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Keywords:

  • bromelain;
  • cysteine protease;
  • intestinal inflammation;
  • intestinal motility;
  • protease-activated receptors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Author Contributions
  8. Competing Interests
  9. References

Background  Bromelain (BR) is a cysteine protease with inhibitory effects on intestinal secretion and inflammation. However, its effects on intestinal motility are largely unexplored. Thus, we investigated the effect of this plant-derived compound on intestinal contractility and transit in mice.

Methods  Contractility in vitro was evaluated by stimulating the mouse isolated ileum, in an organ bath, with acetylcholine, barium chloride, or electrical field stimulation. Motility in vivo was measured by evaluating the distribution of an orally administered fluorescent marker along the small intestine. Transit was also evaluated in pathophysiologic states induced by the pro-inflammatory compound croton oil or by the diabetogenic agent streptozotocin.

Key Results  Bromelain inhibited the contractions induced by different spasmogenic compounds in the mouse ileum with similar potency. The antispasmodic effect was reduced or counteracted by the proteolytic enzyme inhibitor, gabexate (15 × 10−6 mol L−1), protease-activated receptor-2 (PAR-2) antagonist, N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (10−4mol L−1), phospholipase C (PLC) inhibitor, neomycin (3 × 10−3 mol L−1), and phosphodiesterase 4 (PDE4) inhibitor, rolipram (10−6 mol L−1). In vivo, BR preferentially inhibited motility in pathophysiologic states in a PAR-2-antagonist-sensitive manner.

Conclusions & Inferences  Our data suggest that BR inhibits intestinal motility – preferentially in pathophysiologic conditions – with a mechanism possibly involving membrane PAR-2 and PLC and PDE4 as intracellular signals. Bromelain could be a lead compound for the development of new drugs, able to normalize the intestinal motility in inflammation and diabetes.


Abbreviations:
ACh

acetylcholine

BaCl2

barium chloride

DMSO

dimethyl sulfoxide

BR

bromelain

DTT

dithiothreitol

EFS

electrical field stimulation

ENMD-1068

N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine

FBS

fetal bovine serum

HBS

HEPES buffer solution

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NEAA

non-essential amino acid

PAR-1

protease-activated receptor 1

PAR-2

protease-activated receptor 2

PBS

phosphate-buffered saline

PDE4

phosphodiesterase 4

PLC

phospholipase C

STZ

streptozocin

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Author Contributions
  8. Competing Interests
  9. References

Proteolytic enzymes (also called proteases or proteinases) refer to a group of enzymes whose catalytic function are to hydrolyse peptide bonds of proteins. Proteases are divided into five groups on basis of the mechanism of action at the active site (cysteine, serine, aspartic, metallo, and mixed depending on the principal amino acid participating in the catalysis). Cysteine proteases which compose a large class of enzymes from plant, animal, and bacterial sources play important roles in various biologic processes.1–3

Bromelain is a cysteine protease derived from the stem of the pineapple plant, Ananas comosus (L.) Merr. (Family Bromeliaceae). In the United States, BR is sold in health stores as a nutritional supplement to promote digestive health and as a cleansing agent to improve the texture of the skin and to promote the healing of wounds. Bromelain is also commercially available as an anti-inflammatory drug. Preclinical and/or clinical studies have reported anti-inflammatory, immunomodulatory, antitumoral, and wound healing actions from BR.4–6 Bromelain has been shown to have efficacy similar to – or better than – classic non-steroidal anti-inflammatory agents in patients with rheumatologic diseases or ulcerative colitis.7–9 In addition, BR has been also reported to protect animals from experimentally induced diarrhea and diarrhea-induced death. The precise antidiarrheal mechanism of BR is not known; however, BR has shown to inhibit enterotoxigenic Escherichia coli receptor activity, thereby inhibiting bacterial attachment and colonization in the small intestine.10 Moreover, BR has demonstrated to prevent intestinal fluid secretion mediated by secretagogues that act via adenosine 3′:5′-cyclic monophosphate, guanosine 3′:5′-cyclic monophosphate, and calcium-dependent signalling pathways.11

Although the effect of BR on intestinal inflammation and diarrhea has been extensively studied, there is a paucity of reports on the possible effect of BR on intestinal motility. This is an important lack of information as it is well known that motility changes play an important role in intestinal inflammation and diarrhea. Therefore, the aim of our study is to investigate the effect and the mode of action of BR on intestinal motility. For this purpose we have performed in vitro experiments on isolated intestinal tissues and in vivo experiments on intestinal motility, both in physiologic and in pathophysiologic states (i.e., dysmotility due to experimental ileitis and diabetes).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Author Contributions
  8. Competing Interests
  9. References

Animals

Male ICR mice (20–22 g) were purchased from Harlan Italy (San Pietro al Natisone, MI, USA) and housed in polycarbonate cages in isolators under a 12-h light/12-h dark cycle, temperature 23 ± 2 °C, and humidity 60%. Animals, used after 1 week of acclimation, had free access to water and food. All experiments complied with the Italian D.L. no. 116 of January 27, 1992 and associated guidelines in the European Communities Council Directive of November 24, 1986 (86/609/ECC).

