Inflammatory bowel diseases(IBD)are immune mediated chronic inflammatory disorders of the digestive tract,ulcerative colitis(UC)and Crohn’s disease(CD)being the major forms[1].UC is a disorder involving the contiguous inflammation of the lamina propria and disruption of the mucosal barrier of the large intestine with an increased risk of developing colitis associated colorectal cancer(CAC)[2].Although the etiology of IBD remains elusive,recent studies have hinted that the combination of genetic susceptibility factors and dysregulated immune response driven by microbial factors in the enteric environment contribute to the pathogenesis of these diseases.Dysregulated immune response causes infiltration of phagocytic leukocytes into the mucosal interstitium.The large number of activated lymphocytes and non-lymphoid cells,such as macrophages and neutrophils enter the injured mucosa,which conduces to the over production of oxygen free radicals,reactive oxygen species(ROS)that provoke oxidative stress.However,the accumulation of oxidative stress along with the proinflammatory cytokines(eg.TNFα)and inflammation related proteins(eg.COX-2)cause injury to the inflamed target tissue and play an important role in the patho physiology of UC[3].

Animal models of IBD are a valuable tool for investigating the pathogenesis of IBD and for the development of new drugs.DSS induced colitis model is the most widely used,over other animal models of colitis,which closely mimics human UC with symptoms that include weight loss,diarrhea and colonic shortening[4].In addition,this model has been shown to respond to human UC drugs such as sulfasalazine,olsalazine and mesalazine[5].Oral administration of DSS in drinking water results in acute and chronic colitis[4,6].DSS induced colitis model is especially useful for analyzing the contribution of inflammatory mechanisms in colitis[7].

The current therapeutics used for the treatment of human IBD are sulfasalazine,azathioprine,5-aminosalicylic acid,corticosteroids,and immunosuppressive agents that were mainly focusing on immunomodulatory and inflammatory signaling cascade[2,8].However,these drugs have certain restrictions due to their potentially serious side effects.Thus,there is an urgent need for inexpensive alternative drugs that may be equally or more effective on cancer cells but less toxic to normal cells.

Plants possess a long history of utilization in the treatment of various cancer types.Many of the anticancer agents like vincristine and vinblastine that are available in the drug market are derived from natural sources[9].Among traditional medicines,Alpinia,a Zingiberaceae,is a gold mine for the researchers towards the development of potential therapeutics against various diseases like cancer,diabetes and ulcer[10].Alpinia officinarum,a member of ginger family(Zingiberaceae),commonly known as lesser galangal,is widely cultivated as spice in tropical Asia.It is a traditional Chinese medicinal plant,which has been used to control storage pests,as a food additive and for treating a wide range of symptoms including stomach ache,swelling,inflammatory and gastrointestinal ailments[11].Chemical studies of the rhizomes have reported the presence of three groups of important chemical constituents,flavonoids,glycosides and diarylheptanoids[12].Galangin,a member of the flavonol group of flavonoids is the major flavonoid in Alpinia officinarum and the diarylheptanoids are the characteristic compounds.The chemical constituents isolated from Alpinia officinarum,mainly the galangin and group of diarylheptanoids were reported to possess strong antioxidative,anti-inflammatory,antiproliferative,antiplatelet,anti-emetic,antimicrobial and cytotoxic activities[13].In view of the above properties of Alpinia officinarum Hance and its derived metabolites,we investigated the anti-inflammatory effect of HEAO on DSS induced acute and chronic colitis rat model and the antiproliferative effect on HT-29 human colon cancer cell lines.

2.Materials and methods

2.1.Chemicals

DSS(M.W 36–40 kDa)was purchased from MP Biomedicals,USA.Hexadecyltrimethylammonium bromide(HTAB),O-Dianisidine hydrochloride,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)were purchased from Sigma-Aldrich Chemicals(USA).Dulbecco’s Modified Eagle Medium-high glucose(DMEM)and fetal bovine serum(FBS)were obtained from GIBCO BRL Germany.Rabbit monoclonal TNF-α,NF-κB and anti-rabbit HRP conjugated antibodies were purchased from Cell Signaling Technology(Beverly,MA,USA).All the other chemicals used for the study were of the finest Analytical grade.

