The Effect of Stinging Nettle ( Urtica dioica ) Seed Oil on Experimental Colitis in Rats

Abstract

This study investigated the effect of Urtica dioica, known as stinging nettle, seed oil (UDO) treatment on colonic tissue and blood parameters of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. Experimental colitis was induced with 1 mL of TNBS in 40% ethanol by intracolonic administration with a 8-cm-long cannula with rats under ether anesthesia, assigned to a colitis group and a colitis+UDO group. Rats in the control group were given saline at the same volume by intracolonic administration. UDO (2.5 mL/kg) was given to the colitis+UDO group by oral administration throughout a 3-day interval, 5 minutes later than colitis induction. Saline (2.5 mL/kg) was given to the control and colitis groups at the same volume by oral administration. At the end of the experiment macroscopic lesions were scored, and the degree of oxidant damage was evaluated by colonic total protein, sialic acid, malondialdehyde (MDA), and glutathione levels, collagen content, tissue factor activity, and superoxide dismutase and myeloperoxidase activities. Colonic tissues were also examined by histological and cytological analysis. Pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), lactate dehydrogenase activity, and triglyceride and cholesterol levels were analyzed in blood samples. We found that UDO decreased levels of pro-inflammatory cytokines, lactate dehydrogenase, triglyceride, and cholesterol, which were increased in colitis. UDO administration ameliorated the TNBS-induced disturbances in colonic tissue except for MDA. In conclusion, UDO, through its anti-inflammatory and antioxidant actions, merits consideration as a potential agent in ameliorating colonic inflammation.

Introduction

U lcerative colitis is a chronic inflammatory bowel disease (IBD) of the large bowel. Although the etiology of ulcerative colitis remains unknown, it is believed that exaggerated intestinal immune response plays a key role in the pathophysiology of this intestinal disorder. Excessive production of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, IL-8, leukotriene B₄, and platelet-activating factor, the presence of highly activated inflammatory cells such as neutrophils, monocytes, and macrophages, and excessive production of reactive oxygen species are common characteristics of this disease.^1,2

Of the several animal models of intestinal inflammation, the well-characterized haptene reagent trinitrobenzene sulfonic acid (TNBS)-induced colitis resembles human ulcerative colitis in its various histological features, including infiltration of colonic mucosa by neutrophils and macrophages and increased production of inflammatory mediators, including the type 1 helper cell profile of cytokines.³ Treatment with anti-inflammatory or antioxidant agents has been shown to ameliorate the disease symptoms.^4
–6 Steroid hormones, immunosuppressive agents, or salicylic acid derivatives are used to treat IBD with modest results and often with serious side effects.⁷

Urtica L. stinging nettles (Family Urticaceae) are annual and perennial herbs, distinguished with stinging hairs. Among Urtica species, Urtica dioica and Urtica urens have already been known and therefore consumed for a long time as medicinal plants in many parts of the world. The leaf, flower, seed, and root of nettle contain different chemical constituents. Allergic rhinitis, hypertension, gout, hair loss, and mild bleeding (particularly mild menorrhagia) are some of the traditional indications for nettle leaf. Stinging hairs contain histamine, formic acid, acetylcholine, acetic acid, butyric acid, leukotrienes, 5-hydroxytryptamine, and other irritants.^8
–10 In vitro and in vivo studies on nettle extracts have shown a mild anti-inflammatory effect, which may also be effective for treating certain individuals with allergic rhinitis.^11,12 The water extract of nettle has been demonstrated to be a powerful antioxidant against various oxidative systems in vitro, due to phenolic compounds; moreover, this extract had antimycotic and antibacterial activity against Staphylococcus aureus, one of the most common Gram-positive bacteria causing food poisoning.¹³ The biological activities of nettle leaves are assigned to the flavonoidic fraction: the methanolic extract of the aerial parts, containing quercetin and isorhamnetin glycosides, had an immunostimulatory activity on neutrophils, suggesting that it could be useful in treating patients suffering from neutrophil function deficiency and chronic granulomatous diseases; the aqueous methanolic extract of nettle roots has been used in clinics in Europe for the treatment of prostatic hyperplasia; and the seeds and aqueous extract of the aerial parts of U. dioica L. have been occasionally used in Turkey as an herbal medicine by cancer patients.¹⁴ Furthermore, nettle leaves have demonstrated antiplatelet action, useful in the treatment and/or prevention of cardiovascular disease.¹⁵

