INTRODUCTION

A fermented wheat germ extract (FWGE, code name: MSC, trade name: FWGE ), standardized to and characterized by its methoxy-substituted benzoquinone contents and by its qualitative high-performance liquid chromatographic spectra, was developed in Hungary in the early 1990s. In some European countries, this orally applicable preparation has been approved as an over-the-counter medical nutriment (dietary food for special medical purpose) for cancer patients, because it has been demonstrated to have supportive therapeutic effects in the treatment of different human neoplastic diseases.1–5 Although the specific molecule or molecules of the extract responsible for its beneficial health effects has not been identified yet, the molecular targets of this dietary food for special medical purposes are, at least partially, known (FIG. 1).6–11 In a pilot clinical study it has been observed that FWGE, when administered together with anti-inflammatory drugs in patients with severe rheumatoid arthritis (RA), could significantly improve disease outcome.12 It was therefore decided to find out more of the underlying mechanisms by which this extract can set forth these positive effects in RA. Wistar rat adjuvant arthritis (AA) is still a relevant animal model of human RA13 and also a widely used chronic model for studying the anti-inflammatory properties of drugs.14 The onset of symptoms in the noninjected paw (e.g., “secondary” or “immune-mediated” response, which is considered to be the model of RA) occurs between the 8th–12th day and the disease approaches maximal intensity between days 16 and 21 after the injection of the adjuvant.15 The aim of our study was to investigate the mechanism of action of the FWGE in the rat AA model.

Animals

Female Wistar rats (weighing 200 ± 5 g) were used. The animals were kept in plastic cages (max. 5 rats per cage) under controlled illumination (lights on from 07:00–19:00 h), fed with standard rodent food pellets (LATI, G¨od¨oll¨o, Hungary) and given tap water ad libitum. The room temperature and the relative humidity were 23±1◦C and 50±5%, respectively. The experimental protocols had been approved by an institutional expert board (Ethical Committee for Preventing Abuse and Torture of Animals).

Induction and Evaluation of the Degree of AA

The rats were injected subcutaneously in the right hind footpad with 0.5 mg of heat-killed, desiccated, homogenized Mycobacterium butyricum (Difco, Detroit MI, USA) in 0.1 mL of liquid paraffin. Prior to injection of the adjuvant and then 1, 4, 7, 12, 14, 18, and 22 days later, the volumes of both (right = injected and left = noninjected) hind paws (the measure of the primary and secondary reaction, respectively) were determined by plethysmography with the animal under light ether anesthesia. The inhibition of inflammation was calculated by comparing the volumes of the legs to those of the control as 100%

Chemicals

FWGE (Biromedicina, Budapest, Hungary) in different doses, and positive controls (indomethacin [Chinoin-Sanofi, Budapest, Hungary], diclofenac-Na [Sigma-Aldrich, Budapest, Hungary], and dexamethasone phosphate [MSD, West Point WA, USA]) were administered orally (by gastric tube) twice a day (i.e., between 8:30 and 10:30 and between 16:30 and 18:30 h). FWGE and dexamethasone were suspended in distilled water, while indomethacin and diclofenac were suspended in 0.5% carboxymethylcellulose, and all treatments were administered at a dose of 1.0 mL/150 g body weight.