2.2.Collection and Preparation of plant extract

Dried root of A.officinarum was collected from Ayurvedic pharmacy,Chennai and authenticated by a botanist from the Institute of Herbal Botany Plant Anatomy Research Centre,Chennai.The rhizome of A.officinarum was shade dried and ground to a fine powder.The powder obtained was successfully extracted in n-Hexane and filtered.The filtered extracts were dried under reduced pressure in a rotary flash evaporator and used for further studies.

2.3.Animals,induction of colitis and experimental design

Male Wistar rats weighing 150–200 g obtained from The King Institute of Preventive Medicine(Chennai)were used in the study.The animals were housed in a well aerated room under 12-h light/dark cycle for 1 week,prior to the experiment.Food and water were provided ad libitum.All experiments were performed with the approval of the Institute Animal Ethics committee.(IAEC No:15/01/2012)

Table 1 Scoring of Disease Activity Index.

Acute colitis was induced by administration of 5%DSS in drinking water for 5 days.Chronic colitis was induced by administration of 5%DSS for the first 9 days and it was subsequently decreased to 2%for the next 18 days[14].DSS solution was prepared freshly and changed daily.Control rats received normal drinking water alone.

For the induction of acute colitis,the animals were randomly divided into four groups(n=6 rats per group).Group A received normal drinking water and served as control.Group B received HEAO,200 mg/kg body wt/day in olive oil served as drug control.A preliminary dosage fixation study with varying concentrations of HEAO showed 200 mg/kg to be the optimum oral dose in rat,with no long-term toxicity(data not shown).Groups C and D rats received 5%DSS in drinking water for 5 days and Group D rats were co treated with HEAO 200 mg/kg body wt for 5 days.For the induction of chronic colitis,the rats were grouped as same in acute colitis,but the experimental period was extended for 27 days.Group A received normal drinking water and served as control.Group B received HEAO,200 mg/kg body wt/day in olive oil and served as drug control,Groups C and D rats received 5%DSS in drinking water for the first 9 days;it was then decreased to 2%in the following 18 days and Group D rats were co treated with HEAO,200 mg/kg body wt while inducing with DSS.

2.4.Observation of colitis

Throughout the experimental period body weight,food intake,water intake as well as the presence of blood in the stool and water content of the stools were observed every day to assess the severity of colitis.At the end of the experimental period,the rats were fasted overnight and were anesthetized the next morning with ketamine hydrochloride,intravenously(30 mg/kg bw).Blood was collected in heparinized tubes and serum was separated by centrifugation at 4000 rpm for 10 min at 4°C to analyze the hematological profile.The colon and liver tissue samples were collected for further analysis.The length and weight of the colon were measured.Colonic damage was assessed by means of indirect(DAI score)and direct measures(macroscopic and microscopic damage)and myeloperoxidase(MPO)activity.

2.5.Disease activity index(DAI)

DAI was used to assess the severity of the induced colitis.It is the combined score of weight loss,stool consistency and bleeding from day 0 to 5 and 0 to 27 during acute and chronic DSS treatment,respectively.Each of these parameters were assigned a score based on the criteria shown in Table 1,which was used to calculate an average daily DAI for each animal,as described previously[15].

2.6.Relative colonic weight/length ratio

The length of colon was measured from the colo cecal junction to the anal verge.The colons were opened longitudinally and gently washed with phosphate buffered saline(PBS).Excess fluid was removed from the colon and weight per unit length was calculated.

2.7.Haematological analysis and clinical chemistry

Red blood cell counts(RBC)and white blood cells(WBC)were determined as described by Brown[16].Hemoglobin(Hb)concentration was determined as described by Hewitt[17].Packed cell volume(PCV)was determined by micro hematocrit method using capillary tube while total and differential leucocytes count,platelets count were determined as described by Schalm et al[18].Mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH)and mean corpuscular hemoglobin concentration(MCHC)were calculated as described by Schalm et al.[18].Serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),lactate dehydrogenase(LDH)and gamma glutamyl transferase(γGT)were assayed by the standard procedures[19–22].CRP levels were determined using commercial enzymatic kit(Bio-Diagnostic,Cairo,Egypt)

2.8.Determination of colonic myeloperoxidase(MPO)activity

MPO activity was evaluated according to Bradley et al.[23].Colonic MPO was extracted using HTAB.Then,o-dianizidine HCl was oxidized by MPO in presence of hydrogen peroxide.One unit of myeloperoxidase activity is defined as the amount of the MPO present/g tissue weight that caused a change in absorbance of 1.0/min at 460 nm.