Materials and Methods

Animals

Both sexes of Wistar albino rats (weighing 200–250 g) were kept in a light- and temperature-controlled room with 12:12-hour light–dark cycles, where the temperature (22±0.5°C) and relative humidity (65–70%) were kept constant. The animals were fed a standard pellet, and food was withheld overnight before colitis induction. Access to water was allowed ad libitum. Experiments were approved by the Marmara University (Istanbul, Turkey) School of Medicine Animal Care and Use Committee.

U. dioica oil fatty acid analysis

A fatty acid analysis of U. dioica oil (UDO) (manufactured by Talya, Antalya, Turkey) was performed at Department of Pharmacognosy, School of Pharmacy, Anadolu University, Eskisehir, Turkey, by the gas chromatography technique.

Induction of colitis and UDO administration

Animals were fasted for 18 hours before the induction of colitis. With the animal under light ether anesthesia, a polyethylene catheter (PE-60) was inserted into the colon with its tip positioned 8 cm from the anus. To induce colitis (n=6), a single solution of 1 mL of a 30 mg/mL TNBS solution, dissolved in 40% ethanol in saline, was instilled. The rats in the control group (n=5) were subjected to the same procedure with the exception that an equal volume of isotonic saline was substituted for TNBS solution. UDO or saline (2.5 mL/kg) was given orally 5 minutes later than the induction of colitis, and the treatment was continued for the following 3 days. On the fourth day of colitis induction, rats were decapitated, and trunk blood was collected for the assessment of cholesterol, triglyceride, lactate dehydrogenase (LDH) activity, as a marker of tissue injury, and levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. The distal 8 cm of the colon obtained from each animal was initially examined for recording macroscopic damage scores and then stored at −80°C until the determination of collagen content (a fibrosis marker), myeloperoxidase (MPO) activity, an indirect evidence of neutrophil infiltration, malondialdehyde (MDA), an index of lipid peroxidation, glutathione (GSH), a key antioxidant, superoxide dismutase (SOD), an antioxidant, sialic acid (SA), one of the markers of membrane integrity, tissue factor (TF) activity, one of the markers of membrane integrity, and total protein (TP) levels in the colonic tissue.

Assessment of colitis severity

From each animal, the distal 8 cm of the colon was opened longitudinally down its mesenteric borders, cleansed of luminal contents, gently rinsed in saline, and dried on filter paper. The severity of colitis was assessed using macroscopic and microscopic damage scoring and tissue collagen content. For macroscopic scoring of colonic damage, the 8-cm colonic segments were scored according to the following criteria: 0, no damage; 1, localized hyperemia, no ulcers; 2, ulceration without hyperemia or bowel wall thickening; 3, ulceration with inflammation at one site; 4, two or more sites of ulceration/inflammation; 5, major sites of damage extending more than 1 cm along the length of colon; and 6–10, damage extending more than 2 cm along the length of colon, where the score is increased by one for each additional 1 cm.¹⁶

For light microscopic analysis, samples from distal colon were fixed in 10% buffered formalin for 48 hours, dehydrated in an ascending alcohol series, and embedded in paraffin wax. Approximately 7-μm-thick sections were stained with hematoxylin and eosin for general morphology. Stained sections were observed under an Olympus (Tokyo, Japan) model BX50 photomicroscope. Assessment of the colonic injury was performed by using the previously described criteria: damage/necrosis (0, none; 1, localized; 2, moderate; 3, severe), submucosal edema (0, none; 1, mild; 2, moderate; 3, severe), inflammatory cell infiltration (0, none; 1, mild; 2, moderate; 3, severe), vasculitis (0, none; 1, mild; 2, moderate; 3, severe), and perforation (0, absent; 1, present), with a maximum score of 13.¹⁷ All tissue samples were evaluated in a blind fashion by two experienced histologists.

