Mucoprotective and Mast Cell Stabilization Activities of Chromium (D-Phenylalanine)3 in Indomethacin-Induced Chronic Enterocolitis in Rats: In vitro Anti-Cancer Activity Using Caco-2 Cell Line

Introduction

Inflammatory bowel disease (IBD) comprises of Crohn’s disease (CD) and ulcerative colitis (UC). More specifically, CD condition roots to transmural inflammation by disturbing the inner lining of the gastrointestinal tract.¹ The incidence and prevalence of CD critically differ based on environment, geographic area and ethnicity. The maximum prevalence rates of CD were found in North America and Europe with 319 and 322 per one lakh population respectively. Whereas, the disease is considered rare in India with approximately seven per one lakh population. However, the disease is showing its mark in the Indian population due to modern and sedentary life style. CD is a diverse condition with complicated aetiology including genetics and environment factors to promote disease development. In addition, external factors such as smoking, use of NSAID’s, low fibre-high carbohydrate consumption, altered microbiome, alcohol, stress and excessive use of antibiotics that cause damage to the gut flora are responsible for chronic intestinal inflammation.² Further, aggressive immunological responses mediated by both innate and acquired immune cells enhance intestinal permeability and cause epithelial barrier disruption, resulting in microbial translocation and inflammation.³

One of the key etiological factors of various symptoms of IBD, including CD, is oxidative stress. The generation of pro-oxidants in the gastrointestinal tract induces oxidative stress, inflammatory cells and inhibits antioxidant enzyme levels. This causes severe damage to the gastrointestinal tract.⁴ Several immune system disturbances stimulate the higher levels of interleukin-6 (IL-6) and TNF-a release via over-expression of NF-kB. Dysregulation of any of these proteins causes severe inflammation and leads to colorectal cancer progression.⁵ Existing treatment options for IBD are glucocorticoids, amino salicylates, methotrexate and cyclosporine. Nevertheless, safety is the biggest issue with these drugs as they cause severe toxic effects such as seizures, hypertension, low WBC count and kidney damage.^6,7 So, there is an urgent requirement for harmless druggable compound that fights against oxidative-nitrative stress, ulcer development and inflammatory conditions.

In the pharmaceutical and dietary product segments, supplements of chromium-based products are in growing stages in the world market. However, the invention of safer chromium products with enhanced bioavailability and pharmacological action is inspiring. Chromium trivalent ion (Cr3+) is a vital element existing in various food products, crucial for the glucose homeostasis and useful in protein and lipids metabolism.^8,9 The harmless and adequate daily human consumption of Cr3+ is 50 to 200 µg per day. Various chromium complexes were used in the treatment of diabetes, inflammation, oxido-nitrative stress and hepatotoxicity.⁹ Administration of Cr3+ resulted in reduction in pro-inflammatory cytokines & oxidative stress in high dextrose exposed monocytes.¹⁰

Current research work is our persistent effort in searching for bioactive compounds beneficial in the treatment of IBD from the natural sources and plant supplements.^11-14 Furthermore, valuable effects of Cr (D-phe)₃ in indomethacin-induced acute condition similar to Crohn’s disease in humans were reported from our lab.¹⁵ Also, untreated chronic inflammatory bowel disease can lead to colorectal cancer. Taking together all these above facts, the current research effort was premeditated to discover the beneficial effects of title complex in indomethacin-induced chronic enterocolitis and in vitro anti-cancer activity in Caco-2 cells.

Materials and Methods

Chemicals

Analytical-grade chemicals were procured and utilized in the present study. Biochemical kit was purchased from ERBA Diagnostics, Mannheim, Germany.

Synthesis of Chromium complex

The Cr(D-Phe)₃ was synthesised, and characterized as per the reported literature.^15,16 Briefly, Chromium chloride (10 mmoL) and D-Phenylalanine (30 mmoL) were dissolved separately in 50 mL of water. Both the solutions were mixed and refluxed for 4 h at 80°C. A greenish-violet coloured homogenous solution was obtained. This solution was freeze dried and solid was washed repeatedly with acetone to obtain pure complex.

In vitro anti-cancer activity

Cell culture

The cell line (Caco-2) was procured from NCCS, Pune. Caco-2 were maintained in a 25 cm3 flask using DMEM medium containing 10% v/v fetal bovine serum and antibiotics in a humidified atmosphere with 5% carbon dioxide. Cultured flasks of 70-90% confluency with >90% viability were utilized for the experiment.