Experimental Design

In the first set of experiments two arms (A and B) were created. In Arm A FWGE, the positive controls, and placebo were administered per os 24 h prior to the adjuvant subplantar injection (in rats weighing 138 ± 5 g). In Arm B treatments were initiated 14 days prior to the adjuvant subplantar injection (in rats weighing 118 ± 5 g). Both arms consisted of seven groups: control (2 × 1 mL/150 g distilled water); FWGE 2 × 2.5; 2 × 1.0; 2 × 0.25 and 2 × 0.05 g/kg/day; indomethacin 2 × 0.5 mg/kg/day; dexamethasone 2 × 50.0 g/kg/day. Each experimental group consisted of 10–16 rats. Histologic examination was performed on rats sacrificed on day 21. In the second set of experiments (confirmatory study; rats weighing 130 ± 10 g) the study, was done in two steps (dose-finding for equal anti-inflammatory effects and, interactional). Dose-finding: control (2 × 1 mL/150 g distilled water); FWGE 2 × 2.5 and 2 × 1.0 g/kg/day; diclofenac 2 × 0.3; 2 × 1.0; 2 × 3.0; and 2 × 10.0 mg/kg/day; dexamethasone 2 × 1.0; 2 × 3.0; 2 × 10.0; and 2 × 30.0 g/kg/day. Each group consisted of 10 rats. On day 15, two rats of each group were sacrificed for histologic study. Interactional: control (2 × 1 mL/150 g distilled water); FWGE 2 × 2.5 g/kg/day; diclofenac 2 × 1.0 mg/kg/day; dexamethasone 2 × 15.0g/kg/day; FWGE 2 ×1.25 g/kg/day + diclofenac 2 × 0.5 mg/kg/day; FWGE 2 × 1.25 g/kg/day + dexamethasone 2 × 7.5 g/kg/day; FWGE 2 × 1.0 g/kg/day; diclofenac 2 × 0.4 mg/kg/day; dexamethasone 2 × 6.0 g/kg/day; FWGE 2 × 0.5 g/kg/ day + diclofenac 2 × 0.2 mg/kg/day; FWGE 2 × 0.5 g/kg/day + dexamethasone 2 × 3.0 g/kg/day. The rats were sacrificed by exsanguination under Nembutal narcosis on day 24 after the adjuvant injection.

Histologic Studies

The affected joints of the left hind footpad together with the epiphyses of the bones and the surrounding fibrous and muscular tissues were fixed in buffered neutral 4% formalin. Decalcification was performed using EDTA and the samples were embedded into paraffin. Eight m thin longitudinal sections were cut and the sections were stained with hematoxylin and eosin (H+E). In selected positive control and treated cases immunoperoxidase reaction was performed to demonstrate CD4+ and CD8+ T lymphocytes. The antibodies (Santa Cruz Biochemicals, Santa Cruz CA, USA) were applied at 1:100 dilution.

COX-Genes Expression

Real-time polymerase chain reaction (PCR) was carried out to determine gene expressions of COX-1 and COX-2. Whole blood was collected from the aorta into EDTA-containing tubes to prevent coagulation. The samples were lysed with red blood cell lysing buffer (Sigma-Aldrich) and total RNA was extracted from the remaining white blood cells using Tri-Reagent (SigmaAldrich) according to the manufacturer’s protocol. Purity of the nucleic acid templates was elevated by extrapurification with DNA-freeTM (Ambion, Austin TX, USA). One microgram of total RNA was reverse-transcribed from each sample using deoxy (d)-NTPs (0.5 mmol/L each) random primer (final concentration 3 mcM), RNasinTM ribonuclease inhibitor (Promega, Madison WI, USA) (0.4 U/L), DTT (final concentration 10 mM), reverse transcriptase buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, and 15 mM MgCl2) and M-MLV reverse transcriptase (Sigma-Aldrich) (200 U/reaction). Samples were incubated for 50 min at 37◦C and then at 85◦C for 10 min. Primers for quantitative PCR were designed using the Array Designer (Premier Biosoft International, Palo Alto CA, USA).

