1. Introduction
Ulcerative colitis is one of the main forms of inflammatory bowel disease (IBD) affecting the rectum and colon that is characterized by an imbalance in proinflammatory and anti-inflammatory reactivity [
1,
2]. The remarkable rise in IBD worldwide, including in most developing nations, affects millions of individuals and is a major public health issue that may raise the risk of colon cancer [
3,
4]. Several factors contribute to the etiology of UC including microbial, environmental, and genetic factors [
5].
Although the exact pathogenesis of UC is still unknown [
6], it is characterized by relapsing and remitting mucosal inflammation [
7,
8]. In particular, macrophages play a significant role in inflammatory disorders by engendering the cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) and other inflammatory mediators such as nitric oxide (NO) and prostaglandins [
9]. Chronic inflammation has been linked to a lower risk of colitis-associated colorectal cancer by increasing the production of pro-inflammatory cytokines such as IL-6, IL-1ß, IL-17, and TNF-α. Therefore, targeting NF-kB inflammation pathways together with Wnt/ß-catenin signaling may act to control colorectal carcinogenesis [
10]. In fact, it has been reported that the negative regulation of the Wnt signaling pathway by the degradation of β-catenin, a transcriptional coactivator of the Wnt signaling pathway, allows its subsequent translocation to the nucleus and activation of Wnt target genes by associating with LEF-1/TCF proteins [
11].
The standard treatment methods for UC use medications targeting inflammation and the immune system including mesalamine, sulfasalazine, and corticosteroids that, taken alone or in association, contribute to treating this disease [
12]. Unfortunately, these drugs are linked with side effects and patients eventually become refractory or intolerant over time [
13]. Therefore, the research for alternative and/or supplementary treatments among herbal and traditional medicines has been highly motivated [
14,
15]. Recent studies have focused on natural products and supplements obtained from plants with anti-inflammatory effects, low toxicity, and minimal side effects [
16].
Urtica dioica, often known as common nettle, is one of the most commonly used medicinal plants in the world due to its biologically active compounds [
17]. The leaves of this plant have been reported to show hypotensive, anti-inflammatory, hypoglycemic, analgesic, anti-ulcer, antioxidant, antimicrobial, cytoprotective, and anticancer activities [
18,
19]. Some of the chemicals in this plant include lignan, secolignan, norlignan, alkaloid, sesquiterpenoid, flavonoid, triterpenoid, sphingolipid, and sterol [
20,
21]. The trichomes of the nettle contain formic acid, acetyl choline, serotonin, and histamine [
22].
According to various studies, the stinging nettle plant contains biologically active chemicals such as phenols and flavonoids that can help reduce free radical generation by diverse pharmacological properties such as antioxidative and anti-inflammatory properties and may play a role in the prevention of intestinal inflammation. The use of water as a solvent showed the highest total phenolic content values as well as producing a significant effect on the antioxidant capacity of the extracts [
23].
The aim of this study was to evaluate the prophylactic coloprotective action of AEUD on DSS-induced ulcerative colitis via the regulation of inflammatory reactions and antioxidant properties in a colitis rat model.
2. Materials and Methods
2.1. Animals
Healthy adult male Wistar rats (weighing between 180 and 200 g) were purchased from the Society of Pharmaceutical Industries of Tunisia (SIPHAT, Ben-Arous, Tunisia) and acclimatized for 1 week before performing any experiment. All animals were housed under safe laboratory conditions in a temperature- and humidity-controlled room (22–24 °C, 70%) and kept on a 12 h light/dark cycle using hygrometer, thermometer, and timer settings with food and tap water available ad libitum. All animal procedures were performed in accordance with the Guidelines for Care and Use of Animals Laboratory and approved by the Bio-Medical Ethics Committee (CEBM) for the Care and Use of Animals for scientific purpose (JORT454002 (6 May 2021)). Furthermore, all experiments were performed at the same time of day (8 h).
2.2. AEUD Preparation
Leaves of Urtica dioica were collected from Beja, Tunisia, in March 2021 and were identified by Dr. Chokri Hafsi, a Professor at the University of Jendouba. The Voucher specimens (No. SO.325) have been deposited with the herbarium of the Higher Institute of Biotechnology of Beja, Tunisia. After drying in an oven at 50 °C for 48 h, the leaves were ground into fine powder using a blender. An amount of 10 g of the powder mixture was dissolved in 100 mL of bi-distilled water and incubated in a shaker for 24 h. Then, the extract solution obtained was filtered, concentrated in a water bath under vacuo, frozen, and lyophilized. AEUD was used for the phytochemical and mineral determination and in vivo experiments.