Tissue collagen measurement

Tissue samples were cut with a razor blade, immediately fixed in 10% formaldehyde, and then embedded in 0.1 M of paraffin to obtain approximately 15-μm-thick sections. The method of collagen measurement is based on the selective binding of Sirius red and Fast Green to collagen and noncollagenous components, respectively, when the sections are stained with both dyes dissolved in aqueous saturated picric acid.¹⁸ Both dyes were eluted readily and simultaneously with NaOH–methanol, and the absorbances obtained at 540 and 605 nm were used to determine the amounts of collagen and protein, respectively.

Cytological examinations

Colonic tissue samples were smeared over a glass microscope slide and fixed with air. Then they were stained with Giemsa stain¹⁹ and microscopically examined (×100) for showing the presence of necrosis, amorphous and fibrous structures, and leukocyte, bacterium, and fat cell counts.

Biochemical analyses

Measurement of serum LDH activity and cytokine levels

Serum LDH was determined spectrophotometrically using an automated analyzer,²⁰ and levels of TNF-α, IL-1β, and IL-6 were quantified using enzyme-linked immunosorbent assay kits specific for the previously mentioned rat cytokines according to the manufacturer's instructions and guidelines (Biosource International, Camarillo, CA, USA). These particular assay kits were selected because of their high degree of sensitivity, specificity, inter- and intraassay precision, and small amount of plasma sample required for conducting the assay.

Tissue MPO activity

The activity of tissue-associated MPO, a natural constituent of primary granules of neutrophils, was determined in the colonic samples.²¹ The tissue samples (0.2–0.3 g) were homogenized in 10 volumes of ice-cold potassium phosphate buffer (50 mM K₂HPO₄, pH 6.0) containing hexadecyltrimethylammonium bromide (0.5%, wt/vol).

The homogenate was centrifuged at 41,000 g for 10 minutes at 4°C, and the supernatant was discarded. The pellet was then rehomogenized with an equivalent volume of 50 mM K₂HPO₄ containing 0.5% (wt/vol) hexadecyltrimethylammonium bromide and 10 mM EDTA (Sigma, St. Louis, MO, USA). MPO activity was assessed by measuring the H₂O₂-dependent oxidation of o-dianisidine dihydrochloride. One unit of enzyme activity was defined as the amount of the MPO present per gram of protein that caused a change in absorbance of 1.0/minute at 460 nm and 37°C.

Tissue MDA and GSH assays

The MDA levels were measured by the method of Ledwozyw et al. ²² for products of lipid peroxidation. Results were expressed as nanomoles of MDA per milligram of protein. GSH was determined by the spectrophotometric method using Ellman's reagent,²³ and the results were expressed as milligrams of GSH per gram of protein.

TF activity

TF activity of colon tissues was evaluated according to Quick's one-stage method using normal plasma.²⁴ This was performed by mixing 0.1 mL of colon homogenate with 0.1 mL of plasma, with the clotting reaction being started on addition of 0.02 M CaCl₂. All reagents were in the reaction temperature (37°C) before admixture. TF activity was expressed as seconds. Shortened clot formation time shows increased TF activity.

SOD activity

SOD activity in the colon samples was measured according to the previously described method.²⁵ In brief, measurements were performed in cuvettes containing 2.8 mL of 50 mM potassium phosphate (pH 7.8) with 0.1 mM EDTA, 0.2 mM riboflavin in 10 mM potassium phosphate (pH 7.5), 0.1 mL of 6 mM o-dianisidin, and tissue extract. Cuvettes with all their components were illuminated with 20-W Sylvania Gro Lux (Osram GmbH, Munich, Germany) fluorescent tubes that were placed 5 cm above and to one side of cuvettes maintaining a temperature of 37°C. Absorbance was measured at 460 nm, and the result was expressed in units of SOD per milligram of protein.

Total SA and TP levels

To determine total SA levels, the colon homogenate was first incubated at 80°C for 1 hour in diluted sulfuric acid in order to liberate bound SA, and the method of Warren²⁶ was then applied. This method consisted of oxidizing SA with periodate; the reaction was terminated by addition of arsenite and then adding thiobarbituric acid. This resulted in the formation of a red substance extracted in cyclohexanone. Its absorbance was read at 549 nm, and the result was expressed in milligrams of SA per gram of protein.

