Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. Antibodies for p38α MAPK: sc-535, p-p38α MAPK: sc-166182, NF-κB: sc-56735, β-actin: sc-81178, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Opti-4CN substrate kit was acquired from Bio-Rad Laboratories (Hercules, CA, USA). Synthetic substrates for thrombin (S-2238™, H-D-Phe-Pip-Arg-pNa), factor XIa (S-2366, pyroGlu-Pro-Arg-pNa), plasma kallikrein and factor XIIa (S-2302™, H-D-Pro-Phe-Arg-pNA), factor Xa (S-2222™, Bz-Ile-Glu-Gly-Arg-pNa) and factor VIIa (SCP-0248, MeSO2-Cha-Abu-Arg-pNA)) were purchased from Chromogenix (Milan, Italy). Aprotinin (Trasylol®) was purchased from Bayer (São Paulo, Brazil). Ellagic acid, rabbit brain thromboplastin, phospholipids were purchased from Wiener Lab (Rosario, Argentina).
Plant material, cell line, maintenance and viability
A voucher specimen was deposited at the Herbarium of Univates (1066). We stated that the plant name has been checked with http://www.theplantlist.org. RAW 264.7 murine macrophage cell line was obtained from the Rio de Janeiro Cell Bank (BCRJ, # 0212), Brazil. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics. Cells were incubated at 37˚C in a humidified atmosphere containing 5% CO2.
The assessment of cell viability was performed using two methods: Alamar Blue™ and MTT, according to manufacturer’s instruction. Cells were seeded in 96-well microplates and challenged with increasing concentrations of ethanol extract (50, 100 and 200 µg/mL). After 48 h incubation (Alamar Blue) or 24 h (MTT), the absorbance was read at 540 nm.
LPS-induced TNF-α release inhibition
RAW 264.7 cells with or without 1 h extract pre-treatment (25 to 200 µg/mL) were stimulated with LPS (1 µg/mL) for 24 h. TNF-α concentration in the supernatants were quantified by ELISA (Invitrogen) according to manufacturer’s instruction. The results were presented as picograms per milliliter (pg/mL).
Protein extraction and western blot analysis
RAW 264.7 cells were incubated in the presence of different concentrations of the extract (25, 50 and 100 µg/mL) and 1 h later, LPS (1 µg/mL) was added. After 24 h of incubation, cells were washed twice with ice-cold PBS and incubated for 30 min on ice with lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 7.4, 0.1% Triton X100, and 0.1% SDS) supplemented with protease and phosphatase inhibitors. The protein concentration was measured using Lowry method. Fifty micrograms of protein were separated on 10% Tris–glycine SDS-Polyacrylamide gels and transferred onto nitrocellulose membranes using a semidry blotting apparatus. For western blot analysis, the membranes were blocked in 10% milk in TBS-Tween. After overnight incubation with primary antibodies (1/500) (p38α, p-p38α MAPK, NF-κB and β-actin) and 2 h incubation with horseradish peroxidase conjugated secondary antibodies (1/5000), proteins were visualized with the Opti-4CN colorimetric detection kit. Protein band intensities were quantified using the program ImageJ 1.48v.
Animal models of inflammation
The research was conducted in accordance with internationally accepted principles for laboratory animal use and care. All procedures with animals were approved by the Ethical Committee on Animal Research of the Federal University of Ceará, Brazil (CEPA # 71/2012). Male Wistar rats (120-160 g) were used in the study. Rats were kept in the Animal Care Facility with a 12 h light–dark cycle at constant temperature, with free access to water and chow during the study period. The paw edema models were assayed according to Coura et al. (2015) .
Carrageenan-induced paw edema assay
Animals (n=6/group) were given increasing doses (50, 100 and 200 mg/kg BW, for each 100 g of BW it was used 100 µL of DMSO) of M. plinioides extract subcutaneously (sc) into the back of each animal. Saline was used as internal control of the experiment, while the glucocorticoid dexamethasone (1 mg/kg, sc) was used as reference drug. Paw edema was induced by intraplantar injection of 100 μL carrageenan solution (700 μg/paw) into the back of the right hind paw after 1 h of drug administration. The volume of the right paw was measured using a plethysmometer and the results were presented as variations in the paw volume (mL), which were calculated relative to the basal volume (time 0).