Organ preparation and treatment

Mice were killed by asphyxiation with CO2 and segments (1–2 cm) of the terminal ileum were removed, flushed of luminal contents and placed in Krebs’ solution (composition in mmol L−1: NaCl 119, KCl 4.75, KH2PO4 1.2, NaHCO3 25, MgSO4 1.5, CaCl2 2.5, and glucose 11). The isolated organs were placed vertically in a bath filled warm (37 °C) aerated (95% O2 : 5% CO2) Krebs’ solution and set up as described previously.12 The mechanical activity of the longitudinal muscle was recorded isotonically (load 0.5 g) with a transducer connected to a PowerLab data-acquisition system (Ugo Basile, Comerio, Italy). At the beginning of each experiment, the ileum was stimulated with acetylcholine (ACh) (10−3 mol L−1) in order to obtain a maximal contraction (100% contraction). After a minimal 1-h equilibration period, the tissues were subjected to electrical field stimulation (EFS, 8 Hz for 10 s, 400 mA, 1 ms pulse duration using a multiplexing pulse booster by Ugo Basile, Milan, Italy) via a pair of platinum electrodes (situated at a distance of 1.5 cm) placed around the intestine or stimulated with spasmogens, such as ACh (10−6 mol L−1) or barium chloride (BaCl2, 10−4 mol L−1). At the used concentrations, ACh and BaCl2 gave a contractile response which was similar in amplitude to that of EFS. Acetylcholine and BaCl2 were added to the bath and left in contact with the tissue for 30 s and then washed out. The interval between each stimulation was 20 min. After at least three stable control contractions, in the mouse ileum the contractile responses were repeated in the presence of increasing (non-cumulative) concentrations of BR (1–1000 μg mL−1) added 20 min before the contacting stimulus (i.e., after washing the tissue). Preliminary experiments showed that a 20 min contract time was sufficient for BR to achieve the maximal inhibitory effect. Moreover, some experiments were performed using a solution of BR with inactivated proteolytic activity (see Materials and Methods for inactivation of BR proteolytic activity).

In some experiments, the effect of BR on ACh-induced contractions was evaluated in the presence of gabexate (a proteolytic enzyme inhibitor, 15 × 10−6mol L−1), SCH 79797 [a selective protease-activated receptor-1 (PAR-1) antagonist, 15 × 10−6 mol L−1], N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine [ENMD-1068, a selective protease-activated receptor-2 (PAR-2) antagonist, 10−4 mol L−1], rolipram [a selective phosphodiesterase 4 (PDE4) inhibitor, 10−6 mol L−1] and neomycin [a phospholipase C (PLC) inhibitor, 3 × 10−3 mol L−1] (contact time: 2030 min for each drug). The concentrations of these inhibitors/antagonists were selected on the basis of previous published works.12–15 The presence of such inhibitors/antagonists did not affect the reproducibility and the stability of the contractions induced by ACh.

In another set of experiments, we evaluated the effect of BR on ACh-induced contractions in the presence of synthetic peptides (I–VII, 10−7 mol L−1) derived from the extracellular N-terminal peptide domain of murine PAR-2 (from Cys22 to Lys36).

Induction of intestinal inflammation (ileitis) and diabetes

Inflammation was induced as previously described.12,16 Briefly, two doses of croton oil (CO) (20 μL per mouse) in two consecutive days were orally administered to mice and 4 days after the first administration of CO, upper gastrointestinal transit of mice was measured. This time was selected on the basis of a previous work,16 which reported that maximal inflammatory response occurred 4 days after the first treatment.

Insulin-dependent diabetes mellitus was induced in mice by a single intraperitoneal injection of 200 mg kg−1 streptozocin (STZ) (prepared freshly by dissolving it in saline adjusted to pH 4.5 in 0.1 N citrate buffer).17 After a week, the non-fasting blood glucose concentration was determined in blood obtained from the cut tip of the tail using a glucose test kit with an automatic analyzer (Accu-Chek@ Active; Roche, Milan, Italy). The animals were considered diabetic if the non-fasting blood glucose concentration was higher than 300 mg dL−1 (16.7 mmol L−1). Four weeks after the onset of diabetes, animals were tested for upper gastrointestinal transit.

Upper gastrointestinal transit

Upper gastrointestinal transit was measured in control-, CO-, and in STZ -treated mice by evaluating the intestinal location of rhodamine-B-labeled dextran.18 Animals were given fluorescent-labelled dextran (100 μL of 25 mg mL−1 stock solution) via a gastric tube into the stomach. Twenty minutes after administration, the animals were killed by asphyxiation with CO2 and the entire small intestine with its content was divided into 10 equal parts as previously reported in detail.18 The fluorescence in duplicate aliquots of the cleared supernatant was read in a multi-well fluorescence plate reader (LS55 Luminescence spectrometer; Perkin-Elmer Instruments, Waltham, MA, USA; excitation 530 ± 5 nm and emission 590 ± 10 nm) for quantitation of the fluorescent signal in each intestinal segment. From the distribution of the fluorescent marker along the intestine, we calculated the geometric center (GC) of small intestinal transit as follows: GC = P(fraction of fluorescence per segment × segment number), where GC ranged from 1 (minimal motility) to 10 (maximal motility).

Bromelain (1–10 mg kg−1) or vehicle (0.9% NaCl solution) was given intraperitoneally, 30 min before the administration of the fluorescent marker to animals. In some experiments, EDNM-1068 (4 mg kg−1, dissolved in 0.9% NaCl) was given intraperitoneally 30 min before the administration of BR. The dose of EDNM-1068 was selected on the basis of previous works.19

In another set of experiments, BR (100–500 mg kg−1) was given orally 1 h before the administration of the fluorescent marker.

