Male 7-week-old Balb/c mice weighing 18–25 g were purchased from Velaz (Prague, Czech Republic). The animals were maintained under standard conditions of temperature (21±1°C), relative humidity (55±10%), and 12 hours/12 hours light/dark cycle. All mice were housed in specific pathogen-free conditions. All animal experimentations were reviewed and approved by the Ethical Committee of the Institute of Animal Physiology.

After a period of adaptation, weight-matched animals were randomized into five groups: group A (20.2±0.971 g), group B (20.6±0.75 g), and group C (20.71±0.844 g), mice with TNBS-induced colitis treated with three different doses (see Table 1) of thyme and oregano oil combination; group D (21.03±1.025 g), mice with TNBS-induced colitis; group E (18.74±1.189 g), sham-treated mice.

Thyme aromatic oil (Thymi aetheroleum-Ph.Eur. 4) and oregano aromatic oil (Origani aetheroleum) were purchased from Calendula, (Nová L'ubovňa, Slovakia; thyme aromatic oil: lot 5-015-003-10-04; oregano aromatic oil: lot 5-027-007-10-04). The thyme aromatic oil contained about 48% of. p-cymene and 24% of thymol, and the oregano aromatic oil contained about 55% of carvacrol. 2,4,6-trinitrobenzene sulphonic acid (TNBS) was purchased from Fluka Chemie (Buchs, Switzerland). The thyme oil and oregano oil were mixed with the diet at concentrations as shown in Table 1. Both aromatic oils were suspended in edible soya oil (Brölio, Hamm, Germany) and added to powdery commercial rodent diet (diet for laboratory mice and rats SPF, M1; Frantisek Machal, Ricmanice, Czech Republic). In the TNBS and sham groups, edible soya oil was mixed with the powdery rodent diet at a concentration of 1% (wt/wt). Diets were fed ad libitum throughout the experiment, starting 6 days before administration of TNBS.

The mice were anesthetized with ketamine and xylazine, and colitis was induced by intrarectal administration of 120 mg/kg of the hapten reagent TNBS (Fluka Chemie) in 50% ethanol, and they were then kept in a vertical position for 30 seconds. The sham group received 50% ethanol alone using the same technique. The total injection volume was 30 μL. Development of colitis was assessed daily by measurement of body weight. The mortality rate was observed during this study. The mice were killed by cervical dislocation 7 days after TNBS administration. The colons were removed, cut longitudinally, and cleared of fecal material with gentle spray of 0.9% saline solution. The extent of mucosal damage was assessed using the colon macroscopic scoring system adapted from Wallace et al. [33]. Ulceration : (1) focal hyperemia, no ulcer; (2) ulceration, no hyperemia/bowel wall thickening; (3) ulceration, inflammation at one site; (4) ulceration, inflammation at 2 or more sites; (5) major injury > 1 cm; 6–10 major damage > 2 cm. Adhesion: (1) minor (colon easily separated from other tissue); (2) major. Diarrhea: (1); Bowel wall thickening: (1). Representative samples from each experimental group were histologically evaluated. Colon tissues were fixed in 4% formalin in 0.1 M phosphate buffer, dehydrated with increasing concentrations of ethanol, embedded in paraffin, and sectioned. Sections (4–6 μm thick) were mounted on slides, cleared, hydrated, and stained with hematoxylin and eosin. The slides were examined and photographed with an Olympus BX51 microscope (Olympus, Japan). Strips of colonic tissue (15–30 mg from segments most intensively affected by the inflammation) were cut out, immersed in liquid nitrogen, and kept at −70°C until the cytokine measurement.

Total RNA was isolated from the mouse colon (about 15 mg of tissue for each sample) with TRIzol reagent (Invitrogen Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions. Total RNA preparations were then cleaned and DNase I was treated with RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. In order to quantify total RNA extracted from each sample, optical density at 260 nm was measured. The integrity of the RNA was assessed by denaturing agarose gel electrophoresis.