2.9.Evaluation of biochemical parameters

The non-enzymatic antioxidant reduced glutathione(GSH)was determined by the method of Sedlak and Lindsay[24]modified according to the method of Moron et al.[25].

The activities of enzymatic antioxidants-superoxide dismutase(SOD)was assayed by the method of Misra and Fridovich[26],catalase(CAT)activity was performed according to the method of Takahara et al.[27],glutathione peroxidase(GPx)by Rotruck et al.[28],and glutathione-s-transferase(GST)by Habig et al.[29].Lipid peroxide content in tissues was determined by thiobarbituric acid reactive substances(TBARS)as described by Ohkawa et al.[30].The activity staining of SOD and CAT was carried out by the methods of Beauchamp and Fridovich[31]and Sun et al.[32],respectively.

2.10.Histological analysis

Tissue samples from the distal colon and liver were fixed in 10% neutral buffered formalin and embedded in paraffin.For histological evaluation,4-μm thick tissue sections were stained with hematoxylin-eosin and examined under light microscope.

2.11.Immuno histochemistry(IHC)

IHC was performed on paraffin embedded tissue sections of 4-μm thickness by the standard method with slight modifications.The tissue sections were deparaffinized and rehydrated through a graded series of alcohol,followed by PBS/H2O2wash for 10 min to block endogenous peroxidase activity.Nonspecific binding was blocked with 3%BSA in 1X PBS at room temperature for 45 min.The sections were incubated with primary antibodies of NF-κB and TNFα(Mouse monoclonal antibody)over night at 4°C,then incubated with respective HRP labeled secondary antibody for 2 h at room temperature.The signals were received by treating the slides with 3,3′-diamino benzidinetetrahydrochloride(DAB).The slides were then counterstained with haematoxylin and mounted with DPX.

2.12.Cell culture and HEAO treatment

Human colon tumor cell line HT29 was obtained from NCCS,Pune.Cells were maintained in Dulbeccos Modified Eagle Medium supplemented with 10%FBS,1X antibiotic-antimycotic solution in tissue culture flasks at 37°C under 5%CO2and 95%humidified air.

Dose response studies were carried out to determine the suitable dose of HEAO for the inhibition of cell growth and induction of apoptosis.The stock solution of hexane extract was prepared using dimethyl sulfoxide(DMSO)and then diluted in DMEM to get the desired doses.The final concentration of DMSO was less than 0.1%.

2.12.1.Analysis of cell viability

HT-29 cells were plated in 96-well plates with 100 μl medium and were exposed to various concentrations of HEAO(0.5,1,2.5,5,7.5,10 μg)for 3,6,24 and 48 h in triplicates.After incubation,cells were treated with 100 μl MTT solution(final concentration,1.0 mg/ml)at 37°for an additional 4 h.After incubation,the medium was discarded and the formazan crystals were solubilized with 100 μl DMSO and then the absorbance was measured at 540 nm using a microplate reader[33].

2.12.2.RNA extraction and reverse transcription-polymerase chain reaction

mRNA expressions of NF-κB and Cox-2 were determined by reverse transcription polymerase chain reaction.RNA was extracted by using the TRIzol reagent(Invitrogen,Carlsbad,CA,United States)and reverse transcription was performed(Invitrogen)according to the manufacturer’s instructions.The primer sequences used were 5′-CAGAGTTGGAAGCACTCTATGG-3′and 5′-CTGTTTTAATGAGCTCTGGATC-3′for COX-2,5′-GGACTGCCGGGATGGCTTCTAT-3′and 5′-GCTGCTCTTCTTGGAAGGGGTTGT-3′for NF-κb and 5′-GGACTGCCGGGATGGCTTCTAT-3′and5′GCTGCTCTTCTTGGAAGGGGTTGT-3′for β-actin analysis.

2.13.Statistical analysis

All results were expressed as mean±SD for six animals in each group.The data obtained were evaluated with SPSS 16 software.Hypothesis testing methods included one-way analysis of variance(ANOVA)followed by least significant difference(LSD)test.p＜0.05 was considered to indicate statistical significance.