TP level was determined by the method of Lowry et al.,²⁷ using bovine serum albumin as a standard, reading absorbance at 500 nm, and expressing the TP level in milligrams of protein.

Statistics

All data were expressed as mean±SEM values. Statistical analysis was carried out using the Instat statistical package (GraphPad Software, San Diego, CA, USA). Following the assurance of normal distribution of data, groups of data were compared with one-way analysis of variance followed by the Tukey–Kramer post hoc test for multiple comparisons. Values of P<.05 were regarded as significant.

Results

The groups were checked for differences in weight at the beginning and at the end of the experiment, but no differences were found.

UDO fatty acid composition analyses

Table 1 shows the fatty acid composition of UDO. According to the fatty acid analysis palmitic (C_16:0), oleic (C_18:1n-9), and linoleic (C_18:2n-6) acids are the major fatty acids in UDO.

Table 1.

Fatty Acid Composition of U. dioica Oil

Content	%
Myristic acid (14:0)	0.1
Palmitic acid (16:0)	12.7
Palmitoleic acid (16:1)	0.2
Margaric acid (17:0)	0.1
Stearic acid (18:0)	2.9
Oleic acid (18:1)	22.5
Linoleic acid (18:2)	56.4
Linolenic acid (18:3)	0.2
Arachidonic acid (20:0)	0.5
11-Eicosenoic acid (20:1)	0.6
Eicosadienoic (20:2)	3.1
Saturated	16.3
Unsaturated	83.0
Unsaturated/saturated	5.1
Total	99.3

Blood parameters

Serum TNF-α, IL-1β, IL-6, cholesterol, and triglyceride levels and LDH activity significantly increased in colitis. UDO significantly reversed all these parameters (Table 2).

Table 2.

Serum Tumor Necrosis Factor-α, Interleukin-1β, Interleukin-6, Cholesterol, and Triglyceride Levels and Lactate Dehydrogenase Activity

Parameter	Control (n=5)	Colitis (n=6)	Colitis+UDO (n=7)	P (ANOVA)
TNF-α (pg/mL)	6.68±1.06	45.18±9.47^***	16.75±2.89^†††	.001
IL-1β (pg/mL)	10.9±1.7	62.1±6.2^***	25.9±5.7^†††	.001
IL-6 (pg/mL)	23.2±3.5	81.9±8.7^***	32.5±7.3^†††	.001
LDH (U/L)	1,077±149	2,935±209^***	1,567±216^†††	.001
Cholesterol (mg/dL)	148±11	207±9^*	150±15^†	.001
Triglyceride (mg/dL)	155±13	221±13^**	163±12^†	.001

Data are mean±SE values.

P<.05, ^** P<.01, ^*** P<.001 compared with the control group; ^† P<.05, ^††† P<.001 compared with the colitis group.

ANOVA, analysis of variance; IL, interleukin; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor-α; UDO, U. dioica seed oil.

Severity of colonic injury

Macroscopic scoring

Compared with the intact colonic tissue of the control group, intracolonic administration of TNBS increased the macroscopic damage score, whereas UDO significantly reduced the score (Table 3).

Table 3.

Colonic Tissue Total Protein, Lipid Peroxidation, Glutathione, Sialic Acid, and Collagen Levels and Tissue Factor, Superoxide Dismutase, and Myeloperoxidase Activities

