Modulating effect of Biophytum sensitivum extract on rats with acetic acid-induced ulcerative colitis

Abstract

Context: Traditionally, Biophytum sensitivum (L.) DC (Oxalidaceae) is used in Indian medicine to treat diseases include stomachache, convulsions, cramps, inflammation, and ulcer.

Objective: The present study examines the effect of aerial parts of B. sensitivum (methanol extract) on a murine model of ulcerative colitis (UC).

Materials and methods: UC was induced by intracolonic injection of 3% acetic acid in Wistar rats. B. sensitivum (50 or 100 mg/kg b wt) or reference drug sulfasalazine (100 mg/kg b wt) was administrated intra-peritoneally for 5 consecutive days before induction of colitis.

Results: In the present study, we demonstrated for the first time that the administration of B. sensitivum (50 mg/kg b wt) was found to inhibit colitis by lowering macroscopic score (up to 3.66 ± 0.77) and also showed significant reduction (p < 0.01) in lactate dehydrogenase (LDH) and myeloperoxidase (MPO) activities. Furthermore, a significant reduction (p < 0.01) in mucosal content of lipid peroxidation (LPO), glutathione (GSH), superoxide dismutase (SOD), and nitric oxide (NO) confirms that B. sensitivum could significantly inhibit colitis. The study showed significant reduction (p < 0.01) in colonic tumor necrosis factor-α (TNF-α), interleukin-1-β (IL-1β), and IL-6 levels as well as the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) after treatment compared with the colitis control group. The histopathological study also confirms the foregoing findings. Treatment with B. sensitivum was also able to inhibit the activation and translocation of transcription factors, nuclear factor (NF)-κB subunits (p65/p50).

Conclusion: These results suggest that B. sensitivum exhibits protective effect against acetic acid-induced UC.

• Acetic acid
• inflammatory mediators
• pro-inflammatory cytokines

Introduction

Inflammatory bowel disease (IBD) encompasses chronic inflammatory condition of intestine which includes ulcerative colitis (UC) and Crohn’s disease (CD). UC is a chronic, relapsing, immunologically mediated, and most intractable gastrointestinal disease with high risk of colorectal cancer (Eaden & Mayberry, 2002). The precise mechanism involved in pathogenesis of IBD remains unknown. However, it has been reported that immune dysfunction plays a decisive role in the development of UC. There is convincing evidence that imbalances between proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1-β (IL-1β), IL-6, and IL-12 and expression of inflammatory proteins include cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) are believed to play a vital role in modulating the inflammation process (Ardizzone & Porro, 2005). Numerous therapeutic agents for IBD include anti-inflammatory agents and corticosteroids along with certain immunomodulators were used. Although these treatments are effective, they are associated with severe adverse events include diarrhea, cramps, abdominal pain accompanied by fever, and high blood pressure (Xu et al., 2004). Hence, there is a need to develop novel therapeutic options with negligible side effects.

Biophytum sensitivum (L.) DC (Oxalidaceae) has been used in traditional medicine by many people of Asia, especially in Indian medicine (Jirovetz et al., 2004). We previously reported that B. sensitivum possess antioxidant (Guruvayoorappan et al., 2006), anti-inflammatory, anti-angiogenic (Guruvayoorappan & Kuttan, 2007a), antitumor, immunomodulatory (Guruvayoorappan & Kuttan, 2007b) effects, as well as inhibiting nitric oxide (NO) and proinflammatory cytokine production and regulating the expression of iNOS and COX-2 genes in LPS/Con A stimulated macrophages (Guruvayoorappan & Kuttan, 2008). B. sensitivum has been reported to contain flavonoids including luteolin 7-methyl ester, isoorientin, biflavones such as cupressuflavone and amentoflavone, as well as two acids including 4-caffeoylquinic acid and 5-caffeoylquinic acid (Sakthivel & Guruvayoorappan, 2012). Although the activity of B. sensitivum has attracted a great deal of interest, its primary molecular target and its mechanism of action remain to be clarified. So far, to our knowledge, there is no study of the effect of B. sensitivum on UC reported.

In view of the above findings, we sought to investigate the protective effect of B. sensitivum on the development of colonic inflammation in murine models. Acetic acid-induced UC in rats is one of a common model in IBD research, which resembles human UC in histology, eicosanoid production, and excessive oxygen-derived free radicals release by inflamed mucosa (Millar et al., 1996). To test our hypothesis, the present study was undertaken to determine the effect of B. sensitivum against experimental colitis in Wistar rats.