Western blot analysis

Full-thickness ileum from control-, CO-, and STZ-treated mice were homogenized in lysis buffer (1 : 2 w/v) containing 0.5 mol L−1β-glycerophosphate, 20 mmol L−1 MgCl2, 10 mmol L−1 ethylene glycol tetraacetic acid and supplemented with 100 mmol L−1 dithiothreitol (DTT) and protease/phosphatase inhibitors (100 mmol L−1 dimethylsulfonyl fluoride, 2 mg mL−1 apronitin, 2 mmol L−1 leupeptin, and 10 mmol L−1 Na3VO4). Homogenates were centrifuged at 600 g for 5 min at 4 °C; the supernatants were collected and centrifuged at 16 200 g for 10 min at 4 °C. Protein concentrations were determined using the method of Bradford.20 Proteins (50 μg) were subjected to electrophoresis on an SDS 10% polyacrylamide gel and electrophoretically transferred onto a nitrocellulose transfer membrane (Protran, Schleicher & Schuell, Dassel, Germany). The immunoblots were developed with 1 : 1000 dilution for PAR-2 (epitope specificity within amino acids 37–50) (Santa Cruz Biotechnology, Inc., DBA Italia, Segrate, Milano, Italy) and the signal was detected with the ECL System according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Cologno Monzese, Milano, Italy). The membranes were probed with an anti-β-actin antibody, to normalize the results, which were expressed as a ratio of densitometric analysis of PAR-2/β-actin bands.

Bromelain inactivation and evaluation of its proteolytic activity

Inactivated BR was prepared as described by Hale et al.21 Briefly, 15 mg mL−1 BR in phosphate-buffered saline (PBS, pH 8.0) was incubated with 15 mmol L−1 DTT for 40 min at 37 °C. Subsequently, the preparation was incubated for 80 min at 37 °C with 130 mmol L−1 iodoacetamide. Finally, to remove DTT and iodoacetamide, a dialysis (membranes MWCO = 3500) for 22 h against 0.9% saline was performed. This inactivation procedure reduced the activity to less than 5%. The BR integrity was investigated measuring its proteolytic activity against the proteic substrate p-Glu-Phe-Leu-p-nitroanilide (P-ANL) (Sigma, Milan, Italy). Briefly, 100 μL of the BR solution was incubated at 37 °C with 1.6 mL of PBS (pH 6.5) and 0.2 mL P-ANL solution [1 mg mL−1 in dimethyl sulphoxide (DMSO)]. Subsequently, the mixture was incubated at 37 °C for 15 min and then analysed using an UV spectrophotometer (Shimadzu UV 1204, Shimidzu, Milan, Italy) with the wavelength set at 410 nm.

Measurement of [Ca2+]i in Caco-2 cells

Human colon adenocarcinoma Caco-2 cells were purchased from the American Type Culture Collection (LGC Promochen, Milan, Italy) and used between passages 30 and 50. The cells were routinely maintained in 75 cm2 polystyrene flasks in growth media consisting of Dulbecco’s modified eagle medium containing 10% fetal bovine serum, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, 1 mol L−1 Hepes [4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid] 2.5%, non-essential amino acid 1×, 2 mmol L−1l-glutamine at 37 °C in a 5% CO2 atmosphere. The culture medium was replaced every 2 days. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described.22 Intracellular calcium measurement was performed using a modified method adapted from the procedure described by Jacob et al.23 Briefly, after washing in PBS, Caco-2 cells were trypsinized with 0.25% trypsin–EDTA at 37 °C for 5 min, centrifuged at 1000 g for 3 min, and then re-suspended at the concentration of 55 000 cells mL−1 in HEPES buffer solution (HBS) (composition in mmol L−1: NaCl 125, KCl 4, CaCl2 2, l-glutamine 4, glucose 10, Hepes 30) containing Fura-2AM (10 μmol L−1) and ENMD-1068 (5 mmol L−1). After 30 min, some cells were treated with BR (1 μg mL−1) for 20 min. In another set of experiments, cells were preincubated with BR (1 μg mL−1) for 3 min and then challenged with SLIGKV-NH2 (100 μmol L−1). After these treatments, cells were centrifuged at 1000 g for 2 min, and then re-suspended in calcium-free HBS. Intracellular calcium levels were measured using a fluorescent microplate reader (LS55 Luminescence Spectrometer; Perkin-Elmer Instruments, excitation–emission wavelengths of 343/485 nm). The results are expressed as 343/485 nm ratio. The treatments were carried out in triplicate and four independent experiments were performed.

Peptides and N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068) synthesis

The sequences and MS analyses of peptides I–VII are for peptide I: H-Cys-Ser-Arg-Thr-Glu-Asn-NH2 (MW calcd/found: 707.8/708.4); peptide II: H-Ser-Arg-Thr-Glu-Asn-Leu-NH2 (MW calcd/found: 717.8/718.5); peptide III: H-Ser-Arg-Thr-Glu-Asn-Leu-Ala-Pro-Gly-Arg-Asn-Asn-Ser-Lys-NH2 (MW calcd/found: 1542.7/1543.0); peptide IV: H-Arg-Thr-Glu-Asn-Leu-Ala-Pro-Gly-Arg-Asn-Asn-Ser-Lys-NH2 (MW calcd/found: 1455.6/1456.0); peptide V: H-Ser-Arg-Thr-Glu-Asn-Leu-Ala-Pro-Gly-Arg-Asn-Asn-NH2 (MW calcd/found: 1327.4/1327.7); peptide VI: H-Ser-Arg-Thr-Glu-Asn-Leu-Ala-Pro-Gly-Arg-NH2 (MW calcd/found: 1099.2/1099.7); and peptide VII: H-Ser-Arg-Thr-Glu-Asn-Leu-Ala-Pro-NH2 (MW calcd/found: 886.0/886.4).