Parameter	Control (n=5)	Colitis (n=6)	Colitis+UDO (n=7)	P (ANOVA)
TP (mg/g of tissue)	58.70±3.32	61.32±1.89	53.27±1.63	.0603 (NS)
TF activity (seconds)	38.40±0.93	24.67±1.17^**	32.83±3.34^†	.0026
LPO (nmol of MDA/mg of protein)	0.41±0.03	0.59±0.02^**	0.63±0.01^**	.0001
GSH (mg/g of protein)	1.74±0.07	1.38±0.09^*	1.88±0.11^††	.0050
SOD (U/mg of protein)	0.56±0.03	0.44±0.02^**	0.54±0.02^†	.0037
MPO (U/mg of protein/minute)	4.37±0.26	12.80±0.46^**	8.49±0.89^** ††	.0001
SA (mg/g of protein)	7.02±0.2	10.31±0.43^**	8.56±0.12^** ††	.0001
Collagen (μg/mg of protein)	7.18±0.50	11.03±0.34^**	5.6±0.79^††	.0001
Macroscopic score	0.20±0.09	7.17±0.60^**	3.20±0.97^** †	.0001

Data are mean±SE values.

P<.05, ^** P<.01 compared with the control group; ^† P<.05, ^†† P<.01 compared with the colitis group.

GSH, glutathione, LPO, lipid peroxidation; MDA, malondialdehyde; MPO, myeloperoxidase; NS, nonsignificant; SA, sialic acid; SOD, superoxide dismutase; TF, tissue factor (shortened clot formation time shows increased TF activity); TP, total protein.

Microscopic evaluation

Light microscopic evaluation of the control group demonstrated well-designated, regular epithelial lining with abundant goblet cells containing mucus (Fig. 1A). In the colitis group, severe necrosis in the colonic epithelium, partial loss of tubular glands, and intense staining of leukocytes at the submucosa were observed (Fig. 1B). UDO administration regenerated surface and gland epithelium of colonic tissue, but there was still intense staining of leukocytes at the submucosa (Fig. 1C).

FIG. 1.

Micrographs illustrate the histological appearances of colonic tissues in different experimental groups. (A) Control group. Surface (arrow) and gland epithelium (arrowhead) and cells of the gland epithelium (*). Hematoxylin and eosin, ×200. (Inset) ×400. (B) Colitis group. Severe leukocytic infiltration in the submucosa (arrow), high-grade necrosis (arrowhead), prominent desquamation (*), necrotic area, and gland tissue (*). Hematoxylin and eosin, ×200. (Inset) ×400. (C) Colitis+UDO group. Intensive erythrocytic and leukocytic infiltration in the submucosa (arrowhead), ameloration of the surface (arrow) and gland epithelium (*), and inflamation in the capillaries (double-headed arrow). Hematoxylin and eosin, ×200.

Cytological examinations

Epithelial cells, amorphous structure, few leukocytes, fat cells, and collagen fibers were observed in the control group (Fig. 2A). In comparison with the control group, the reduction in amorphous structure was more pronounced in the colitis group. Also, the increases in leukocytes and collagen fibers were prominent in the colitis group (Fig. 2B). In the UDO-administered colitis group, the density of collagen fibers and leukocytes were decreased, and amorphous structure was found to be increased (Fig. 2C).

FIG. 2.

Appearance of colon tissue in different experimental groups. (A) Control group. In colon tissue, a few amorphous structure (*), epithelial cells (arrowhead) and leukocytes (arrow) were observed. May Grünwald–Giemsa, ×400. (B) Colitis group. In colon tissue, severe collagen fibers (arrows) and leukocytes (arrowhead). May Grünwald–Giemsa, ×400. (C) Colitis+UDO group. In colon tissue, increased amorphous structure (*), a few and irregular collagen fibers (arrow), and leukocytes (arrowhead) were observed. May Grünwald–Giemsa, ×400.

Tissue collagen content

The collagen content in the colonic tissue of the colitis group was markedly increased compared with the control group. UDO administration significantly decreased the collagen content (Table 3).

Colonic tissue parameters

TF (shortened clot formation time shows increased TF activity) and MPO activities, LPO, and SA level significantly increased, whereas GSH level and SOD activity significantly decreased in the colitis group. UDO administration to the colitis group significantly decreased TF and MPO activities and SA level and significantly increased SOD activity and GSH level (Table 3).

Discussion

The results of the present study demonstrate that daily UDO administration to the rats markedly improves TNBS-induced colonic lesions, as confirmed by macroscopic examination and biochemical assays. In the present study, consistent with other studies,^28,29 serum TNF-α, IL-1β, and IL-6 increased in the TNBS-induced colitis model. This inflammatory status has been reversed by UDO administration.