Materials and methods

Chemicals and reagents

Sulfasalazine was procured from Wallace Pharmaceuticals, Goa, India. COX-2 and iNOS polyclonal antibody were purchased from Abcam, Cambridge, MA, and Thermo Scientific, Waltham, MA, respectively, lactate dehydrogenase (LDH) assay kit was procured from Biovision, Milpitas, CA. Myeloperoxidase (MPO), tumor necrosis factor (TNF-α), and iNOS kits were obtained from USCN Life Science, Wuhan, Hubei. Interleukins, IL-1β, and IL-6 were purchased from KOMA Biotech, Daejeon, Korea, and COX-2 kit from Shangai Bluegene Biotech, Shangai, China. All other chemicals used were analytical grade.

Animals

Male Wistar rats (7–8 weeks of age, 150–180 g) were obtained from the Kerala Veterinary and Animal Sciences University (Mannuthy, Thrissur, Kerala, India). All rats were maintained in a controlled sterile environment maintained at a constant temperature (24 ± 2 °C), 50% relative humidity, and a 12 h light/dark cycle. All rats had ad libitum access to standard diet pellets (Sai Durga Feeds, Bangalore, India) and filtered water. All experiments performed here were based on the rules and regulations assigned by the committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, Government of India, and had the approval of Institutional Animal Ethics Committee (IAEC/KU/BT/2012/001), Karunya University.

Collection of plant material

The fresh aerial parts of the plant were collected in the month of August 2011 from Amala Ayurveda Pharmacy, Thrissur, India. The plant was identified by Dr. C.I. Jolly at Fr. Gabriel Herbarium of Amala Ayurvedic Hospital and Research Centre, Thrissur, Kerala, and the voucher specimen (ACRC-Ox-07) was retained in the Department of Biotechnology, Karunya University, Coimbatore, India. The remainders of the harvested plant samples were washed thoroughly with water and shade-dried at room temperature.

Preparation of extract

The shade-dried aerial parts of the plant were subjected to mechanical size reduction. The powdered material was then extracted with methanol in a Soxhlet apparatus. Traces of the solvent were ultimately removed by evaporation and the final extract concentrated using a vacuum rotatory evaporator. The yield of the extract was 12%. The crude extract thus obtained, as a thick semisolid mass, was stored in the refrigerator for use in the various experimental protocols. For in vivo studies, the extract as well as sulfasalazine was re-suspended in 1% gum acacia and administrated intra-peritoneally. Based on our previous studies regarding toxicity, a dose of B. sensitivum (50 and 100 mg/kg b wt) was chosen for use in all the experiments in the present study (Guruvayoorappan & Kuttan, 2007a).

Experimental design

Rats were divided into five groups, each consisting of 12 animals. The Group I served as the normal control, Groups II, III and IV were subjected for the induction of UC by intra-colonic injection of 2 ml of 3% acetic acid. Group II served as a UC control group. Groups III and IV were treated with B. sensitivum (50 and 100 mg/kg b wt, respectively) and Group V was treated with a standard drug sulfasalazine (100 mg/kg b wt) intra-peritoneally for 5 consecutive days before induction of UC with acetic acid.

Induction of experimental colitis in rats

All animals were kept fasting overnight, with access to water ad libitum and anesthetized by ether inhalation before induction of colitis. For this procedure, 2 ml of acetic acid (3%, v/v, in 9% saline) were infused for 30 s using a polyethylene tube (2 mm in diameter), inserted through rectum into the colon up to a distance of 8 cm. Blood and colon were collected 24 h later, after sacrificing the animals. For separation of serum, blood samples were centrifuged at 500 × g (10 min) and stored at −20 °C. Portions of colonic specimens were dissected out, washed with ice cold phosphate buffered saline (PBS) (pH 7.2) and kept in 10% formalin for macroscopic, histopathology, and immunohistochemistry studies. The remaining portions of colonic specimens were used for biochemical studies (Jagtap et al., 2004).

Assessments of colitis

Determination of the effect of B. sensitivum on macroscopic scoring during UC

The severity of colitis was evaluated by an independent observer who was blinded to the treatment. For each animal, the distal 10 cm portions of the colon were removed and cut longitudinally and then cleaned with physiological saline to remove fecal residues. Macroscopic inflammation scores are assigned based on the clinical features of the colon using an arbitrary scale ranging from 0 to 10 as follows: 0 = no damage, 1 = focal hyperemia (water oozes out), 2 = ulcerization without hyperemia or bowel wall thickness, 3 = ulcerization with inflammation at one site, 4 = ulcerization with inflammation at two sites, 5 = major sites of inflammation >1 cm along the organ with redness, 6 = major sites of inflammation >2 cm along the organ with redness, 7 = major sites of inflammation >3 cm along the organ with redness, 8 = major sites of inflammation >4 cm along the organ with redness, 9 = major sites of inflammation >5 cm along the organ with redness and bleeding, and 10 = major sites of inflammation >6 cm along the organ with redness, swelling, and bleeding (Jagtap et al., 2004).