Compounds IVII were prepared by solid phase peptide synthesis using a continuous-flow instrument employing an on-line UV monitoring (Milligen/Biosearch 9050, Novato, CA, USA). The stepwise syntheses were carried out by Fmoc chemistry on 0.1 mmol of a Rink amide MBHA resin (0.78 mmol g−1 substitution grade). The resin was swelled in DMF for 1 h and packed in the reaction column. tert-Butyl was used as a side chain protecting group for serine, threonine, and glutamic acid, trityl was used for asparagine and cysteine while tert-butoxycarbonyl (Boc) and 2,2,5,7,8- pentamethylchromane-6-sulfonyl were used for lysine and arginine, respectively. Nα-Fmoc amino acids were used in a fourfold excess using TBTU/HOBt/NMM, as coupling and capping was accomplished with 20% acetic anhydride in DMF. Cleavage of N-Fmoc protecting group, using 25% piperidine (v/v) in DMF, was monitored at each stage by measuring the absorbance of the liberated N-(9-fluorenylmethyl) piperidine. After completion of the synthesis each protected peptide was cleaved from the resin, and the amino acid side chains were simultaneously deprotected by treatment with 10 mL of TFA/H2O/Et3SiH (88 : 5 : 7) mixture for 2 h at room temperature. The resin was removed by filtration and washed with TFA (2 × 1 mL), the filtrate and washings were combined and evaporated in vacuo, and the oily residue was triturated with ethyl ether (10 mL). The crude material was purified by preparative high-performance liquid chromatography (HPLC) on a Vydac C18 column (15–20 μm, 22 × 5000 mm) employing water-acetonitrile (containing 0.1% TFA) linear gradients. All peptides were purified to over 95% homogeneity as judged by analytical HPLC performed using a Vydac C18 column (5 μm, 4.6 × 250 mm) employing the following conditions: eluent A, 0.05% TFA (v/v) in water; eluent B, 0.05% TFA(v/v) in acetonitrile; the employed linear gradients are reported in Table 1. Structural verification of the final desired peptides IVII was achieved by amino acid analysis (Table 1) and mass spectrometry.

Table 1.   Analytical data of peptides I–VII
CompoundsAmino acid analysis*HPLC
SRTEDLAPGK
  1. *The method used for hydrolysis does not allow the recovery of cysteine. The method used for hydrolysis completely converted asparagine to aspartic acid. The analytical HPLC were run on a reversed-phase Vydac C18 column (5 μm, 4.6 × 250 mm) using the following gradient system: A, 0.05% TFA in CH3CN; B, 0.05% TFA in H2O, UV detection at 220 nm, flow rate 1 mL min−1. §Gradient from 0% A to 15% A over 25 min. Gradient from 0% A to 50% A over 25 min. **Gradient from 5% A to 20% A over 30 min.

I1.000.961.020.980.958.20§
II1.030.981.061.030.911.0111.88
III1.921.900.981.012.910.960.981.030.991.0214.03**
IV0.971.950.991.042.940.990.990.941.020.9113.51**
V1.011.910.971.012.880.940.920.991.0414.60**
VI0.931.930.980.900.911.020.970.951.0115.33**
VII0.920.981.020.980.991.041.000.9814.80**

Molecular weights of final peptides were assessed by electrospray ionization mass spectrometry on an Applied Biosystems API 2000 apparatus. Amino acid analyses were carried out using PITC methodology (Pico-Tag; Waters-Millipore, Waltham, MA, USA). Reversed-phase purification was routinely performed on a Waters Delta Prep 4000 system equipped with a Waters 484 multi-wavelength detector on a Vydac C18 silica (15–20 μm, 22 × 5000 mm) HPLC column. The operational flow rate was 60 mL min−1. Homogeneity of the products was assessed by analytical reversed-phase HPLC using a Vydac C18 column (5 μm, 4.6 × 250 mm), UV detection at 220 nm, flow rate 1 mL min−1. The column was connected to a Rheodyne Model 7725 injector, a Waters 600 HPLC system, a Waters 486 tunable absorbance detector set to 220 nm, and a Waters 746 chart recorder.

ENMD-1068 was synthetized (purity 98%) as follows: piperazine (1 eq), 3-methylbutanoic acid (1 eq), EDC·HCl (1.2 eq), and HOBt (1.2 eq) were dissolved in anhydrous DMF (20 mL) and stirred under N2 at room temperature. Once the reaction was complete, as judged by TLC, water (25 mL) and ethyl acetate (15 mL) were added and both layers separated. The aqueous layer was extracted with ethyl acetate (3 × 15 vol) and the combined organic layers were washed with brine, dried over Na2SO4 and filtered. After solvent removal the intermediate was purified by column chromatography (mixtures ethyl acetate-heptane), affording the pure 3-methyl-1-(piperazin-1-yl)butan-1-one (yield 80%). 3-Methyl-1-(piperazin-1-yl)butan-1-one (1 eq), Boc-aminocaproic acid (1 eq), EDC·HCl (1.2 eq), and HOBt (1.2 eq) were dissolved in anhydrous DMF (20 mL) and stirred under N2 at room temperature. Once the reaction was complete, as judged by TLC, the solvent was removed and the residue dissolved in ethyl acetate (15 mL) was extracted with 10% citric acid, 5% NaHCO3, and brine. The combined organic layers were dried over Na2SO4 and filtered. The N-Boc protected derivative, obtained as a solid after solvent removal, was dissolved in 10 mL of anhydrous HCl in dioxane (4.0 mol L−1) and stirred at room temperature for few hours. Once the reaction was complete, the solvents were removed under vacuo to afford ENMD-1068·HCl, as a powdery solid in quantitative yield.