Cell viability and cytotoxicity evaluation

Cell viability assay was estimated by MTT test. Live cells produce mitochondrial lactate dehydrogenase, which converts yellow coloured MTT into dark blue coloured formazan and its absorbance at 570 nm is relative to the viable cell counts.¹⁷ Briefly, Caco-2 (20,000 cells /well) cells were seeded into 96-well plates for proliferation for about 24 h. Following incubation, cells were treated with varying concentrations of Cr(D-Phe)₃ (31.25, 62.5, 125, 250 and 500 mM) and the reference drug Camptothecin (5.7 mM) was used as a positive control and were incubated in a carbon dioxide incubator for 48 hours. Then, cells were treated with MTT and the absorbance at 570 nm was measured using a microplate reader.

In vivo experiments

Animals

Prior to the start of the trial, male Wistar rats (180–220 g) were accustomed for seven days in our animal house as per the standards. The investigational procedure was permitted by the Institutional Animal Ethics Committee (SSCPT, IAEC, Clear/171/2017-2018 dated 22.07.2017) as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. The effective dose of title complex was chosen as per our earlier reported acute oral toxicity test and animal experimentations.^14,15

Indomethacin-induced chronic enterocolitis

All the rats were randomized as per body weight and were separated into five-groups containing six rats each. Group 1 was assigned as normal control and received only vehicle (2% v/v Tween-80, p.o), Group 2 was assigned as colitis control and treated orally with indomethacin only (1.5 mg/kg, BID), Group 3 and 4 received orally Cr(D-Phe)3 at the dosage of 30 & 60 µg/kg respectively and indomethacin twice daily, while Group 5 received oral sulfasalazine (100 mg/kg) and indomethacin twice daily.

Indomethacin was given to the respective group of rats twice daily (BID) for fourteen days.¹⁸ Rats were pre-treated with two dosages of chromium complex and one dosage of sulfasalazine as mentioned above for three days earlier to indomethacin administration and this was continued till the end of treatment protocol. Food and water intake were measured every day for all fourteen days. One-day after the complete treatment protocol, rats were sedated with thiopentone sodium and blood specimens were collected by retro-orbital puncture from each animal. Serum was parted and used for lactate dehydrogenase (LDH) estimation.¹⁹ Animals were sacrificed by overdose of anaesthesia. The whole GI tract and mesentery were excised. The stomach, jejunum and ileum were isolated and cut open for macroscopic scoring according to the published scoring pattern.^20,21 Further, 2 cm of ileum samples were preserved in 10% formalin for microscopic damage evaluation using H & E stain.¹⁵ The remaining samples of ileum were wrapped in aluminium foil and stored at -20° C before processing for the biochemical analysis.

Mesenteric mast cell degranulation

In order to measure the damaging impact of indomethacin on mast cells, mesentery was dissected out carefully using bent forceps and taken in petri plates containing Ringer-Locke solution (37°C). Then, the mesenteric pans were mounted carefully on a glass slide and were air-dried at room temperature for fixation. Slides were stained with 0.01% toluidine blue in 4% v/v aqueous formalin solution. The stained cells were then immersed in xylene for 10 mins and finally rinsed 2 or 3 times with acetone and were observed under microscope 45X. A total of 100 mast cells from different visible areas were observed for the number of intact and degranulated cells and percentage protection was calculated using the formula.²²

% Protection of mast cells = (Total number of mast cells – Total number of degranulated cells) / (Total number of mast cells) × 100

Preparation of tissue extracts for biochemical estimation

Ileum tissue samples were blotted to remove wet contents and weighed. Then the samples were chopped and homogenized at 3500 rpm in an ice-cold potassium phosphate buffer (0.06 M) 10% w/v and the suspensions was centrifuged at 6,000 revolutions per minute for 20 min. Supernatants were used for the measurement of tissue nitrite,²³ total protein,²⁴ malondialdehyde (MDA), a marker of lipid peroxidation,¹² catalase²⁴ and reduced glutathione.²⁵ Portion of inflamed tissue was separately assayed for myeloperoxidase (MPO) quantification.^11,26

Results

In vitro anti-cancer activity

MTT assay revealed that a rise in Cr(D-Phe)₃ concentration decreases the viability of cells cultured for 48 h (Figure 1). IC₅₀ (half maximal inhibitory concentration) of tested chromium complex was found to be 722.40 mM as calculated by log regression equation method. Whereas, cytotoxic drug, Camptothecin (5.7 mM) restricted the cell viability to 46.89 ± 0.003 percentage when incubated for 48 h. 