Determination of the effect of B. sensitivum on colon wet weight and spleen weight during UC

The wet weight of the colon was assessed by the following method. A 5 cm segment of the distal colon, 3 cm proximal to the anus was resected, the lumen rinsed with ice-cold saline and weighed. Results are expressed as mg/cm of wet weight of colon, mean ± SD from six samples of each group. The spleens were also obtained from animals in each group and their weights were measured.

Determination of the effect of B. sensitivum on colonic vascular permeability

In separate experiments, the vascular permeability of the colon was measured by a modified method of Erickson et al. (1992). Separately, six animals from each group were used to measure the colonic vascular permeability. Vascular permeability was measured in rat colon 30 min after induction of colitis by intra colonic administration of acetic acid (3%). The rats were anesthetized, a femoral vein catheter was inserted, and 1% Evans blue in 0.9% saline was injected in a volume of 0.2 ml/100 kg b wt; 30 min later, the rats were sacrificed by decapitation. The colons were removed, opened longitudinally, and rinsed in saline. Approximately 200 mg segments (colon) were taken, weighed and put into formamide in a shaking bath at 37 °C for 48 h. The amount of Evans blue extracted into formamide from the tissue sample was measured spectrophotometrically at 620 nm. The amount of extravasated dye was calculated from the standard curve. Tissue Evans blue concentrations were expressed as microgram per gram wet weight (Erickson et al., 1992).

Determination of the effect of B. sensitivum on oxidative stress marker enzymes

Sample from the colon was homogenized in 10 mmol Tris-HCl buffer (pH 7) and the homogenate was used for the measurement of NO (Green et al., 1982), lipid peroxidation (LPO) (Ohkawa et al., 1979), glutathione (GSH) (Moron et al., 1979), and superoxide dismutase (SOD) (Kakkar et al., 1984). The blood samples collected separately in non-EDTA coated tubes were used for the separation of serum to determine the serum NO level.

Determination of the effect of B. sensitivum on cytokines (TNF-α, IL-1β, and IL-6 level), inflammatory mediators (iNOS and COX-2 level), colonic MPO activity, and serum LDH levels

Colon sample from each animals were collected, weighed, and tissue homogenate were prepared using Tris-HCl buffer (pH 7), centrifuged at 500 × g for 10 min and the supernatant used for the measurement of TNF-α, iNOS, MPO (USCN Life Science, Wuhan, Hubei), IL-1β, IL-6 (KOMA Biotech, Seoul, Korea), COX-2 (Shanghai Bluegen Biotech, Shanghai, China) using standard sandwich Enzyme-Linked Immunosorbent Assay (ELISA) kit specific for murine cytokines according to the manufacture’s instruction. The blood samples collected from all animals were centrifuged, serum separated, and used to determine lactate dehydrogenase (LDH) activity using Biovision kit, Milpitas, CA.

Preparation of nuclear extracts

Colon tissues were homogenized in cold PBS, and then were centrifuged at 500 × g for 5 min at 4 °C. The resulting supernatants were discarded. The cell pellet was resuspended in ice cold cell lysis buffer (200 µl; pH 7.9) containing HEPES (10 mM), MgCl₂ (1.5 mM), KCl (10 mM), phenylmethyl sulfonyl fluoride (1 mM), dithiothreitol (DTT) (1 mM), Nonidet P40 (0.5%), and EGTA (1 mM) followed by centrifugation at 500 × g for 15 min. The cell pellet was resuspended in double the volume of lysis buffer and the cells were disrupted by repeated single rapid stroke using a sterile syringe. The nuclear pellet was resuspended in extraction buffer (200 µl) containing HEPES (20 mM), glycerol (25%), MgCl₂ (1.5 mM), NaCl (420 mM), PMSF (0.1 mM), and DTT (1 mM) and incubated in ice for 30 min. The nuclear suspension was centrifuged at 500 × g for 15 min at 4 °C, and the supernatant (nuclear extract) was frozen in aliquots at −80 °C for the transcription factor profiling (Pratheeshkumar et al., 2012).

Transcription factor profiling NF-κB (p65/p50)

Transcription factor profiling was done using the Cayman TM transcription factor kit (Cayman^TM, Ann Arbor, MI). The kit provided rapid, high-throughput detection of specific transcription factors, namely subunits of NF-κB such as p65 and p50. Using an ELISA based format, the transcription kit detected the DNA bound transcription factors. Bound transcription factors in the DNA were detected by specific primary antibodies towards NF-κB p65 and NF-κB p50 sub-units. A horseradish peroxidase-conjugated secondary antibody was then used to detect the primary antibody. The enzymatic product was then measured using an ELISA reader and the percentage inhibition was calculated using the formula: 100 – ([OD of treated/OD of control] × 100), where OD is the optical density (Pratheeshkumar et al., 2012).