Statistical analysis

Data are mean ± SEM. Comparisons between two sets of data were made by Student’s t-test for paired data. When multiple comparisons against a single control were made, one-way analysis of variance was used, followed by Dunnett’s multiple comparisons test. Non-linear regression analysis for all concentration-response curves were performed (graph pad instat program version 4.01; GraphPad Software Inc., San Diego, CA, USA) and analysis of variance (two-way) was used to compare different concentration–effect curves. A P-value less than 0.05 was considered significant. The IC50 (concentrations that produced 50% inhibition) and the Emax (maximal effect) were used to characterize the potency and efficacy of BR, respectively. The IC50 and Emax values (geometric mean ± 95% confidence limits) were calculated using the graph pad instat program version 4.01.

Drugs

Acetylcholine chloride, BaCl2, atropine, tetrodotoxin, rolipram, neomycin, DTT, Fura 2-AM, STZ, MTT, and CO were purchased from Sigma, while gabexate mesylate, SCH 79797 dihydrochloride, and SLIGKV-NH2 were obtained from Tocris (Eching, Germany). Peptides and ENMD-1068 were synthesized in our laboratories (see above). All solvents were purchased from Carlo Erba (Rodano, Milan, Italy) while reagent grade materials (used without further purification) were purchased from Bachem (Bubendorf, Switzerland), Inalco-Novabiochem (Milan, Italy), and Aldrich (Milan, Italy). All reagents for cell culture were obtained from Sigma and Microglass Heim (Naples, Italy).

Acetylcholine, BaCl2, atropine, tetrodotoxin, neomycin, DTT, and peptides were dissolved in distilled water. Rolipram was dissolved in DMSO to give 10−5 mol L−1 stock solution and subsequent dilutions were made in distilled water. Bromelain, ENMD-1068, and SLIGKV-NH2 were dissolved in distilled water (for in vitro experiments) or in 0.9% NaCl solution (for in vivo experiments). Drugs were added in volumes less than 0.01%in vitro and given in the amount of 0.1 mL per mouse in vivo. The drug vehicles had no effect on the responses under study, both in vitro and in vivo.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Author Contributions
  8. Competing Interests
  9. References

Effect of bromelain on acetylcholine-, barium chloride- or electrical field stimulation-induced contractions

Electrical field stimulation (EFS, 8 Hz for 10 s, 400 mA, 1 ms pulse duration) produced a contractile response of mouse ileum which was 52.1 ± 3.25% (n = 8) of the maximal contractile response (100% contraction) produced by 10−3 mol L−1 ACh. EFS-induced contractions were abolished by tetrodotoxin (3 × 10−7 mol L−1) or atropine (10−6 mol L−1), thus suggesting that these contractions were mediated by the release of ACh from enteric nerves. Tetrodotoxin did not modify the contractions induced by either ACh (10−6 mol L−1) or BaCl2 (10−4 mol L−1), while atropine abolished the ACh (but not BaCl2)-induced contractions (data not shown).

Bromelain (1–1000 μg mL−1) significantly and in a concentration-dependent manner inhibited the contractions evoked by ACh, BaCl2, or EFS (Fig. 1). A significant effect was achieved starting from 100 μg mL−1 concentration. No significant differences were observed in the inhibitory effect of BR on ACh-, BaCl2-, or EFS-induced contraction (Fig. 1).

image

Figure 1.  Inhibitory effect of bromelain (1–1000 μg mL−1) on the contractile response induced by exogenous ACh (10−6 mol L−1), BaCl2 (10−4 mol L−1), or electrical field stimulation (EFS, EFS, 8 Hz for 10 s, 400 mA, 1 ms pulse duration) in the isolated mouse ileum. Each point represents mean ± SEM of six to eight experiments.

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Involvement of the proteolytic activity of bromelain in its effect on intestinal motility

Inactivation of BR, by reduction and alkylation processes, completely abolished the inhibitory effect of BR (Fig. 2A). Moreover, gabexate (a proteolytic enzyme inhibitor, 15 × 10−6 mol L−1) significantly reduced the antispasmodic effect of BR on mouse ileum (Fig. 2A). Gabexate (15 × 10−6 mol L−1), given alone, reduced (52.3 ± 6.11% reduction. P < 0.001, n = 8) the contractions induced by ACh.

image

Figure 2.  Acetylcholine-induced contractions in isolated mouse ileum: (A) effect of bromelain (BR) (1–1000 μg mL−1) alone (vehicle), proteolytically inactive BR and BR in the presence of gabexate (15 × 10−6 mol L−1); (B) effect of BR alone (vehicle) and in the presence of ENMD-1068 (10−4 mol L−1) or SCH79797 (15 × 10−6 mol L−1); (C) effect of BR alone (vehicle) and in the presence of synthesized peptide ligands (10−7 mol L−1); (D) effect of BR alone (vehicle) and in the presence of rolipram (10−6 mol L−1) and neomycin (3 × 10−3 mol L−1). Each point represents mean ± SEM of six to eight experiments. aP < 0.001 vs vehicle (significance between the two dose–response curves).