2.3. AEUD Phytochemical and Mineral Analysis
A phytochemical characterization of AEUD was made by determining the total phenolic compounds according to the colorimetric method of Folin–Ciocalteu. Briefly, 500 µL of the extract was added to 10 mL of water and 0.5 mL of Folin–Ciocalteu reagent. After 5 min, 8 mL of 7.5% sodium carbonate solution was added. The reaction was kept in the dark for 2 h and was measured at 765 nm using a UV-visible detector spectrophotometer. Gallic acid was applied as a standard, and the results were expressed in milligram gallic acid equivalent per gram dry matter (mg GAE/g DM) [
24].
The extract solution (0.5 mL) was mixed with 500 μL of 50% Folin–Ciocalteu reagent. The mixture was then allowed to stand for a 2–5 min period followed by the addition of 1.0 mL of 20% sodium carbonate. After 10 min incubation at room temperature, the mixture was centrifuged for 5 min (1000×
g), and the absorbance of the supernatant was measured at 730 nm. The total tannin content was expressed as mg of tannic acid equivalents/g DM [
25].
The total flavonoid content was detected using the AlCl
3 colorimetric method. In fact, 1 mL of the sample was mixed with 1 mL of 2% AlCl
3 solution. After 15 min incubation at room temperature, the optical density of their action mixture was evaluated at 430 nm. Quercetin was used as a reference standard and the total flavonoid content was expressed as milligram quercetin equivalent per gram dry matter (mg QE/g DM) [
26]. The total sugar level was determined using a previous procedure [
27].
Atomic spectroscopy was used to detect the contents of magnesium (Mg), zinc (Zn), iron (Fe), manganese (Mn), molybdenum (Mo), and copper (Cu) in AEUD.
2.4. Experimental Procedure
The study was continued for 21 days and a total of 36 rats were divided into six groups, each consisting of six animals, including: Group 1: normal control given only saline solution with oral intake of NaCl (0.9%, 5 mL/kg, b.w.); Group 2: the colitis group receiving DSS (5%) in the drinking water; Group 3: the reference group, MESA was administered to the rats at 100 mg/kg by gavage from day 0 to 21; Groups 4, 5, and 6: AEUD given at 50, 100, and 200 mg/kg once a day by gavage route for 21 days.
Ulcerative colitis was induced in rats by administering 5% DSS in the drinking water from day 15 to 21, except for the control group. During DSS treatment, stool consistency, the presence of blood in the feces, body weight, and food intake were examined and documented daily. After 21 days of experiment, animals were anesthetized to avoid any kind of stress which could distort the results and sacrificed by decapitation. The entire colon was measured; then, a portion of the colon tissue was stored in 10% buffered formalin for histopathological analysis and the remaining colon tissue was stored at −80 °C for further biochemical analysis.
2.5. Evaluation of Clinical Colitis and Colonic Weight and Length Measurement
The assessment of clinical colitis included daily monitoring of disease activity score determined on the basis of stool consistency, blood in the stool, and weight loss during exposure to DSS. The relevant specific criteria that were used to calculate the DAI are presented in
Table 1 [
28]. The samples of the large intestine were weighed and the colon lengths were measured.
2.6. Biochemical Assays
Blood samples were collected in lithium heparin tubes and then plasma was obtained by centrifugation (4000 t/min/4 °C for 15 min) and stored at −80 °C until analysis. Plasma levels of C-reactive protein (CRP), amylase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood sugar, urea, and creatinine were measured using automated enzymatic assays (ProXL). Potassium (K+) and sodium (Na+) were measured using a Cornley AFT-300 Electrolytes Analyzer (Precimed, China).
2.7. Determination of Hematological Parameters
Hematological parameters including hemoglobin (Hb), hematocrit (Hct), red blood cells (RBCs), white blood cells (WBCs) as well as hematological indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean cellular hemoglobin concentration (MCHC) were commonly analyzed using an electronic automate (HORIBA-ABX Pentra XL 80 (Bioplus, China)).