Histopathological assessment of colitis

Colonic specimens were fixed in 10% formalin in PBS, embedded in paraffin, after several steps to induce dehydration in alcohol, sections of 4 μm thickness were prepared and stained with hematoxylin and eosin (H&E). Thereafter, histopathological analysis was carried out using an EVOS-xl CORE light microscope (AMG, Bothell, WA). All samples were analyzed in a blinded manner. A certified histopathologist performed all analyses/interpreted the observed outcomes.

Immunohistochemical study

The colonic tissues were fixed in 4% neutral formalin, dehydrated with increasing concentrations of ethanol, embedded in paraffin, and sectioned. Sections (5 µm thick) were mounted on slides, cleaned, and hydrated. The sections were treated with a buffered blocking solution (3% bovine serum albumin in PBS for 15 min. Then, the sections were co-incubated with primary antibody for COX-2 or iNOS at a dilution of 1:400 in PBS (v/v), at room temperature for 1 and 24 h, respectively, followed by washing with PBS and co-incubated with secondary antibody at a dilution of 1:500 in PBS (v/v), at room temperature for 1 h. Thereafter, sections were washed as before and with Tris-HCl 0.05 M, pH 7.66, and then co-incubated with a 3,3′-diaminobenzidine solution in darkness, at room temperature for 10 min. The sections were washed with Tris-HCl, stained with hematoxylin according to standard protocols, mounted with glycerin, and observed in an EVOS-xl CORE light microscope (AMG, Bothell, WA).

Statistical analysis

All data values are expressed as mean (±SD). Statistical analyses were performed using a one-way analysis of variance (ANOVA) followed by Dunnett’s test, using Graphpad InStat version 3.0 (Graphpad Software, San Diego, CA). Results from the rats with UC treated with B. sensitivum (50 and 100 mg/kg b wt) and sulfasalazine were considered statistically significant compared with those from UC control hosts.

Results

Effect of B. sensitivum on clinical and macroscopic evaluation of the colonic lesions, colon wet weight, and spleen weight

Acetic acid caused severe macroscopic edematous inflammation in the colon; 24 h after administration, as assessed by the high score of colonic damage with increased wet weight of colon. Treatment with B. sensitivum (50 and 100 mg/kg b wt) ameliorated the effect of acetic acid evidenced by assessment of low macroscopic score and significantly decreased the increase in colon wet weight as well as spleen weight. Figures 1 and 2 represent the effect of acetic acid, B. sensitivum treatment, and the reference drug sulfasalazine, in comparison with normal rats on macroscopic examination, colon wet weight, and spleen weight.

Figure 1. Macroscopic appearance of rat colonic mucosa from each group.

Figure 2. Effect of B. sensitivum (50 and 100 mg/kg b wt) and sulfasalazine (100 mg/kg b wt) on macroscopic evaluation of the colonic lesions, colon wet weight, and spleen weight. Treatments were administered once daily for 5 consecutive days before induction of colitis. Values shown are mean ± SD. Values were significantly different from ulcerative colitis (no-drug-treated) control (**p < 0.01).

Effect of B. sensitivum on colonic vascular permeability during UC

Vascular integrity of the colon was assessed by Evan’s blue administration. An increase in the vascular permeability was observed 30 min after acetic acid exposure. Maximal extravasation (61.90 ± 4.12 µg/g wet weight of colon) was observed in rats with UC (untreated) as depicted in Figure 3. Administration of B. sensitivum (50 and 100 mg/kg b wt) significantly reduced vascular integrity evidenced by reduced Evan’s blue value to 33.70 ± 3.35 and 27.45 ± 1.23 µg/g wet weight of colon, respectively, whereas sulfasalazine also significantly reduced Evan’s blue value to 33.23 ± 3.50 µg/g wet weight of colon.

Figure 3. Effect of B. sensitivum (50 and 100 mg/kg b wt) and sulfasalazine (100 mg/kg b wt) on colonic vascular permeability of rats with acetic acid-induced ulcerative colitis. Vascular integrity of the colon was assessed by Evans blue administration. An increase in the vascular permeability was observed 30 min after acetic acid exposure. Values shown are mean ± SD. Values were significantly different from ulcerative colitis (no-drug-treated) control (**p < 0.01).