Pro-inflammatory cytokines play a key role in the pathophysiology of IBD,²⁹ and anti-TNF-α antibodies are therapeutically used in some severe forms of IBD.³⁰ Many plant extracts have been occasionally used in Turkey as an herbal medicine for the treatment of IBD. UDO is one of these herbal medicines.

Several mechanisms of action for U. dioica have been discussed. This includes the suppression of cytokine production via an inhibition of nuclear factor-κB activation by nettle leaf extract.¹¹ In IBD models, the inhibition of nuclear factor-κB or the pro-inflammatory cytokine TNF-α by an antisense oligonucleotide strategy led to an improvement in the disease.³¹ Moreover, Roschek et al.³² have reported that the nettle extract has broad in vitro anti-inflammatory activities and that the synergistic interactions of the many functional bioactive compounds present in this nettle extract address multiple steps in the pro-inflammatory cascade. Increased SA concentrations have been reported during inflammatory processes, probably resulting from increased levels of richly sialylated acute-phase glycoprotein.³³ SA is the generic term for a family of acetylated derivatives of neuraminic acid. They are suggested to be major participants in many biological functions. In accordance with the above finding, the present study showed an increased colonic SA level in the colitis group. UDO administration significantly decreased the colonic SA level. From this point of view, UDO showed its anti-inflammatory effect in blood, and this result is reflected in the colonic SA level.

A role for MPO-derived oxidants in the development of tissue injury has been suggested in IBD. Considerable neutrophil accumulation in conjunction with increased MPO activity is seen in intestinal mucosal lesions of patients with IBD. Results from animal models further support the contention that neutrophils contribute to tissue injury in IBD.³⁴ In our observation, elevated MPO levels in colonic tissues indicate that neutrophil accumulation contribute to the colitis-induced oxidative injury, and UDO appears to have a preventive effect through the inhibition of neutrophil infiltration. It was previously demonstrated that U. dioica leads to the release of nitric oxide, which then inhibits the adhesion and aggrevation of neutrophil leukocytes.³⁵ The decrease in neutrophil recruitment in response to U. dioica could be mediated by nitric oxide. In the present study, increased colonic MPO activity in the colitis group supports the notion that the neutrophils are at least one source of oxidants. Stimulated polymorphonuclear leukocytes and macrophages as well as peroxisomes appear to be the main endogenous sources of most of the oxidants produced. As leukocytes are the producers of pro-inflammatory mediators and a major source of reactive oxygen radicals in the inflamed colon mucosa,³⁶ infiltration of leukocytes into the mucosa has been suggested to contribute significantly to the tissue necrosis and mucosal dysfunction associated with colitis.³⁷ An acute inflammatory phase impairs lipoprotein metabolism, causing alterations in the lipid and lipoprotein levels even in IBD patients.³⁸ IL-6 and C-reactive protein are closely related and together play a role in general lipid metabolism, inhibiting adipocyte lipoprotein lipase activity.^39,40 To determine the lipid alterations occurring in intestine inflammation, we quantified serum cholesterol and triglyceride levels in TNBS-induced colitis. In the present study serum cholesterol and triglyceride levels increased in the colitis group; UDO administration to this group markedly decreased the cholesterol and triglyceride levels. This result was linked with the improved inflammation state in TNBS-induced colitis. Moreover, hypercholesterolemia enhances the free radical generation in various ways,⁴⁰ and the formation of oxygen free radicals, such as superoxide anion radical (O₂ ⁻) or peroxynitrite (ONOO⁻), is postulated to be derived from different cellular sources in the vasculature and in parenchymatous tissues plays a significant role in the pathogenesis of many other diseases besides cardiovascular diseases (i.e., cancer and inflammatory disorders) as well.⁴¹ Thus, increased oxidative stress and impairment of the antioxidant defenses by the deleterious effect of reactive oxygen metabolites contribute to the pathogenesis of colitis. In the present study the colonic tissue MDA level, an indicator of lipid peroxidation, significantly increased in TNBS-induced experimental colitis. The increased MDA content might have resulted from an increase of reactive oxygen species as a result of oxidative stress in the rats after TNBS administration. UDO administration also increased the colonic MDA level. The reason for such elevation of lipid peroxidation with the UDO administration may be due to the superoxide overproduction after some xenobiotic has been consumed. In the present study unsaturated fatty acid content was found to be higher than the saturated fatty acid content of UDO. In general, it is accepted that the higher the degree of unsaturation of an oil, the more susceptible it is to oxidative deterioration. This high unsaturated fatty acid content (83%) may have caused the increase in MDA level after UDO administration to the colitis group. Palmitic acid (12.7%), oleic acid (22.5%), and linoleic acid (56.4%) contents of UDO were higher than the other fatty acid contents. Moreover, it was reported that oleic acid oxidizes at a rate 50 times slower than linoleic acid.⁴² It is often followed by singlet oxygen dismutation and produces hydrogen peroxide, which is easily converted later into the reactive •OH. Both single oxygen and OH radicals have a high potential to initiate the free radical chain reactions of lipid peroxidation. Thus the increased MDA level could also be partly explained by its high linoleic acid level.