The RNA (0.75 μg from each sample) was reverse transcribed at 42°C for 1 hour in 30 μL containing 300 units of Superscript II Rnase H^-reverse transcriptase (Invitrogen Life Technologies), 7 μM anchored oligo dT13VN, 50 mM Tris-HCl pH 8.3, 3 mM MgCl₂, 75 mM KCl, 10 mM DTT, 500 μM dNTPs (dATP, dTTP, dCTP, dGTP), 60 units RNase OUT (recombinant ribonuclease inhibitor, Invitrogen Life Technologies), and 0.75 μg acetylated BSA. The reaction was terminated by heating at 95°C for 5 minutes. To check for the presence of genomic DNA contamination in the RNA preparations, reverse transcriptase negative control (no reverse transcriptase in the reaction) was carried out in parallel, using RNA pool prepared from aliquots of all RNA samples. The pool of colon RNA obtained from aliquots of all samples served as standard RNA. The relative standard curve was generated using Mx 3000P 2.0 software (Stratagene, La Jolla, Calif).

PCR reactions were carried out in a 20 μL final volume in duplicates using SYBRGreen I as a fluorescent detection dye. The reactions contained 0.8 μL of cDNA (corresponding to 20 ng of sample total RNA), one unit of platinum Taq DNA polymerase (Invitrogen Life Technologies), SYBRGreen I in final dilution of 1 : 25000 (Sigma-Aldrich, Munich, Germany), 30 nM ROX (passive reference dye for correction of non-PCR-related fluctuations in fluorescence signal, Stratagene), 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl₂(except for the β-actin reaction, where 1.5 mM MgCl₂ was used), forward and reverse primers in final concentration of 0.25 μM (for IL-1β, IL-12b, TNFα, IFN-γ, HPRT, and SDHA), or 0.5 μM (for IL-6, IL-10, GM-CSF, and β-actin; see Table 2 for full gene names). Oligonucleotide primers used in the experiment were designed in our previous work [34], and their sequences are shown in Table 2. PCR amplification was performed in the real-time PCR system Mx 3000P (Stratagene). After an initial step at 95°C for 2 minutes (DNA denaturation and hot-start DNA polymerase activation), 40 cycles with the following thermocycling conditions were carried out: 94°C for 30 seconds, specific annealing temperature for 30 seconds, 72°C for 30 seconds, and specific temperature at which the fluorescence was acquired (“acquiring temperature”) for 30 seconds. Measurement of fluorescence at an elevated temperature (“acquiring temperature,” a few degrees of Celsius below the melting temperature of the specific PCR product) enables elimination of the fluorescence signal produced by incidental short nonspecific PCR products. Amplification specificity was then checked by generation of a melting curve using 41 cycles with temperature increments of 1°C (starting with 55°C) and a fluorescence measurement in each cycle. Specific annealing and acquiring temperatures are shown in Table 2.

To ensure the correctness of the quantification, we normalized cytokine expression to the expression of three housekeeping genes. Firstly, expression stability of several housekeeping genes was tested using geNorm software [35]. Subsequently, the normalization factor for each sample was calculated (by geNorm software) as the geometric means of the relative amounts of the three most stable housekeeping genes—HPRT, SDHA, and β-actin (see Table 2 for full gene names). Finally, the relative amount of cytokine mRNA in each sample was divided by the normalization factor of the sample.

Colon tissue samples were homogenized in ice-cold PBS containing protease inhibitor cocktail for use with mammalian cell and tissue extracts (P8340, Sigma-Aldrich), and the homogenates were then centrifuged at 12000 xg at 4°C for 15 minutes. Total protein amounts in the tissue supernatants were determined using Bradford protein assay [36] with BSA employed as the standard. IL-1β and IL-6 amounts were determined using an ELISA kit, according to the manufacturer's recommendation (Pierce-Endogen, Rockford, Ill, USA).