Effect of B. sensitivum on oxidative stress marker enzymes during UC

Acetic acid-induced colitis resulted in increased NO level in both serum and tissue (colon) in comparison with control animals (60.82 ± 1.60 versus 14.54 ± 0.52 µmol in serum and 21.09 ± 0.42 versus 6.90 ± 0.51 µmol in tissue respectively). Administration of B. sensitivum (50 and 100 mg/kg b wt) and sulfasalazine showed a significant reduction in NO level compared with the acetic acid-induced colitis group (Figure 4). Regarding the redox state, the UC group showed a significant decrease in colonic non-enzymatic (GSH) (789.90 ± 29.65 nmol/g of wet tissue) and enzymatic (SOD) defense systems (80.35 ± 5.43 U/mg protein); however, these levels were enhanced by treatment with B. sensitivum (50 and 100 mg/kg b wt) or sulfasalazine treated animals towards the value of naive hosts. Regarding LPO status, acetic acid induced non-treated animals shows significant increase (7.83 ± 0.55 nmol/mg protein), whereas in treatment with B. sensitivum (50 and 100 mg/kg b wt) or sulfasalazine produced a marked decrease in the LPO level (4.33 ± 0.37, 3.93 ± 0.48 and 3.50 ± 0.41 nmol/mg protein, respectively).

Figure 4. Effect of B. sensitivum (50 and 100 mg/kg b wt) and sulfasalazine (100 mg/kg b wt) on oxidative stress markers colonic glutathione (GSH), superoxide dismutase (SOD), lipid peroxidation (LPO), and nitric oxide (NO) in both tissue and serum during ulcerative colitis. Values shown are mean ± SD. Values were significantly different from ulcerative colitis (no-drug-treated) control (**p < 0.01).

Effect of B. sensitivum on cytokines TNF-α, IL-1β, and IL-6 levels during UC

The effect of B. sensitivum (50 and 100 mg/kg b wt) on pro-inflammatory cytokine levels during UC is illustrated in Figure 5. Colonic TNF-α, IL-1β, and IL-6 level were significantly higher (135.41 ± 6.35, 86.43 ± 7.32, and 112.74 ± 4.48 pg/mg tissue, respectively) than the corresponding value in naive hosts (27.21 ± 1.82, 37.99 ± 5.11, and 30.51 ± 2.04 pg/mg tissue, respectively). This increase in colonic TNF-α, IL-1β, and IL-6 level were significantly mitigated by treatment with either B. sensitivum (50 and 100 mg/kg b wt) or the sulfasalazine group compared with acetic acid-induced colitis group. B. sensitivum at a dose of 100 mg/kg b wt) was effective in reducing these cytokine levels.

Figure 5. Effect of B. sensitivum (50 and 100 mg/kg b wt) and sulfasalazine (100 mg/kg b wt) on colonic tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6) level of rats with acetic acid-induced ulcerative colitis. Treatments were administered once daily for 5 consecutive days before induction of colitis. Values shown are mean ± SD. Values were significantly different from ulcerative colitis (no-drug-treated) control (**p < 0.01).

Effect of B. sensitivum on inflammatory mediators iNOS and COX-2 levels during UC

The effect of B. sensitivum (50 and 100 mg/kg b wt) on pro-inflammatory mediators, iNOS and COX-2 level, are shown in Figure 6(A and B). Colonic iNOS and COX-2 level were significantly higher (73.98 ± 3.80 and 91.34 ± 6.72 ng/mg tissue, respectively) than the corresponding value in normal animals (13.70 ± 0.66 and 23.69 ± 3.69 ng/mg tissue, respectively). Treatment with B. sensitivum (50 and 100 mg/kg b wt) significantly decreased these inflammatory mediators up to (29.93 ± 1.46, 44.1 ± 8.83, and 25.49 ± 3.83, 38.56 ± 4.91 ng/mg tissue, respectively). B. sensitivum showed significantly better results compared with the standard drug, but sulfasalazine also produced a marked decrease in these levels compared with the acetic acid-induced colitis group.

Figure 6. Effect of B. sensitivum (50 and 100 mg/kg b wt) and sulfasalazine (100 mg/kg b wt) on colonic inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), myeloperoxidase (MPO), and serum lactate dehydrogenase (LDH) level of rats with acetic acid-induced ulcerative colitis. Treatments were administered once daily for 5 consecutive days before induction of colitis. Values shown are mean ± SD. Values were significantly different from ulcerative colitis (no-drug-treated) control (**p < 0.01).

Effect of B. sensitivum on colonic MPO activity and serum LDH level during UC

Determination of MPO level was used as an indicator of colonic infiltration with polymorphonuclear leukocytes. The MPO level was found to be higher in acetic acid-induced colitis group (91.85 ± 4.92 ng/g tissue), whereas the treatment with B. sensitivum (50 and 100 mg/kg b wt) significantly reduced the MPO level (31.13 ± 1.39 and 30.89 ± 2.80 ng/g tissue, respectively) towards the value of normal hosts (18.74 ± 1.04 ng/g tissue). Serum LDH level was significantly raised after the administration of the acetic acid (2046.69 ± 149.18 U/L) compared with naive hosts (577.60 ± 65.24). Treatment with B. sensitivum (50 and 100 mg/kg b wt) significantly reduced the increase in the serum LDH level (670.28 ± 94.67 and 618.94 ± 145.63 U/l) whereas the standard drug sulfasalazine was found reduce MPO and LDH level during UC as shown in Figure 6(C and D).