Dextran-induced paw edema assay
Sterile saline (0.9%, 100 μL) or M. plinioides ethanol extract (50, 100 and 200 mg/kg BW) were sc administered into the back of the rats 1 h before dextran (500 μg/paw)-induced edema by injection into the right hind paw. Variations in the paw volume (mL) were calculated relative to the basal volume (time 0).
Inhibition of mast cell proteolytic activity
A suspension enriched in mast cells was obtained by peritoneal lavage of rats pre-stimulated with carrageenan. Mast cells were purified after centrifugation in 70% Percoll gradient solution as described by Kovarova et al. (2013). The mast cell suspension was macerated in PBS, centrifuged at 3000 g for 15 min and the protein extract obtained was 1:5 diluted in the same buffer and incubated with different concentrations of the extract (0-600 μg/mL). The residual activity of the trypsin-like enzymes released from mast cells was measured by the addition of a synthetic chromogenic substrate (BAPNA - L-benzoyl-arg-pNa). Kinetics of p-nitroaniline formation were monitored at 405 nm in 14 s intervals for 30 min. Results were expressed as the initial velocity of trypsin-like enzymes (mOD/min).
Effects of M. plinioides extract on hemostatic system
Human venous blood or rabbit, rat and bovine blood were collected from healthy subjects in 1:10 (v/v) 3.8 % trisodium citrate and centrifuged at 1500 X g for 10 min to obtain plasma. The anticoagulant activity of M. plinioides ethanol extract (0 – 1200 µg/mL) was verified by determination of the following coagulation parameters in plasma: recalcification time (RT), activated partial thromboplastin time (aPTT) and prothrombin time (PT). Evaluation of these parameters was performed using commercial kits following manufacturer’s instructions (Wiener Lab, Rosario, Argentina). The kinetic of all assays were conducted using a 96-well microplate spectrophotometer (SpectraMax 190, Molecular Devices Co., Sunnyvale, CA, USA) at 650 nm equipped with temperature control and shaking systems as previously described (Ribeiro, 1995). Results were expressed as coagulation time (measured in seconds).
Amidolytic activity of intrinsic pathway factors (FXIIa, FXIa and kallikrein) in plasma
The inhibitory capacity of M. plinioides ethanol extract on the amidolytic activity of factors XIIa, XIa and kallikrein was measured directly in the plasma using chromogenic substrates and specific inhibitors for each factor. Human plasma was treated with 2 mM acetic acid and diluted 1:10 in 20 mM Tris-HCl buffer, pH 7.5. Subsequently, 50 μL of the diluted plasma was incubated with M. plinioides extract (90 μg/mL) for 10 min at 37 °C and then the intrinsic factors were activated by the addition of 20 μL of ellagic acid. The residual XIa, XIIa and kallikrein activities produced during reactions in the presence or absence of the extracts were determined in three different experiments: (i) The activity of factor XIa was measured by the addition of 2 mM of the substrate S-2366 (pyroGlu- Pro-Arg-pNA) in the presence of aprotinin (50 μM) and a Bowman-Birk trypsin inhibitor (SBTI, 100 nM), (ii) the activity of factor XIIa formed was measured by the addition of 2 mM of substrate S-2302 (H-D-Pro-Phe-Arg-pNA) in the presence of aprotinin (50 μM), and (iii) the activity of the kallikrein formed was measured by adding S-2302 (2 mM) in the presence of SBTI (100 nM). In all cases the kinetics of p-nitroaniline formation were monitored (405 nm) at a time interval of 14 s during a total time of 30 min on the microplate reader spectrophotometer (SpectraMAX 190, Molecular Devices, Sunnyvale, CA, USA). Results are expressed as percentage of amidolytic activity.