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Effect of bromelain on acetylcholine-induced contraction in the presence of PAR-1 and PAR-2 antagonists and peptide ligands

Fig. 2B shows the effect of BR on ACh-induced contractions in the presence of ENMD-1068 (a PAR-2 antagonist, 10−4 mol L−1) or SCH79797 (a PAR-1 antagonist, 15 × 10−6 mol L−1). ENMD-1068 significantly reduced the inhibitory effect of BR on ACh-induced contraction (Fig. 2B). By contrast, SCH 79797 did not affect the antispasmodic effect of BR (Fig. 2B).

Peptide I, II, and VII, but not peptides III, IV, V, and VI (all at the 10−7 mol L−1 concentration), significantly reduced the effect of BR on ACh-induced contraction (Fig. 2C).

When given alone (i.e., in the absence of BR) SCH 79797 (15 × 10−6 mol L−1) reduced (28.2 ± 3.02%, n = 8) the contractions induced by ACh while ENMD-1068 (10−4 mol L−1) and peptides I–VII did not modify ACh-induced contractions.

Effect of bromelain on acetylcholine-induced contractions in the presence of phospholipase C and phosphodiesterase 4 inhibitors

Fig. 2D shows the effect of BR on ACh-induced contractions in the presence of rolipram (a selective PDE4 inhibitor, 10−6 mol L−1), neomycin (a PLC inhibitor, 3 × 10−3 mol L−1) and verapamil (10−7 mol L−1). Rolipram and neomycin significantly shifted on the right, the inhibitory curve of BR on ACh-evoked contractions (Fig. 2D). When given alone (i.e., in the absence of BR) rolipram (10−6 mol L−1) increased (44.7 ± 3.78% increase, n = 8, P < 0.01) the ACh-induced contractions. Neomycin, used alone, did not affect the action of ACh.

Effect of bromelain on intracellular calcium levels

Bromelain (1 μg mL−1) (P < 0.01) increased [Ca2+]i in Caco-2 cells and this effect was significantly (P < 0.05) reduced by ENMD-1068 (5 mmol L−1) (Fig. 3). In addition, the human PAR-2–activating peptide SLIGKV-NH2 (100 μmol L−1), corresponding to the tethered ligand, increased [Ca2+]i in vehicle-, but not in BR (1 μg mL−1)-treated Caco-2 cells (Fig. 4). Bromelain, ENMD-1068 and SLIGKV-NH2, at the concentrations used in this assay, did not affect cell viability (data not shown).

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Figure 3.  Effect of bromelain (BR) (1 μg mL−1) alone and in the presence of ENMD-1068 (5 mmol L−1) on intracellular calcium concentration ([Ca2+]i) in Caco-2 cells. Bars represent the mean ± SEM of four experiments. aP < 0.01 vs control; bP < 0.05 vs BR.

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Figure 4.  Effect of SLIGRL-NH2 (100 μmol L−1) alone (vehicle) and after bromelain preincubation (1 μg mL−1 for 5 min) on intracellular calcium concentration ([Ca2+]i) in Caco-2 cells. Results are expressed as a percentage of the control response of vehicle-treated cells. Bars represent the mean ± SEM of four experiments. aP < 0.001 vs vehicle.

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Transit in control mice

Bromelain, administered intraperitoneally, at the dose ranging from 1 to 10 mg kg−1, reduced significantly and in a dose-dependent manner the intestinal transit, as indicated by a lower GC value compared to control (Fig. 5A). The effect was significant starting from the 1 mg kg−1 dose. By contrast, BR, given orally (100–500 mg kg−1) was inactive (GC: control 5.0 ± 0.26; BR 100 mg kg−1 4.9 ± 0.12; BR 250 mg kg−1 5.2 ± 0.24; BR 500 mg kg−1 4.8 ± 0.18; n = 10–12 animals for each experimental group). The PAR-2 antagonist ENMD-1068 (4 mg kg−1), which, used alone, did not affect motility (GC: control 5.0 ± 0.26; ENMD-1068 5.6 ± 0.49, n = 8–10 animals for each experimental group) counteracted the inhibitory effect of BR (3 mg kg−1) on intestinal transit (Fig. 5B).

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Figure 5.  (A) Effect of intraperitoneally injected bromelain (BR) (1–10 mg kg−1) on intestinal transit in control mice and (B) effect of intraperitoneally injected BR (3 mg kg−1) alone or in the presence of ENMD-1068 (4 mg kg−1) on intestinal transit in control mice. Transit was expressed as the geometric center of the distribution of a fluorescent marker along the small intestine (see Materials and Methods section). Bars represent the mean ± SEM of 10–12 animals for each experimental group. aP < 0.01 and bP < 0.001 vs control, cP < 0.01 vs 3 mg kg−1 BR alone.

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Transit in croton oil- or streptozocin-treated mice

According to previous studies,12,16,18 administration of the inflammatory agent CO produced a significant increase (P < 0.01) in intestinal transit (Fig. 6A,B). Bromelain, given intraperitoneally (1–10 mg kg−1) or orally (100–500 mg kg−1), reversed the increase in motility induced by CO (Fig. 6A,B). The inhibitory effect of BR (BR, 500 mg kg−1, per os) was reverted by the PAR-2 receptor antagonist ENMD-1068 (4 mg kg−1) [GC: control 5.2 ± 0.22; CO 6.7 ± 0.33a; CO + BR 5.3 ± 0.35b; CO + BR + ENMD-1068 8.1 ± 0.18c. aP < 0.01 vs control, bP < 0.05 vs CO, and cP < 0.001 vs CO + BR; n = 8 for each experimental group]. ENMD-1068 alone did not significantly affect transit in CO-treated mice (GC: CO 6.7 ± 0.46; ENMD-1068 6.5 ± 0.44).