The Kruskal-Wallis test and the Mann-Whitney U test were used for the estimation of macroscopic damage scores. The chi-square test was used to assess differences in mortality rate. The Student t test was used for the comparison of differences in body weight. The Kruskal-Wallis test was used for the comparison of differences in cytokine expression between groups and the Mann-Whitney U test was used to compare differences between the group of untreated colitic animals and other groups of animals. Values of p<.05 were considered as significant.

As shown in Figure 1, administration of TNBS caused a dramatic decrease in body weight (almost 20% after 3 days); body weight was recovered gradually from day 4 but not fully to the initial weight in day 7. Mice receiving 50% ethanol without TNBS (control sham group) showed only slight and transient loss of body weight. In mice of group B (colitic animals treated with 0.2% thyme and 0.1% oregano oils), body weight was recovered gradually from day 3 when it became higher than the body weight of untreated colitic animals (group D); on day 7, the body weight of animals in group B reached a level near to that of the control mice (sham group). The body weight of mice in group A (colitic animals treated with 0.4% thyme and 0.2% oregano oils) and group C (colitic animals treated with 0.1% thyme and 0.05% oregano oils) did not differ significantly from that of untreated colitic animals (group D).

The mortality rate of mice with TNBS-induced colitis (group D) was 53.3%, while that of the control sham group was 0% (see Figure 2). The mortality rate in group A (colitic animals treated with 0.4% thyme and 0.2% oregano oils) was 50% and in group C (colitic animals treated with 0.1% thyme and 0.05% oregano oils) was 62.5%, which are comparable to that found in group D. Treatment with the combination of 0.2% thyme and 0.1% oregano oils (group B) decreased the mortality rate to 33.3% (which was still not significantly different from that of mice with TNBS-induced colitis).

Macroscopic damage scores of mice in group D were significantly higher than those of mice in the control sham group. Treatment with the combination of 0.2% thyme and 0.1% oregano oils (group B) significantly lowered the macroscopic damage scores in comparison to untreated colitic animals (group D). Animals in groups A and C showed no significant changes in macroscopic damage scores compared with mice in group D (see Figure 3).

Representative samples of colon after hematoxylin and eosin stainings are shown in Figure 4. There is no evident histological modification in sham mice (E). In mice with TNBS-induced colitis (D), there is a wide range of histopathological changes including necrosis of epithelium, destruction of glands, and infiltration of inflammatory cells in the mucosa and submucosa (up to 50% of colon section). The samples from mice treated with 0.2% thyme and 0.1% oregano oils (B) show intermediary histopathological changes (up to 30% of colon section).

Relative amounts of IL-1β, IL-6, GM-CSF, and TNFα mRNAs (for full gene names, see Table 2) were significantly higher in animals with TNBS-induced colitis (group D) than in the control sham-treated animals (see Figure 5). Treatment of the colitic animals with the combination of thyme and oregano oils significantly lowered the amount of IL-1β mRNA, using all three tested doses of the aromatic oils (see Figure 5). The amount of IL-6 mRNA in group B (colitic animals treated with 0.2% thyme and 0.1% oregano oils) was significantly lower than that in the group D, whereas the decrease of IL-6 mRNA level in groups A and C (colitic animals treated with 0.4% thyme and 0.2% oregano oils or with 0.1% thyme and 0.05% oregano oils) was not statistically significant (see Figure 5). A similar effect was found for GM-CSF and TNFα but the difference between group B and group D did not reach statistical significance (p=.064 and p=.053, resp.; see Figure 5). We found no significant changes in mRNA levels of two other cytokines (IL-10 and IFNγ, data were not shown).

As shown in Figure 6, the amounts of IL-1β and IL-6 proteins were significantly higher in the animals with TNBS-induced colitis (group D) than in the control sham group. The concentrations of these proteins were significantly reduced in group B (colitic animals treated with 0.2% thyme and 0.1% oregano oils) compared to group D. The levels of IL-1β and IL-6 proteins in groups A and C (colitic animals treated with 0.4% thyme and 0.2% oregano oils or with 0.1% thyme and 0.05% oregano oils) did not differ significantly from those found in group D (see Figure 6).