In vivo indomethacin-induced chronic enterocolitis model

Feed and water intake

Oral administration of indomethacin twice daily for fourteen days exhibited severe anorexia and adipsia in rats as evident by a significant (p <0.001) decrease (~ 2 fold) in feed intake (10.57 ± 0.00 g/day) and water consumption (13.00 ± 0.19 mL/day) when compared to normal control. Both the oral dosages of Cr(D-Phe)3 showed a substantial increase (p <0.001) in feed and water intake, thus ameliorated the effect of anorexia and adipsia induced by indomethacin (Table 1).

Macroscopic scoring of distal jejunum and proximal ileum

Figure 2 depicts the representative picture of ileum and jejunum portions of an experimental group of rats. The oral administration of indomethacin twice dialy for 14- days exhibited oedematous inflammation, increase in ulceration along with severe damage of mucosal layer throghout the jejunum and ileum. Macrosocpic scoring of indomethacin alone treated rats exhibited suggestively increased (p <0.001) inflammation and ulceration as evident by higher macroscopic score (~9-folds) related to normal group (Table 1). Oral administration of chromium complex (30 µg/kg) exhibited 2.5-fold decrease in macroscopic score of ileum (1.60 ± 0.51) and 1.5-fold decrease for jejunum (2.57 ± 0.38). Whereas, oral administration of higher dose (60 µg/kg) showed significantly (p <0.001) diminished macroscopic scores with percentage protection of 83.80% in ileum and 100% in jejunum specimens when compared to indomethacin alone group. In addition, standard drug sulfasalazine significantly (p <0.001) reduced the macroscopic scoring of ileum, thus validated and confirmed the induction in the model (Table 1).

Severity of stomach ulcers

Twice-daily oral administration of indomethacin (1.5 mg/kg) produced destructive effects on stomach and produced severe ulceration, mucosal eruptions and gross lesions (Figure 3). Severity of ulcers in rats treated with only indomethacin displayed substantial (p <0.001) rise in the number of ulcers (2.25 ± 0.37) when compared to normal rats (0.38 ± 0.018). Both the doses of Cr(DPhe)3 treatment revealed a substantial drop in (p <0.01 and p <0.001 respectively) severity of ulcers (1.00 ± 0.00 & 0.38 ± 0.18) as compared to indomethacin alone group. The percentage protection of chromium complex (30 & 60 µg/kg) treated group was found to be 55.56 & 83.12% respectively. In addition, sulfasalazine exhibited significant (p <0.001) decrease in stomach ulcers (0.88 ± 0.13) compared to indomethacin group (Table 1).

Mesenteric mast cell degranulation

Administration of graded doses of indomethacin to rats for fourteen-days exhibited significantly higher (p <0.001) percentage of degranulated mast cells (70.00 ± 1.12%) in mesenteric pans compared with the normal control group (30.75 ± 1.82%). A considerable reduction (p <0.001) was seen after oral administration of the complex (30 & 60 g/kg) in the percentage of degranulated cells and it was brought back to near normal values. Further, the higher dose of chromium complex showed 69.93% protection against indomethacin-induced mast cell degranulation and the effect was found to be comparable with that of sulfasalazine (69.06%) (Table 2).

Biochemical parameters

Serum LDH levels

The oral administration of indomethacin exhibited noteworthy (p <0.05) increase (~ 2-folds) in levels of serum LDH (805.7 ± 152.0 IU/L) compared to normal control group. Oral administration of both the doses of chromium complex exhibited 1.8-fold and 2.6-fold decrease in serum LDH levels (428.9 ± 22.35 & 303.6 ± 20.63 IU/L) with p <0.05 and p <0.01 significance levels correspondingly when compared to indomethacin alone animal set. In addition, treatment with sulfasalazine (100 mg/kg) exhibited significant reduction in LDH in serum (303.6 ± 51.37 IU/L) (Table 3). 