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Figure 6.  Effect of (A) intraperitoneally injected bromelain (BR) (1–10 mg kg−1) and (B) orally administered BR (100–500 mg kg−1) on intestinal transit in croton oil (CO)-treated mice. Transit was expressed as the geometric center of the distribution of a fluorescent marker along the small intestine (see Materials and Methods section). Bars represent the mean ± SEM of 10–12 animals. aP < 0.01 vs control; bP < 0.05; cP < 0.01; and dP < 0.001 vs CO. Insert shows the different potency of BR in reducing transit in physiologic and inflammatory conditions.

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Four weeks after a single administration of STZ (200 mg kg−1), transit was significantly increased in mice. Bromelain (1–10 mg kg−1, i.p.) counteracted the increase in motility induced by the diabetogenic agent [GC: control 5.3 ± 0.09; STZ 6.3 ± 0.17a; STZ + BR 1 mg kg−1 6.2 ± 0.43; STZ + BR 3 mg kg−1 3.0 ± 0.48b; STZ + BR 10 mg kg−1 1.5 ± 0.09b; aP < 0.05 vs control; and bP < 0.001 vs STZ; n = 8 for each experimental group].

PAR-2 expression in croton oil- and streptozocin-treated mouse ileum

Western blot analysis revealed the expression of PAR-2 in ileal tissues of normal-, CO-, and STZ -treated animals (Fig. 7). However, the densitometric analysis indicated a significant (P < 0.001) decrease in the expression of PAR-2 in the inflamed tissues (Fig. 7). By contrast, the expression of PAR-2 was not significantly modified in the ileal tissues of STZ-treated mice (Fig. 7).

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Figure 7.  PAR-2 expression in ileum of control (vehicle)-, croton oil (40 μL per mouse)-, and streptozocin (200 mg kg−1)-treated mice (see Materials and Methods for details). Bars represent the mean ± SEM of three experiments. aP < 0.05 vs control.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Author Contributions
  8. Competing Interests
  9. References

Clinical studies have shown that BR, a cysteine protease obtained from pineapple stems, was effective – presumably through an anti-inflammatory mechanism – in reducing a number of symptoms, including diarrhea, associated with inflammatory bowel disease (IBD).9,24 This is consistent to animal experiments in which it has been shown that BR prevented the intestinal fluid accumulation due to cholera toxin and Escherichia coli,10,11,25 and the damage in the IL-10-deficient murine model of intestinal inflammation.26 Here, we provide evidence that BR affects directly rodents smooth muscle ileum contractility in vitro and reduces intestinal transit in vivo. Furthermore, BR was more potent in pathophysiologic states – such as inflammation or diabetes – than in healthy animals.

In vitro studies

We have shown that BR inhibited intestinal spasms in the isolated ileum. The inhibitory effect of BR was non-selective as this proteolytic compound inhibited with a similar potency the contractions induced by ACh (which acts directly on muscarinic receptors located on smooth muscles), BaCl2 (which enters the intracellular space of the smooth muscle and directly stimulates the contractile mechanism), and by EFS (which acts releasing ACh from myenteric nerves). This non-selective profile of inhibition probably indicates that BR acts directly on smooth muscle contractility (i.e., postjunctional effect) rather than on processes involved in neurotransmitter(s) release. Therefore, to investigate the mechanism of BR-induced contractility inhibition we performed further experiments on the contractions induced by ACh.

To evaluate whether the antispasmodic effect of BR was related to its proteolytic activity, we analyzed the effect of inactivated BR on ACh-induced contractions. Our results showed that the antispasmodic effect of BR requires proteolytic activity, as a treatment with a proteolytically inactive BR did not affect the intestinal motility. Further evidence about the importance of the proteolytic activity in the mode of action of BR comes from the experiments with the proteolytic enzyme inhibitor gabexate, which reduced the inhibitory effect of BR on ACh-induced contractions.

In the gastrointestinal tract there are specific receptors that can be activated by proteases, including the protease-activated receptors (PARs), which comprise a family of four subtypes (PAR-1, PAR-2, PAR-3, and PAR-4) all activated by serine proteases.27,28 These receptors are activated by proteolytic cleavage of a receptor-bound, amino-terminal tethered ligand domain, which is then able to bind to the receptor and to initiate intracellular signalling. PAR-1 and PAR-2 are widely expressed in smooth muscle cells; however their role in motility modulation is very complex, depending upon the animal species and region of the gut studied.29,30 In the isolated mouse small and large intestine, PAR-1 and PAR-2 agonists elicit transient relaxation followed by contraction.31–35 In our experiments the selective PAR-2 antagonist ENMD-1068, but not the selective PAR-1 antagonist SCH 79797, reduced the inhibitory effect of BR. These data provide an indirect evidence about an involvement of PAR-2 on the antispasmodic effect of BR.