3.Results

3.1.General observations of DSS induced acute and chronic colitis

The consumption of food,water intake,the symptomatic colitis parameters such as weight loss and DAI score were monitored daily throughout the experimental period.Animals given varying concentrations of DSS in their drinking water developed Acute and Chronic colitis without mortality.There were no significant differences in water intake among the control and experimental groups.Food intake did not show any variation between the control and drug control group during the experimental period.The food consumption of the DSS treated group of animals in acute colitis decreased progressively,up to 50%,day by day and for chronic colitis the same trend was observed till day 6 and then it increased progressively and reached the initial value at the end of the experimental period.When compared to the colitis group,the DSS+HEAO treated animals consumed higher amount of food.

Fig.1.Effect of HEAO on DSS-induced acute colitis in rats.A:disease activity index(DAI)changes,B:macroscopic appearance,C:Relative colonic weight/length ratio,D:CRP level and E:MPO level.Values are expressed as mean±SD.a:DSS versus control,b:DSS versus DSS+HEAO.“*”denotes a statistically significant difference at p＜0.05,and ns indicates a non significant difference.

Fig.2.Effect of HEAO on DSS-induced Chronic colitis in rats.A:disease activity index(DAI)changes,B:macroscopic appearance,C:Relative colonic weight/length ratio,D:CRP level and E:MPO level.Values are expressed as mean±SD.a:DSS versus control,b:DSS versus DSS+HEAO.“*”denotes a statistically significant difference at p＜0.05,and ns indicates a non significant difference.

The control and drug treated animals(in acute and chronic groups)showed normal growth with a moderate increase in their body weight.All rats with DSS induced colitis progressively showed weight loss and manifested with bloody diarrhea.Administration of HEAO during DSS induction prevented this weight loss.The degree of severity of the DSS-induced colitis in rats was determined by assessing the DAI score as shown in Table 1.DAI started to increase significantly from day 2,whereas the DAI score was significantly decreased by oral HEAO supplementation in DSS+HEAO treated rats,compared with DSS induced animals(Figs.1 and 2A).

Table 2 Hematological and serobiochemical parameters of DSS induced Acute and Chronic colitic rats.

Table 3 Serum marker enzymes in DSS induced acute and chronic colitic rats.

3.2.Morphology and relative weight/length ratio of Colon

Morphological examination of colon in acute and chronic group animals showed visible thickening of the colon wall and hemorrhagic erosions in the DSS group.It has been reported that the length of the colon is inversely linked to the severity of DSS-induced colitis.The treatment of DSS caused a reduction in colon length and significant increase in the relative colonic weight/length ratio in both acute and chronic groups compared to control animals.Cotreatment with HEAO markedly reduced macroscopic damage in DSS induced animals and decreased the colon weight/length ratio.HEAO alone had no effect on the colonic weight/length ratio compared to control animals(Fig.1B,C and Fig.2B,C).

3.3.Haematological analysis and clinical chemistry

Haematological parameters of the acute and chronic colitis group are represented in Table 2.DSS treated animals showed a great variation when compared with the control.These animals showed signs of anemia indicating severe loss of blood with reduction in the haematological parameters such as red blood cells(RBC),hemoglobin(Hb),packed cell volume(PCV),Total count(TC),mean corpuscular hemoglobin(MCH),mean cell volume(MCV)and platelet count.There was an increase in the levels of lymphocytes and neutrophils.There was no significant difference in the levels of the rest of the haematological parameters analysed.DSS+HEAO treated animals showed a significant increase in the levels of haematological parameters such as RBC,Hb,PCV,TC,MCH,MCV and platelet count.There was a significant reduction in the parameters such as lymphocyte and neutrophil levels.This shows that the there was no sign of anemia in the rats,which were treated with HEAO.

The acute phase reactive protein(CRP)level in serum of both the colitis induced groups was very high when compared to the control group.DSS treated animals showed a significant increase,whereas,HEAO administration was effective in treating inflammation by significantly reducing the CRP level(Fig.1D and Fig.2D).

Fig.3.Activity staining of superoxide dismutase(SOD)and CAT in the colon of acute and chronic experimental colitis animals.Lane 1-control,Lane-2 Drug control group,Lane 3 DSS induced colitis,Lane 4-DSS+HEAO.A significant decrease of SOD and CAT(lane 3)activity in DSS induced acute and chronic colon tissues was seen.Lane-4 shows the increased SOD and CAT activity in colon of the DSS+HEAO treated rats.