Effect of B. sensitivum on Transcription factor profiling NF-κB (p65/p50) during UC

The effect of B. sensitivum (50 and 100 mg/kg b wt) on the activation of transcription factors during UC is presented in Table 1. Measurement of DNA-bound transcription factor NF-κB (p65/p50) was identified by the primary antibody. A horseradish peroxidase-conjugated secondary antibody was then used to detect the primary antibody. Finally the enzymatic product was measured using ELISA reader. B. sensitivum (100 mg/kg b wt) could inhibit the activation and nuclear translocation of NF-κB subunits, p65 (79.16%) and p50 (64.06%), whereas sulfasalazine also exhibited p65 (71.52%) and p50 (58.19%), respectively.

Table 1. Effect of B. sensitivum (50 and 100 mg/kg b wt) on the translocation of transcription factors NF-κB (p65/p50) subunits.

Transcription factors	Ulcerative colitis control (OD)	Ulcerative colitis  + sulfasalazine (100 mg/kg b wt) (OD)	Ulcerative colitis  + B. sensitivum (50 mg/kg b wt) (OD)	Ulcerative colitis  + B. sensitivum (100 mg/kg b wt) (OD)	% inhibition by sulfasalazine treatment (100 mg/kg b wt)	% inhibition by B. sensitivum (50 mg/kg b wt) treatment	% inhibition by B. sensitivum (100 mg/kg b wt) treatment
NF-κB p65	0.576 ± 0.023	0.164 ± 0.010**	0.129 ± 0.011**	0.120 ± 0.026**	71.52%	77.60%	79.16%
NF-κB p50	0.665 ± 0.025	0.278 ± 0.029**	0.241 ± 0.038**	0.239 ± 0.029**	58.19%	63.75%	64.06%

NF-κB, nuclear factor kappa B; OD is the optical density. Nuclear extracts from colonic tissue were prepared separately and subjected for transcription factor assay as per manufacturer’s instruction using Cayman ^TM transcription factor kit (Cayman^TM, MI**). Values shown are mean ± SD. Values were significantly different from ulcerative colitis (no-drug-treated) control (**p < 0.01).

Effect of B. sensitivum on histopathological assessment

Histological investigation revealed that the administration of acetic acid triggered transmural necrosis in layers of the bowel wall, infiltration of inflammatory cells was observed in the mucosa, mucosal edema and loss of epithelial cells were detected in colitis control animals (no-drug treated). Administration of B. sensitivum (50 and 100 mg/kg b wt) or sulfasalazine treated animals significantly mitigated the cell damage, reduced mucosal injury, edema, and reduced infiltration of inflammatory cells was observed in comparison with the colitis control animals as displayed in Figure 7.

Figure 7. Histological colonic mucosal sections of normal rat: (A) normal mucosa with intact epithelial surface and acetic acid-induced colitis; (B) massive necrotic destruction of epithelium. (C and D) Pre-treatment with B. sensitivum (50 or 100 mg/kg b wt) and (E) sulfasalazine (100 mg/kg b wt) attenuated the extent and severity of cell damage.

Effect of B. sensitivum on expression of iNOS and COX-2 immunostaining

Immunohistochemical investigation revealed that the inflammatory mediator, iNOS expression was mainly observed on neutrophils and smooth muscle cells with a sparse distribution in the epithelial cells in normal animals (Figure 8). iNOS was unregulated in acetic acid-induced colitis control animals (particularly localized in the infiltrated inflammatory cells) and treatment with B. sensitivum (50 and 100 mg/kg b wt) or sulfasalazine reduced the overexpression of iNOS which was induced by the administration of acetic acid. Correspondingly, another inflammatory mediator, i.e., expression of COX-2, was scarcely found in the surface epithelium and mononuclear cells of lamina and propria of mucosa in normal animals. The expression of COX-2 was found to be elevated in the acetic acid-induced colitis animals, whereas treatment with B. sensitivum (50 and 100 mg/kg b wt) or sulfasalazine exhibited diminished level of COX-2 expression in acetic acid induced colitis animals as presented in Figure 9.

Figure 8. (A) Immunohistochemical localization of iNOS in normal control, (B) positively stained brown granules for iNOS were significantly increased in both number and intensity in colonic tissue of acetic acid treated rats. (C and D) B. sensitivum (50 or 100 mg/kg b wt) and (E) sulfasalazine (100 mg/kg b wt) treated reduced colonic iNOS expression of acetic acid-treated rats.