2.8. Assessment of DSS-Induced Oxidative Stress in the Colonic Tissues
Colonic lipid peroxidation was determined by measuring MDA using the double heating method. In brief, aliquots of colonic homogenates were mixed with a BHT–TCA solution that contained 1% BHT (
w/
v) dissolved in 20% TCA (
w/
v) and centrifuged at 1000×
g for 5 min at 4 °C. The supernatant was mixed with 0.5 N HCl and 120 mM TBA in 26 mM Tris and heated at 80 °C for a duration of 10 min. The absorbance of the resulting chromophore was determined at 532 nm after cooling. The levels of MDA were determined using an extinction coefficient for the MDA–TBA complex of 1.56 × 105 M
−1 cm
−1 [
29].
For the determination of glutathione peroxidase (GPx), 1 mL of reaction mixture containing 0.2 mL colonic homogenate supernatant, 0.2 mL (0.1 M) phosphate buffer pH 7.4, 0.2 mL GSH (4 mM), and 0.4 mL H2O2 (5 mM) was incubated at 37 °C for 1 min and the reaction was stopped by addition of 0.5 mL TCA (5%, w/v). After centrifugation at 1500× g for 5 min, an aliquot (0.2 mL) of the supernatant was combined with 0.5 mL of 0.1 M phosphate buffer pH 7.4 and 0.5 mL of DTNB (10 mM) and the absorbance was read at 412 nm. The GPx activity was expressed in nanomolar of GSH consumed per minute per milligram of protein.
The superoxide dismutase (SOD) activity level was obtained using modified epinephrine assays. At alkaline pH, the superoxide anion (O
2−) causes the auto-oxidation of epinephrine to adrenochrome while competing with this reaction: SOD decreases the formation of adrenochrome. An SOD unit is the quantity of extract that inhibits the rate of adrenochrome formation by 50%. The enzyme extract was added to 2 mL reaction mixture containing 10 µL bovine catalase (0.4 U/µL), 20 µL epinephrine (5 mg/mL), and 62.5 mM sodium carbonate/ bicarbonate buffer pH 10.2. Absorbance changes were observed at 480 nm [
30].
The catalase (CAT) activity was evaluated by measuring the initial rate of hydrogen peroxide (H
2O
2) disappearance at 240 nm. The reaction mixture contained 33 mM H
2O
2 in 50 mM phosphate buffer at pH 7.0 and the activity of CAT was calculated using the extinction coefficient of 40 mM
−1 cm
−1 for H
2O
2 [
31,
32].
The colonic mucosal H
2O
2 level was determined. Briefly, hydrogen peroxide reacts with p-hydroxybenzoic acid and 4-aminoantipyrine in the presence of peroxidase leading to the formation of quinoneimine that has a pink color detected at 505 nm [
33].
2.9. Histopathological Analysis
Colonic tissue specimens from the distal portion were collected, washed with ice-cold saline solution, and fixed in phosphate-buffered formalin (10%). Then, specimens were embedded in blocks of paraffin, sliced into 3 to 5 μm sections, stained with hematoxylin and eosin (H&E), and assessed for mucosal damage, ulceration, erosions, hemorrhage, and necrosis by a pathologist in a blinded manner under light microscopy equipped with a color video camera for digital imaging [
34].
2.10. Statistical Analysis
All values were evaluated as mean ± standard error of the mean. The statistical significance of differences between groups was measured using SPSS statistical program software version 20 using one-way analysis of variance with post hoc Tukey’s multiple comparison test. A value of p < 0.05 was considered significant.
4. Discussion
UC is an inflammatory disorder of the colon with a complicated etiology. 5-ASA, often known as mesalazine, is one of the most commonly used medicines in the treatment of UC. However, these treatments have the potential to have negative side effects. Consequently, there is growing interest in using the numerous natural components found in traditional herbs. In this respect, the prophylactic coloprotective action of the total extract of Urtica dioica leaves was investigated in DSS-induced UC in rats.
Firstly, we evaluated the phytochemical and mineral composition of AEUD which has been used as a natural remedy for ages [
35]. The current results exhibit that AEUD is rich in bioactive compounds including phenolic compounds, tannins, flavonoids, and sugar. Furthermore, AEUD contains high mineral levels such as those of magnesium, iron, and zinc. These data are similar to those of previous studies which confirm that stinging nettle’s leaves are becoming more well-known because they contain a wide range of chemical components such as flavonoids, phenolic compounds, organic acids, minerals, and vitamins [
36], as well as tannins, fatty acids, volatile compounds, polysaccharides, isolectins, sterols, terpenes, and proteins [
37,
38].