The levels of the serum marker enzymes(AST,ALT,ALP,LDH and γ-GT)were observed to be higher in acute and chronic colitis of DSS treated groups and lower in groups treated with HEAO when compared to their controls(Table 3).

A significant decrease in the levels of non-enzymatic antioxidant GSH and enzymatic antioxidants such as SOD,CAT,GPX,and GST were evident in DSS induced rats when compared with control.HEAO administration to DSS induced animals significantly increased the levels of these antioxidants as compared with DSSinduced group(Table 4).The same trend was observed in the activity staining of SOD and CAT in colon tissue(Fig.3)

Table 4 Antioxidant status in DSS induced acute and chronic colitic rats.

Fig.4.A and C.Histological findings in the colon of Acute and Chronic colitis in rat(original magnification x40).a,b colon sections from control and Drug control animals showing normal tissue architecture of colon with no inflammatory infiltration.c:Mucosal injury produced after DSS administration for 5,25 days,respectively,with loss of crypts and ulceration as well as severe inflammatory cell infiltration.d:Morphological alterations associated with DSS induced Acute and Chronic colitis was reduced upon HEAO administration(200 mg/kg/day).Microscopic damage produced by DSS was decreased by HEAO administration,facilitating mucosal healing,reducing inflammatory cells infiltration.Fig.4B and D.Histological findings in the liver of Acute and Chronic colitis in rat(original magnification x40).a,b:Liver cells of control,Drug control group showing normal hepatic architecture in both Acute and Chronic groups.c:Morphological alteration associated with DSS induced Acute and Chronic colitis showing displaced nucleus to the periphery and Parenchymal inflammatory infiltration.d:Morphological alterations associated with DSS induced Acute and Chronic colitis were reduced upon HEAO administration.HEAO decreased the microscopic damage produced by DSS.(CV-central vein).

3.4.Myeloperoxidase activity

HEAO showed no effect on the colonic MPO activity compared to control rats.The colonic MPO activity was greatly increased by about 2fold in acute and 3fold in chronic colitis following administration of DSS compared to control rats.Co-treatment with HEAO during DSS administration significantly reduced the colonic MPO activity compared to DSS-treated animals(Fig.1E and Fig.2E).

3.5.Histological assessment of colon and liver tissues of control and experimental animals

Fig.4A and C of control and drug control group showed normal architecture of colon.The colonic sections of the DSS induced animals showed typical inflammatory changes in colonic architecture such as crypt and surface epithelial cell loss as well as inflammatory cell infiltration.In addition,a complete destruction of the epithelial architecture was observed.The DSS+HEAO treated group showed an attenuation in the severity of colon injury,a higher integrity of mucosal architecture,epithelial loss and the histological damage were significantly reduced as compared to the DSS group(Fig.4A and C).

The hepatocytes in control and drug treated groups were normal with intact structures.Liver from DSS induced group of acute and chronic colitis showed moderate degrees of steatosis.Liver sections showed loss of parenchymal cells and infiltrations of inflammatory cells(Fig.4B and D).Displaced nucleus to the periphery of the hepatocytes was occasionally found in the livers.The DSS+HEAO treated group restored the normal architecture of liver as compared to the induced group.

Fig.5.Effect of HEAO on the expression of NF-κB(A and C)and TNF-α(B and D)in the colon of DSS induced Acute and Chronic colitis(original magnification x40).(Fig.5A and C):a,b:NF-κB protein expression in Control,Drug control group.c:DSS induced Acute and Chronic group-the number of NF-κB immunoreactive positive cells was significantly more than that of normal controls.d:DSS+HEAO treated groups of Acute and Chronic colitis-the number of NF-κB immunoreactive positive cells in this group was significantly less than that of DSS controls.(Fig.5A and C):a,b:TNF-α protein expression in control,Drug control groups of Acute and Chronic colitis(original magnification x40).c:DSS induced Acute and Chronic groups-the number of TNF-α immunoreactive positive cell was significantly more than that of normal controls.d:DSS+HEAO treated groups of Acute and Chronic colitis-the number of TNF-α immunoreactive positive cells in this group was significantly less than that of DSS controls.