Tissue MPO levels

Administration of indomethacin to group of rats for fourteen-days showed significant (p <0.001) increase in levels of MPO (33.27 ± 5.46 U/g of tissue) as related to the normal control group rats (1.88 ± 0.70 U/g of tissue). Oral administration of chromium complex and sulfasalazine resulted in a noticeable decrease (p <0.001) in MPO levels by ~ 3.7-folds and 4.8-folds respectively as compared to indomethacin alone group (Table 3).

Tissue nitrite levels

The graded doses of indomethacin twice daily for 14- days for group of rats exhibited significantly (p <0.001) higher (~ 2.5-folds) levels of tissue nitrite than the normal untreated rats. Pre-treatment with higher dose of chromium complex and sulfasalazine showed substantial amelioration (p <0.001) in tissue nitrite by ~ 2.8-folds and 2.2-folds respectively (Table 3).

MDA levels

Administration of indomethacin significantly elevated (p <0.01) the levels of MDA (8.22 ± 1.61 nM/g of tissue) by -2.5-folds compared to normal control group. Oral administration of chromium complex exhibited 3 & 3.5-folds decrease in the levels of MDA (2.56 ± 0.38 & 2.18 ± 0.40 nM/g of tissue respectively). However, oral treatment of complex showed better protection by reducing MDA than the standard sulfasalazine treatment (2.89 ± 0.48 nM/g of tissue) (Table 3).

GSH levels

The inducer control group of rats exhibited a significant (p <0.01) reduction of GSH (2.87 ± 0.57 µM/g of tissue) as associated with normal rats (4.25 ± 0.12 µM/g of tissue). Oral administration of chromium complex exhibited improvement in the levels of GSH in both the tested doses (4.71 ± 0.17 & 4.95 ± 0.38 µM/g of tissue respectively) when compared to indomethacin treated rats. Pre-treatment of test complex exhibited improved defense against oxidative stress by increasing the levels of GSH compared to sulfasalazine treatment (4.61 ± 0.10 µM/g of tissue) (Table 3).

Catalase activity

The catalase content in the tissues of indomethacin treated rats was found to be decreased -11-folds compared to untreated animals. Pre-treatment with both the dosages of chromium complex exhibited a substantial (p <0.05; p- 0.01) improvement in catalase content by around 6.5 and 11 times (Table 3).

Histopathalogical evaluation of ileum

Table 4 represented the histopathological scoring pattern of all the groups in terms of mild, moderate and severe damage. Distal ileum sections of normal control rats demonstrated the normal architecture of the intestinal epithelium and wall (Figure 4G1). Distal ileum sections of indomethacin alone treated group showed goblet cell depletion, oedema, ulcer and inflammatory infiltrations concentrated below the epithelial layer (Figure 4G2). Treatment with Cr(D-Phe)3 (30 µg/kg) repaired the mucosal epithelial layer with mild extent of necrosis and degree of inflammatory cell infiltration and no crypt damage was observed (Figure 4G3). Pre-treatment with chromium complex (60 µg/kg) repaired the mucosal epithelial layer resting on the thin muscularis layer with negligible inflammatory cell infiltration and regular villi with no crypt damage with increased epithelial cell numbers (Figure 4G4). Sulfasalazine treated rats showed intact villi and crypts (Figure 4G5) with slight ulceration and inflammatory cells along with the submucosal layer.

Discussion

Indomethacin is a non-steroidal anti-inflammatory agent used to induce enterocolitis resembling the human Crohn’s disease. Indomethacin is a COX-1 and COX2 inhibitor. The dose and duration of indomethacin administration were chosen to produce small intestinal damage similar to those caused by NSAIDs in humans. Non-fasted rats were given indomethacin (1.5 mg/kg) orally twice daily for fourteen-days to achieve the goal. Inhibition of particularly COX-1 can lead to mucosal erosions by suppression of mucoprotective PGE1, PGE2 and prostacyclin.²⁷ In addition, COX-2 derived PGs are also crucial for tissue integrity, mucosal damage repair, and inflammation management.²⁸ Therefore, the progress of small intestinal damage arises only when both COX-1 and COX-2 were suppressed. Mucosal erosions, mucosal erythema, subepithelial haemorrhage, and ulcerations can all be caused by long-term usage of NSAIDs.^27,28 Furthermore, the contribution of gastrointestinal bacteria and their products to the overall chronic inflammation is undeniable.²⁸