In order to better explore BR mode of action on PAR-2, we have synthesized several peptides (I–VII) derived from the fragment Cys22-Lys36 of the extracellular N-terminal PAR-2 sequence. Bromelain has a broad substrate specificity and hydrolyzes a great variety of synthetic and natural substrates extending from synthetic low molecular mass amides and dipeptides up to high molecular substrates such as fibrin, albumin, casein, angiotensin II, bradykinin.5,36. Several studies reported that BR preferentially cleaves glycyl, alanyl, and leucyl bonds; on the other hand, substrate specificity studies performed using combinatorial libraries of small fluorogenic substrates showed a strong preference of BR for Arg at both the P1 and P2 sites.37,38 These data, in conjunction with the observation that SLIGRL-NH2, a well-known PAR-2 agonist, did not exert any action on the BR inhibitory effect (data not shown), prompted us to synthesize peptides I–VII, in order to verify if the BR’s cleavage site on the PAR-2 amino-terminal extracellular domain might be different from that of trypsin. We have shown that longer peptides, such as compounds III (14 aa), IV (13 aa), V (12 aa), and VI (10 aa), did not modify the antispasmodic action of BR. Smaller peptides, such as compounds I (6 aa), II (6 aa), and VII (8 aa) were able to reduce the antispasmodic effect of BR, probably by preventing the binding to the receptor and its following activation. Interestingly, it has been reported that several synthetic peptides induce receptor activation in a different way than the PAR-2 tethered ligand.39–41

A further aim of our work was to investigate the intracellular signalling pathway that follows the activation of PAR-2 by BR. Compared with PAR-1, intracellular signal pathways associated with PAR-2 have been poorly investigated. Nevertheless, it is well known that PAR-2 activation by trypsin leads to activation of the Gq/G11–PLCβ pathway that induce an increase in intracellular calcium concentration.42–44 In order to evaluate the possible contribution of such pathway in BR-induced PAR-2 activation we have performed experiments with a selective inhibitor of PLC. Our results have shown that neomycin (a PLC inhibitor) reduced the inhibitory effect of BR on ACh-induced contractions, thus suggesting the involvement of Gq/G11–PLCβ pathway on BR action.

Although not all studies yield similar results, PAR-2 activation may modulate intracellular cAMP levels.45,46 In vascular tissues, the effect of PAR-2 is modulated by PDE4. PDE4, which is involved in smooth muscle contraction, specifically hydrolyses cAMP.47 We have shown that the PDE4 inhibitor rolipram reduced the antispasmodic effect of BR, suggesting an involvement of cAMP in BR action.

Because activation of PAR-2 results in an increase in [Ca2+]i, we performed further experiments in order to evaluate the effect of BR on [Ca2+]i. We found that BR increased [Ca2+]i in Caco-2 cells and this effect was reduced by the selective PAR-2 antagonist ENMD-1068. In addition, preincubation of Caco-2 cells with BR strongly reduce the response to SLIGRL-NH2. Together, these data further support the concept that BR could act by cleaving PAR-2.

In vivo studies

Because intestinal antispasmodic drugs may reduce intestinal motility in vivo and because the PAR-2 is involved in the control of intestinal transit, we investigated the effect of BR and the possible involvement of PAR-2 on BR’s action on intestinal transit in mice. BR, given intraperitoneally at doses ranging from 1 to 10 mg kg−1 reduced intestinal transit in control mice. By contrast, oral administration of BR (upto 500 mg kg−1) was ineffective. This is not surprising as BR is a proteolytic enzyme that is mostly destroyed by gastric acid. Incidentally, in order to prevent it from being destroyed by gastric juice, BR is clinically used in enteric-coated tablets.

As BR has been clinically evaluated in IBD patients, we also investigated its effect on motility in a mouse model of intestinal inflammation. We used the irritant compound CO, which has been extensively studied to induce hypermotility associated with inflammation in mice.12,16 We found that BR, either orally (100–500 mg kg−1) or intraperitoneally administered (1–10 mg kg−1), reduced hypermotility-induced inflammation, being the effect more pronounced in the inflamed gut rather than in the physiologic conditions. Collectively, our in vivo studies suggest that: (i) BR was more active after intraperitoneal than oral administration, and (ii) inflammation increased the potency of BR (see Fig. 6, insert).

The inhibitory effect of BR on intestinal transit, both in physiologic and pathophysiologic states, was reverted by ENMD-1068, thus confirming in vivo the possible involvement of PAR-2. Bromelain was also more active (compared to control mice) in a non-inflammatory model of intestinal dysmotility (i.e., diabetic mice),48 thus excluding the possibility that the higher potency of BR is due to a systemic anti-inflammatory effect. Possible sites of action of BR include epithelial cells, neuronal elements, and myocytes, in which PAR-2 is expressed. The PAR-2-containing cells can be reached by BR via systemic circulation or directly through the gastrointestinal tract (for BR escaping gastric inactivation).

It is also very unlikely that the higher potency of BR is due to PAR-2 hyper-expression because we found – in accordance to other studies49– a down-regulation of such receptor in the inflamed intestine. Further studies are needed to verify if the decreased PAR-2 protein expression is due to proteolysis or to changes in mRNA expression.

It is noteworthy that an increased PAR-2 expression has been observed in other non-intestinal experimental models of inflammation such as joint inflammation and rheumatoid synovium.19,50

We have shown for the first time that a cysteine protease, namely BR, exerts inhibitory effects on intestinal motility. The effect involves PLC and PDE4, and possibly PAR-2. From a clinical point of view, our study provides a further mechanism which may help to explain the efficacy of BR in reducing diarrhea in IBD patients.

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Author Contributions
  8. Competing Interests
  9. References

FB designed the research study and wrote the paper; RC, BS, FF, GA, GDR, MM, BR and IF performed the research; FC designed the research study; AAI analyzed the data and wrote the paper.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Author Contributions
  8. Competing Interests
  9. References