Figure 9. (A) Immunohistochemical localization of COX-2 in normal control; and (B) positively stained brown granules for COX-2 were significantly increased in both number and intensity in colonic tissue of acetic acid-treated rats. (C and D) B. sensitivum (50 or 100 mg/kg b wt) and (E) sulfasalazine (100 mg/kg b wt) treated reduced colonic COX-2 expression of acetic acid treated rats.

Discussion

In the present study, we demonstrated for the first time that the administration of B. sensitivum effectively attenuates acetic acid-induced UC in rats through its anti-inflammatory and antioxidant activities. Administration of B. sensitivum attenuated colitis as shown by reduction in macroscopic score as well as decreased wet weight of colon compared with colitis control animals. It is well known that splenic atrophy is associated with a high complication rate in colitis patients (Cho et al., 2011), in the same way, administration of B. sensitivum extract exposed protecting effect by decreased spleen weight in animals with colitis. Enhanced vascular permeability seems to be the rate limiting step in mucosal damage (Viera et al., 2000) and thus prevention of increase in vascular permeability of colon by B. sensitivum may play an active role in the protecting effect of colon.

Oxidative stress plays a significant role in initiation and progression of UC (Kruidenier & Verspaget, 2002). Accumulation of the major free radical in tissue, i.e., superoxide anion (), which is converted into H₂O₂ by the enzymatic defense system SOD which is involved in protecting tissue against oxidative damage. In our study, we observed a significant increase in the SOD activity in the colitis animals, possibly for compensating the damage caused by administration of the acetic acid in the colon (Oktyabrsky & Smirnova, 2007). Further activated neutrophils produce this superoxide anion () through NADPH oxidase, which reduces molecular oxygen to the radical through the enzyme myeloperoxidase. This neutrophil enzyme catalyzes the formation of potent cytotoxic oxidants such as hypochlorous acid from H₂O₂ and chloride ions (Hagar et al., 2007). Thus, colonic MPO activity which serves as an index of neutrophil activation was evidently reduced in B. sensitivum treated colitis animals indicates the inhibition of neutrophil infiltration in the colonic mucosa (Van der Veen et al., 2009). Sufficient experimental and clinical evidence suggests that depletion of GSH was observed in tissues when antioxidants were neutralized by liberated oxygen derived free radicals and this reduction in GSH may lead to increase in the MDA content, an end product of LPO which ultimately results in the oxidative damage (Alzoghaibi et al., 2007). GSH acts as a key component in protecting tissues against damage caused by free radicals. In our results, an increase in the GSH activity and inhibition of LPO in the colitis group treated with B. sensitivum were observed, probably to compensate the colitis-induced oxidative injury (Arafa et al., 2009; Berkhout et al., 2006). Thus, reduced LPO and MPO activities along with the increase in the SOD and GSH activities, as well as histopathological finding of decreased cellular infiltration resulting treatment with B. sensitivum may indicate its potential anti-inflammatory and antioxidant effects in the prevention of acetic acid-induced UC.

Various inflammatory mediators released by activated mucosal immune cells were suspected to participate in IBD includes NO, eicosanoids, and cytokines such as TNF-α and IL-1. These cytokines stimulate the phagocytic leukocytes to express iNOS abundantly leads to the synthesis of micromolar quantities of NO, which can be deleterious to the cells through the formation of NO-reactive products and used as a measurement for toxicity marker (Hagar et al., 2007). In our study, the level of serum and mucosal NO in the inflamed colon was significantly increased with enhanced expression of iNOS. Experimental and clinical evidence clearly shows that enzymes COX-2 and iNOS are predominantly expressed at sites of inflammation and this activation leads to the production of perilous inflammatory mediators which might contribute to intestinal damage, and additionally, it was reported that iNOS will act in synergy with COX-2 to promote the inflammatory response (Itzkowitz, 2006). Additionally, immunohistochemistry analysis in the present study shows decreased expression of iNOS and COX-2, which clearly indicates that the administration of B. sensitivum was found to decrease the NO level via down-regulating iNOS enzyme and also it significantly reduced COX-2 expression which demonstrates its anti-inflammatory mechanism in acetic acid-induced UC (Goldstein et al., 2000). Cumulative evidence from our previous expression studies revealed that B. sensitivum could down-regulate the expression of iNOS and COX-2 in LPS or Con A-stimulated macrophages (Guruvayoorappan & Kuttan, 2008). In contrast, a significant reduction in the LDH activity (potential marker of intestinal damage) was observed in the colitis group administrated with B. sensitivum (50 and 100 mg/kg b wt), a result that also indicates the repair of colonic tissue damage. LDH activity in serum is used as an indicator of cell membrane integrity and also as a measurement of cytotoxicity because this cytosolic enzyme is released into the blood stream when cell damage or lysis occurs during apoptosis or necrosis (Hagar et al., 2007).