In the present study, chronic administration of indomethacin for fourteen-days produced hypermia, haemorrhagic lesions, severe ulceration, bowel wall thickening and gross lesions throughout the ileum and jejunum. Pre-treatment with the chromium complex improved the defence against harmful effects brought on by indomethacin and restored the mucosal integrity in both jejunum and ileum segments than the standard drug (sulfasalazine). Thus, the mucoprotective activity of the title compound may be due to the enhanced action or synthesis of PGE-2 and antimicrobial activity. Inflammation in the GI tract produces inflammatory mediators and these cells migrate to mesentery via mesenteric veins causing thrombosis in veins and degranulation of mesenteric mast cells. Extensive mast cell degranulation was seen in the rat mesentery after indomethacin administration by oral route. Pre-treatment with chromium complex increased the number of intact mast cells in the mesentery, reducing the amount of degranulated mast cells. This clearly suggests the antihistaminic activity of Cr(D-Phe)3 by mast cell stabilization activity.

Oral administration of indomethacin increases the production of free radicals in intestinal brush border, leading to mitochondrial dysfunction. These free radicals directly damage the cells in tissues and causes elevation of lactate dehydrogenase enzyme in blood stream.¹¹ An increase in LDH in blood indicates a shift towards anaerobiosis, which results in increased lactic acid generation. Oral administration of chromium complex significantly decreased the elevated serum LDH levels, thus protecting tissues from free radical injury. The enzyme myeloperoxidase (MPO) is secreted by phagocytes in neutrophils and is a marker of inflammation.^11,18 The MPO activity in the ileal and jejunum portions was significantly elevated in the indomethacin treatment group. Pre-treatment with chromium complex exhibited a decrease in the MPO levels and it indicates reduction in neutrophil infiltration and ultimately the inflammation. Further, this decrease in neutrophil migration was also confirmed with histopathological studies.

Endogenous antioxidant levels in tissues are low when the antioxidant ability of mucus membrane is diminished as a result of production of free radicals. The severity of the intestinal oxidative stress and inflammation was indicated by the increased levels of lipid peroxidation found in the indomethacin-treated group. The extent of lipid peroxidation was substantially decreased in the Cr(D-Phe)₃ treatment groups and improved colonic oxidative balance. Lipophilic free radical like nitric oxide regulates homeostasis in a variety of biological processes. Nitric oxide and L-citrulline are produced when the enzyme nitric oxide synthase oxidises L-arginine. The reactive nitric oxygen species (RNOS) are responsible for oxidative stress and are pathogenic mediators in IBD. Oral administration of chromium complex reduces the increased levels of tissue nitrite to near normal values, thus confirming the protection against nitrative oxidative stress. On the other hand, Cr(D-Phe)₃ treatment exhibited increased levels of endogenous enzymatic (catalase) and non-enzymatic antioxidant (GSH) activity. This strongly reconfirmed the protective effect of title compound in oxidative stress conditions.

Histopathological observations demonstrated that indomethacin administration causes severe ulceration, oedema and the depth of necrosis was continuous along ileum. Patchy hyperaemia and abundant inflammatory cell infiltration was found in the intestinal wall compared to the normal control group. Oral administration of chromium complex repaired the altered architecture and inhibited the migration of infiltration of neutrophils to the site of intestinal wall.

Patients who have chronic inflammatory bowel illnesses like Crohn’s syndrome are more likely to develop colorectal cancer. About 3% of individuals with Crohn’s for over 10 years developed colorectal cancer.²⁹ Treatment of chromium complex to Caco-2 showed reduction in cell viability, confirming its cytotoxicity.

Conclusion

Cr(D-Phe)₃ pre-treatment protected against inflammatory and oxidative stress indicators caused by chronic indomethacin administration. The present study revealed that the observed protective effects of chromium complex can be credited to its anti-inflammatory, antimicrobial, antioxidant and mast cell stabilising abilities. Furthermore, the title complex was found to reduce Caco-2 cell viability corroborating the protective role of title complex in the treatment of colorectal cancer.

Conflicts of Interest

Nil

Acknowledgement

The authors are thankful for the financial funding provided by Rajiv Gandhi University of Health Sciences (RGUHS), Bangalore, Karnataka, for research grant-2017-18 (Project code 17P010 and Ref # RGU / ADV. RES /BR /001 /2017-18 Dated 21/12/2017).