Amidolytic activity of extrinsic pathway factors (FXa and thrombin) in plasma
The inhibitory capacity of M. plinioides ethanol extract on the amidolytic activity of FXa and thrombin was measured directly in plasma using specific chromogenic substrates and human plasma deficient for each factor. To measure the inhibitory capacity of the extract on factor Xa, prothrombin-deficient human plasma (50 μL) was activated with tissue factor (0.17 mg/mL) in the presence of CaCl2 (7 mM) and incubated for 10 min at 37 °C with M. plinioides extract (90 μg/mL). The amidolytic activity of factor Xa produced during incubation time was measured by addition of 2 mM of substrate S-2222 (Bz-Ile-Glu (γ-OR) -Gly-Arg-pNA). To measure the inhibitory capacity of the extract over thrombin, factor X-deficient human plasma was diluted 1:10 in 20 mM Tris-HCl buffer, pH 7.5. Then, 50 μL of the diluted plasma was activated with Oxyurannus scutellatus snake venom (10 μg/ mL) in the presence of CaCl2 (7 mM) and incubated for 10 min at 37 °C with the M. plinioides extract (90 μg/mL). The amidolytic activity of the thrombin produced during the incubation time was measured by adding 2 mM of the substrate S-2238 (H-D-Phe-Pip-Arg-pNA). In both cases the kinetics of p-nitroaniline formation were monitored (405 nm) at a time interval of 14 s during a total time of 30 min on the microplate reader spectrophotometer (SpectraMAX 190, Molecular Devices, Sunnyvale, CA, USA). Results were expressed as a percentage of amidolytic activity.
FVa activity in plasma
The inhibitory capacity of the M. plinioides extract on the activity of FVa was evaluated by the prolongation of PT in FV deficient plasma. For this purpose, the extract (90 μg/mL) were incubated with human normal plasma (50 μL) or factor V deficient plasma (50 μL) for 10 min at 37 °C. The extrinsic pathway was activated by the addition of rabbit brain thromboplastin (100 μL) in the presence of CaCl2 (10 mM) and phospholipids. The experiments were carried out as previously described, with reaction kinetics being monitored at 650 nm for 5 min with readings at time intervals of 5 s. The results are expressed as coagulation time in seconds and represent the mean ± standard error of three independent experiments. The absence PT prolongation in FV-deficient plasma indicates the presence of a specific inhibitor of factor Va.
FVIIa activity in plasma
The amidolytic activity of FVIIa was measured directly in the plasma using its specific chromogenic substrate. For this purpose, factor X deficient human plasma (50 μL) was activated with tissue factor (0.17 mg/mL) in the presence of CaCl2 (7 mM) and incubated for 10 min at 37 °C with M. plinioides extract (90 μg/mL). The amidolytic activity of FVIIa produced during incubation time was measured by the addition of 2 mM SCP-0248 substrate (MeSO2-Cha-Abu-Arg-pNA). The kinetics of p-nitroaniline formation were monitored (405 nm) at a time interval of 14 s during a total time of 30 min on the microplate reader spectrophotometer (SpectraMAX 190, Molecular Devices, Sunnyvale, CA, USA).
FXa and thrombin inhibition
The ability of M. plinioides extract to block FXa and thrombin activities was measured by prothrombin activation and thrombin-induced fibrinogen clotting, respectively. Briefly, purified FXa (3.4 μg/mL) was incubated with M. plinioides extract (0- 600 μg/mL) for 10 min at 37 °C. Purified prothrombin (34 μg/mL) was then added in the presence of CaCl2 (7 mM) and incubation was maintained for a further 20 min. Thrombin formed during the activation process was monitored by the addition of fibrinogen (2 mg/ml, final concentration) at 650 nm. For thrombin-induced fibrinogen clotting assay purified thrombin (2 μg/mL) was incubated with M. plinioides extract at different concentrations (0 - 900 μg/mL) for 10 min at 37 °C. Fibrin formation was monitored by the addition of fibrinogen (2 mg/ml) at 650 nm. Results were expressed as percentage of FXa and thrombin inhibition. IC50 values were determined by exponential nonlinear regression analysis.
All experiments were performed at least in triplicate and data were presented as mean ± SEM. Data were analyzed and graphed using Prism 5.0 (Graphpad Software, Inc). Statistical significance was determined using one-way ANOVA with Dunnett’s or Tukey’s correction for multiple comparisons or unpaired two-sided Student’s t-test. A P value <0.05 was considered statistically significant.