 The sequences of COX-1 primers were: 5 -GTC TGA TGC TCT TCT CCA CGA-3 and 5 -ATG AAC GGA TGC CAG TGA TAG A-3 . The sequences of COX-2 primers were: 5 -GTG GTG AAT GTA TGA GCA TAG GAT-3 and 5 -GTG TAG TAG GAG AGG TTG GAG AA-3 . The real-time PCR analysis for mRNA expression of COX-1 and COX-2 was standardized by coamplifying these genes with the housekeeping gene -actin (primers were: 5 -GTG GGG CGC CCC AGG CAC CCA-3 and 5 -GTC CTT AAT GTC ACG CAC GAT TTC-3 ). The real-time PCR reaction was run on the iCycler iQTM (Bio-Rad, Hercules CA, USA) using standard conditions: optimized concentration of primers (final concentration: 200 nM), IQTMSYBRTM Green Supermix (Bio-Rad) (100 mM KCl, 40 mM Tris-HCl, pH 8.4, 0.4 nM of each dNTP, 6 mM MgCl2, 50 U/mL iTaq DNA polymerase, SYBR Green I, 20 nM fluorescein) and 2 L cDNA. The starting quantity of gene expression in the sample was determined by comparison with a standard curve generated from a dilution series of template DNA (rat Wilms’ tumor, RWT) of known concentration (6.25e2, 1.25e2, 2.5e, 5.0, 1.0, where e is the base of the natural logarithm). A no-template control (containing water) was used as a negative control for every different primer pair. Relative gene expression was calculated by dividing the starting quantity of gene expression (normalized to the housekeeping gene) in treated (indomethacin, dexamethasone, and three different FWGE doses) rats by that in the untreated controls.

Statistical Analysis

Statistical analyses were performed by Prism 2.01 software (GraphPad, San Diego CA, USA). Differences in paw volume or body weight were analyzed with one-way analysis of variance (ANOVA) followed by Neuman–Keuls post hoc test. Differences were considered significant at P < 0.05. Data are given as means ± SE.

RESULTS

In the first set of experiments the effects of FWGE, indomethacin, and dexamethasone on the injected and noninjected paw volume, and histology of the joints were examined. Small oral doses of FWGE (2 × 0.05 and 2 × 0.25 g/kg/day) did not influence the progression of AA. However, treatment with higher dosages (2 × 1.0 and 2 × 2.5 g/kg/day) significantly suppressed the swelling and inhibited the development of the “secondary” (immune-mediated) response in the left paw. Treatments with positive control reference drugs like dexamethasone (2 × 50.0 g/kg/day) or indomethacin (2 × 0.5 mg/kg/day) also effectively suppressed the swelling and the appearance of the immunemediated response. Statistically significant anti-inflammatory activity of the FWGE was determined on days 14, 18, or 22 after the subplantar injection of Mycobacterium butyricum in the right hind paw. FIGURE 2 shows the effects of treatments on the noninjected paws (considered to be the model of immunemediated arthritis) on days 18 and 22 in arm A of the study, and on days 32 and 35 in arm B. It is important to note that days 32 and 35 in arm B correspond to days 18 and 22 (following the initiation of AA) in arm A, respectively. There were no statistically significant differences between the AA-inhibitory efficacies of the 24 h (arm A) or 14 day (arm B) pretreatments. The efficacies of treatments on the injected paws were also detected, but were not as obvious as on the noninjected paws (data are not shown). Histological examinations of the joints of the left hind paws in untreated control rats suffering from AA revealed severe inflammatory changes of the synovium and surrounding tissues (FIG. 3A). 

Combination of FWGE (1.25 g/kg/day) and diclofenac (0.5 mg/kg/day) resulted in a weak inflammatory reaction characterized only by scanty lymphocytic infiltration. However, 15 g/kg dexamethasone as a single therapy could only moderately decrease the inflammation, and the combination of dexamethasone (7.5 g/kg/day) and FWGE (1.25 g/kg/day) was even less effective (FIG. 7B). Lower doses of FWGE (1.0 g/kg/day), dexamethasone (6 g/kg/day) and also of diclofenac (0.4 mg/kg/day) showed moderate anti-inflammatory efficacies and so did the combination of FWGE (0.5 g/kg/day) and dexamethasone (3.0 g/kg/day). The combination of FWGE (0.5 g/kg/day) and diclofenac (0.2 mg/kg/day), however, decreased the inflammation to a minimum level (FIG. 7C).

DISCUSSION

As far as we know, this is the first report on a wheat-derivative with experimental anti-inflammatory efficacy. In the first set of our experiments it was shown that FWGE had potent anti-inflammatory properties, which are likely to be mediated by cyclooxygenase inhibition.