3.6.Effect of HEAO on NF-κB and TNF-α expression in DSS induced acute and chronic inflammation

Fig.5 shows the immunohistochemical staining of NF-κB(Fig.5A and C)and TNF-α(Fig.5B and D)in colonic tissues.The results showed increased expressions of the proinflammatory mediators NF-κB and TNF-α in the DSS induced rats.In HEAO co-treated rats,decreased expression of these inflammatory mediators was observed when compared with DSS induced acute and chronic colitic rats.

3.7.Effect of HEAO on Cell viability,apoptosis and mRNA expression of NF-κB and COX-2 in cultured HT-29 cells

The cells were treated with different doses of HEAO(0.5,1,2.5,5,7.5,10 μg),and cytotoxicity was determined by the MTT assay.The results of the cytotoxicity of HEAO in HT-29 cells are presented in Fig.6.With increasing concentrations of HEAO,cell death increased.As indicated in Fig.6A,there was gradual decrease in the viability of HT-29 cells with increasing doses of HEAO.MTT assay revealed a dose and time dependent response with regard to the cytotoxicity of HEAO with an IC50value of 42.41%,49.2%,49.81%and 56.37%in 2.5,1.0 μg/0.2 ml at 3rd,6th hour and 0.5 μg/0.2 ml at 48 h,respectively.As observed from the Fig.6B,HEAO treated cells showed marked reduction in the mRNA expression levels of NF-kB and COX-2 as compared to untreated cells(Fig.6B).

4.Discussion

Ulcerative colitis(UC)is a chronic inflammatory bowel disease(IBD)associated with an immune mediated disorder of the intestine,which occurs as a result of dysregulated immune response in genetically predisposed individuals due to various environmental conditions[34]The pathological consequences of IBD with increased cell proliferation and evasion from apoptosis thereby ultimately contribute to carcinogenesis.Overproduction of inflammatory mediators,such as tumor necrosis factor(TNF-??)and NF-κB,is strongly associated with the pathogenesis of IBD.This increased inflammatory reaction generates excess ROS,activation of proteolytic enzymes and cytokines which lead to tissue damage.

The animal models of colitis resemble a human IBD in the aspects of histological and immunological changes[7].Administration of DSS to the experimental animals through drinking water is a widely accepted model to study the pathogenesis of UC.Simply changing the concentration of DSS results in acute and chronic colitis and shares clinical and histopathological characteristics as similar to human UC[4,15].Conventional drugs used for treatment of IBD are mostly anti-inflammatory agents,including corticosteroids,and 5-amino salicylic acid(5-ASA)and its derivatives such as sulfasalazine.However,these drugs are considered as double-edged swords due to the generation of additional oxidative stress,which may result in hepatotoxicity and adverse side effects as well[35].Alpinia officinarum,a botanical cousin to ginger,has been traditionally used in medicine for treating inflammatory and gastrointestinal disorders.This directed our attention towards addressing the possible protective role of the HEAO on DSS-induced ulcerative colitis in rats and in vitro studies on HT-29 cells.

Fig.6.Effect of HEAO on cell viability(A)in cultured HT-29 cells.Cells were treated with HEAO at different concentrations(0.5,1,2.5,5,7.5,10 μg)for 3,624 and 48 h.From the MTT assay,the estimated IC 50 value of HEAO in HT-29 cells was found to be 1 μg/0.2 ml at 3,6,24th hour and 0.5 μg/0.2 ml at 48 h,respectively.(B)mRNA Expression of β-actin(a),cox-2(b)and Nf-κB(c)in HT-29 colon cancer cells treated with HEAO.Agarose gel showing products after 24 h of HEAO treatment in HT-29 cells.Densitometric value of cox-2(b)and Nf-κB(c)normalized with β-actin is presented as“fold change”as compared with untreated HT-29 cells.Values are expressed as mean±SD(n=3).Lane 1-0 μg HEAO/0.2 ml.Lane 2-0.5 μg HEAO/0.2 ml.Lane 3-1 μg HEAO/0.2 ml.Lane 4-2.5 μg HEAO/0.2 ml.