Pro-inflammatory cytokines play a vital role in the pathophysiology of IBD. There is substantial evidence that excessive production of TNF-α, IL-1β, and IL-6 in inflamed mucosa contributes significantly to the development of tissue injury in UC. Among this, the most significant pleiotropic cytokine, TNF-α, released by activated mononuclear cells induces the production of other cytokines such as IL-1β and IL-6 (Hagar et al., 2007). In the present study, consistent with others (Amini-Shirazi et al., 2009), colonic TNF-α, IL-1β, and IL-6 increased in the acetic acid-induced colitis animals. This inflammatory status was reversed by B. sensitivum (50 and 100 mg/kg b wt) administration than that of sulfasalazine treated. These findings are supported by our previous studies that have been shown by B. sensitivum can reduce the levels of these pro-inflammatory mediators in lipopolysaccharide (LPS) or concanavalin-A (Con-A) stimulated macrophages both in vitro and in vivo (Guruvayoorappan & Kuttan, 2007a,b, 2008).

Recent reports have shown the use of mitogen-activated protein kinases (MAPKs) pathway inhibitors attenuated colitis, representing a possible application of drugs in the IBD treatment. It is well known that p38 MAPK plays a vital role in the regulation of these pro-inflammatory cytokines involved in the mucosal tissue devastation of IBD patients (Coskun et al., 2011). In addition, our results are in agreement with the previous studies which indicated that flavonoids and their metabolites may interact with MAPK signaling pathways, thereby flavonoids such as amentoflavone and cuppressuflavone present in the B. sensitivum might interact with the proteins involved in the MAPKs pathway by a direct inhibitory effect which represents a specific mechanism by which B. sensitivum extract may prevent colitis (Kuo et al., 2011). Earlier studies have shown evidence that TNF-α and IL-1 cause the activation and translocation of NF-κB into the nucleus (Bauerle & Henkel, 1994). In the current study, B. sensitivum (50 and 100 mg/kg b wt) significantly reduced colonic TNF-α indicating that B. sensitivum has an anti-inflammatory effect probably due to its powerful antioxidant properties and due to NF-κB inhibition. NF-κB, the key transcription factor which mediates inflammatory signaling, is one of the promising targets for developing anti-inflammatory agents (Karin & Greten, 2005). NF-κB exists mainly as a heterodimer composed of subunits of the Rel family, p50 and p65. NF-κB is one of the critical transcription factors that regulate the transcription of many genes associated with UC, mediates transcriptional activation of various inflammatory mediators (iNOS and COX-2), and also promotes expression of various pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α (Kundu & Surh, 2004). In resting stage, NF-κB normally localizes to the cytoplasm, where it is bound by IκB proteins. During inflammatory stimulus, IκB is phosphorylated by IκB kinase, subsequently degraded by proteasome, then get released and translocates into the nucleus, where it triggers the transcription of multiple genes involved in inflammatory cascade (Wang et al., 2011). The present study confirms that B. sensitivum could inhibit the activation and nuclear translocation of NF-κB subunits p50 and p65. These results show that the administration of B. sensitivum effectively overwhelms inflammation in the colon through inhibition of NF-κB signal transduction pathways. Thus blockade of NF-κB signal transduction pathways might be one of the major mechanisms underlying the prevention of acetic acid-induced UC by B. sensitivum extract.

In summary, our data confirm that the administration of B. sensitivum exerts a protective effect in acetic acid-induced UC in rats mediating via modulation of oxidant/antioxidant balance in colonic tissue, inhibition of production of inflammatory mediators, pro-inflammatory cytokines, and inhibition of NF-κB signal transduction pathways. Nevertheless, the precise molecular mechanism by which B. sensitivum extract mediates anti-inflammatory activity remains to be determined. Further investigations are in progress in our laboratory to evaluate the effect of these pharmacologically active flavonoid isorientin, biflavones amentoflavone, and cupressuflavone from B. sensitivum with potential for the use in anti-IBD therapy, and to elucidate their associated mechanisms of therapeutic action.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was funded by Department of Science and Technology (DST), Government of India, New Delhi (Ref no, SR/FT/LS-030/2009). We are grateful and thank for their financial support and for Senior Research Fellowship (SRF).

Acknowledgements

The valuable suggestions from Dr. M. Patrick Gomez, Director, School of Biotechnology and Health Sciences and Dr. Jannet Vennila, Head, Department of Biotechnology, Karunya University, are gratefully acknowledged.