In accordance with previous reports,^43,44 our results also showed depleted tissue GSH and decreased SOD activity in experimental colitis, a finding that clearly shows the oxidant damage in colonic tissue in experimental colitis. Although UDO administration did not seem to ameliorate the lipid peroxidation of the colonic tissue, it reversed low GSH levels and SOD activity. This finding shows the increase of antioxidant defense system activity by UDO administration in the TNBS-treated rats. Although the antioxidant properties of UD have been reported in various studies,^45
–47 our finding related to its enhancing antioxidant defense system activity on colonic tissue in TNBS-induced colitis was novel. Turkdogan et al.⁴⁷ suggested that hepatoprotective properties of U. dioica were possibly through immunomodulator and antioxidant activities. Similar to these results, Kanter et al.⁴⁶ also suggested that U. dioica decreases the lipid peroxidation and liver enzymes (aspartate aminotransferase) and increase the antioxidant defense system activity in the CCl₄-treated rats in the doses used.

In the present study markedly increased colonic tissue collagen content in the colitis group shows enhanced tissue fibrinolytic activity. Enhanced fibrinolytic activity can create obstructions and produce increased toxic waste products surrounding the colonic cells within the colonic tissues. UDO administration to the colitis group significantly decreased the colonic collagen content, reflected in the decreased tissue fibrinolytic activity. Additionally, in cytological examinations, the increases in leukocytes and collagen fibers were prominent in the colitis group. In comparison with the control group, reduction in amorphous structure was more pronounced in the colitis group. The densities of collagen fibers and leukocytes were decreased, and amorphous structure was found to be increased with UDO administration. Increased amorphous structure reflects the ameliorated disorder of the extracellular matrix structure in colonic tissue by UDO administration. Moreover, increased serum LDH activity of colitis group decreased with UDO administration. This result can also show the repaired colonic tissue damage by UDO administration.

TF activity is another parameter that indicates the tissue damage. It is a component of cell membrane, and its activity has been measured by prothrombin time test. TF activity can easily be changed by the alterations in membrane composition, heating, changing in pH, or lipid peroxidation of the membrane due to oxidative stress^48,49 as TF is not a stable protein. It can also promote the activation and/or release of many pro-inflammatory and pro-coagulant mediators, whereas inflammatory cytokines and other factors can stimulate TF production.^50,51

In the present study, TNBS-induced colitis increased TF activity of colonic tissue. This increase can be attributed to inflammation of the colonic membrane. UDO administration decreased TF activity in colonic tissue. It can be assumed that the anticolitis effect of UDO also reflected TF activity in colonic tissue.

Conclusion

The results of the current study suggest that UDO, through its anti-inflammatory and antioxidant actions, merits consideration as a potential agent in ameliorating colonic inflammation.

Footnotes

Acknowledgment

This study was supported by the Marmara University Research Foundation (project number SAG-C-YLP-030180-0003).

Author Disclosure Statement

We have no competing financial interests from this manuscript.

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