In our study,exposure to DSS in drinking water for 5 and 27days,induced acute colitis and the chronic colitis with an increased DAI score and colon weight/length ratio.Cooper et al.,[15]have reported a significant correlation between the DAI score and pathological changes in DSS induced acute and chronic colitis.DSS induced colitic animals exhibited a significant weight loss,developed bloody diarrhea and anemia marked by lower levels of RBC and Hb since anemia and reduction in the length of the colon are considered as the cardinal signs of IBD[36].HEAO significantly attenuated this DSS induced weight loss,DAI score and colonic shortening and prevented the anemia due to rectal bleeding.Reduction in the score of DAI indicates that HEAO prevents the initial progression of UC.During DSS induced colitis,the antioxidant status of the colonic tissue was altered and resulted in the increased production of reactive oxygen species(ROS).According to a previous study on the antioxidant enzyme profile in a DSS induced model,a marked depletion of the cellular antioxidant GSH was observed[37].Clinical studies on IBD also revealed the imbalance in antioxidant status[38].The antioxidant profile was restored by HEAO treatment,which suggested its anti-oxidant property.

MPO is an enzyme that is found predominantly in neutrophils,a good marker of inflammation and tissue injury and is used as an index of inflammatory response.Its activity in the colon is linearly related to neutrophil infiltration[39,40].MPO activity was lower in HEAO treated animals as compared to DSS induced groups.Hence the decreased MPO levels,suggested the anti-inflammatory activity of HEAO with reduction in neutrophil infiltration.The circulating levels of CRP reflect the ongoing inflammation and tissue damage much more accurately than other biochemical parameters.Serum CRP as a biomarker in the course of UC has been reported previously.The CRP level in serum of induced group was elevated when compared to the control.This is due to the inflammation induced by DSS.Masoodi et al.[41],reported a significant correlation between severity of UC and serum CRP levels and it was observed to be 100%specific.HEAO treatment reduced the CRP levels in the serum suggesting a positive therapeutic effect on UC.

Histological examination of colonic sections revealed an altered architecture of colon mucosa with typical inflammatory changes in colonic architecture such as crypt and surface epithelial loss as well as infiltration of inflammatory cells and complete destruction of the epithelial architecture.The severity of the disease increases with chronicity.These macroscopic and microscopic alterations observed in our study were in agreement with numerous studies reported on DSS induced UC models in rats[7].

NF-κB has a specific role in coupling inflammation to cancer by regulating the functions of inflammatory gene expression such as TNF-α and IL-6.DSS has a direct toxic effect on the epithelium and destroys the mucosal barrier and causes the elevated secretion of pro-inflammatory cytokines such as TNF-α[42].Immunohistochemical findings of TNF-α expression are in agreement with the elevated level in DSS treated animals.Upregulation of inflammatory mediators during DSS induced oxidative stress is strongly associated with the pathogenesis of IBD.

Our results indicate that HEAO treatment reduces the viability percentage of HT-29 human colon cancer cells in a time and concentration dependent manner.This confirmed the cytotoxic and anti-proliferative effect of HEAO.It is emphasized that there was a close relationship between apoptosis and proliferation in malignant neoplastic cells.Increased expression of NF-κB and COX-2 has been reported in many colorectal tumors and adenocarcinomas.COX-2 expression increases from normal to progression of cancerous state.There are multiple lines of compelling evidences supporting that COX-2 plays a role in the development of tumors.Thus,inappropriate up regulation of COX-2 prolongs the survival of malignant or transformed cells and leads to phenotypic changes associated with metastatic potential.Our result clearly indicates that the HEAO markedly inhibited NF-κB and COX-2 mRNA expressions in HT-29 cells.From the invitro results,it is seen that HEAO effectively attenuated the proliferation of HT-29 cells and inhibited the expression of inflammatory mediators.

5.Conclusion

The results of this study clearly indicate that,cotreatment of HEAO at 200 mg/kg/day could ameliorate the morphological and biochemical features of DSS-induced acute and chronic colitis.This was evidenced by a decrease in weight loss,DAI score,macroscopic and microscopic damage caused by DSS administration.HEAO treatment was effective by improving hematocrit values,antioxidant enzymes and by reducing neutrophil infiltration as evidenced from decreased MPO activity and reduced histopathological damage.In addition,HEAO down-regulated the levels of inflammatory mediators in the colonic tissues of DSS-administered animals and in HT-29 human colon cancer cells.

This study shows that cotreatment with HEAO was effective against DSS induced colitis by regulating the production of proinflammatory mediators.Taken together,all these findings showed the antioxidant and anti-inflammatory potency of HEAO against DSS induced acute and chronic colitis paradigm.Hence,our findings suggest that HEAO may have a positive therapeutic effect in the treatment of ulcerative colitis.