Secondly, the current study was designed to investigate the protective effects of AEUD in a DSS-induced ulcerative colitis model. The DSS–induced colitis model is one of the most widely used models. It can potentially cause damage to intestinal epithelial cells (IECs) [
39]. The resulting injury has symptoms and characteristics similar to UC in humans [
40]. In this regard, rats treated with DSS in their drinking water demonstrated a significant decrease in clinical parameters such as food intake, water consumption, body weight gain, and colonic length/weight due to severe tissue edema, necrosis, goblet cell hyperplasia, and inflammatory cell infiltration [
41], but an increase in the DAI. MESA and AEUD treatment significantly improved these symptoms. Previously, many studies have shown the benefits of pure polyphenols or polyphenol-rich extracts in preventing colonic length/weight decreases in colitis rats, which is indicative of the therapeutic efficacy of prospective anti-ulcerative medicines [
42,
43].
DSS control rats had mild to severe mucosal and submucosal inflammation, inflammatory cell infiltration, edema, and some ulcerations as compared to normal tissue sections. On the other hand, the colonic tissues of the rats treated with AEUD (50 mg/kg) were determined to have normal histological structure.
When compared to the colitis group, the MESA or AEUD (100 mg/kg and 200 mg/kg) pretreated groups demonstrated mild erosion, mild focal mucosal inflammation, and mild submucosal inflammation. A previous investigation confirms that the administration of dextran alone does not result in any symptomatology in mice; it is actually the sulfate groups that are responsible for DSS toxicity [
44]. In fact, DSS is responsible for altering the intestinal barrier integrity which disturbs the intestinal microbiome and homeostasis of intestinal immunity. The activation of the intestinal immune system and the migration of inflammatory cells into the intestine contribute to the maintenance of inflammation and intestinal lesions [
45,
46]. Many studies have demonstrated that the administration of several antioxidant agents reduces the severity of DSS-induced epithelial damage [
47], mucosal inflammation, and erosion of surface epithelial cells [
48,
49].
AST and ALT levels were increased by DSS administration; however, AEUD reverted this rise in the DSS-treated rats. Cellular enzyme leakage into plasma is a well-known indicator of hepatic injury in conjunction with liver damage. Increased levels of these enzymes are reliable indicators of liver function because they show increased permeability, injury, and/or necrosis of hepatocytes [
50].
CRP and WBCs are commonly used as markers of inflammation. We found an increase in these parameters in DSS-treated mice. In contrast, AEUD demonstrated great efficacy in preventing DSS-enhanced inflammatory mediators. In fact, CRP acts as an opsonin and activates complement, which causes the phagocytosis of bacterial and nuclear material. Therefore, CRP plays a crucial role in the innate immune system of the host and in the defense against autoimmunity [
51]. High serum CRP levels in UC correlate well with disease activity and other inflammatory indicators such as WBCs. In a retrospective single-center cohort study, a higher WBC count at diagnosis was found to be associated with colectomy, underscoring the importance of WBCs in UC [
52]. Many studies have found clinical cases of acute idiopathic pancreatitis and chronic pancreatitis related to IBD [
53]. These findings demonstrated that pretreatment with AEUD or MESA reduced hyperamylasemia in DSS-treated rats. Increased permeability of the inflamed mucosa may be the cause of the pancreatic enzyme rise found in more severe or active disease; this is a mechanism previously suggested in persons with intestinal infarction who also have elevated serum amylase levels [
54].
We found that AEUD significantly reduced the elevated glycemic level due to its ability to control blood sugar [
55]. According to several studies, nettle enhances the release of insulin, which decreases blood sugar levels. This was demonstrated by examining diseased and healthy rats following intraperitoneal treatment with
Urtica dioica aqueous extract [
56].
The leaves of
Urtica dioica have been reported to show anti-inflammatory properties and can be used to treat persistent inflammatory conditions. In this context, previous studies showed that biosynthesis of the arachidonic acid cascade enzymes, particularly the cyclooxygenases COX-1 and COX-2, was inhibited by leaf extracts, which reduced the formation of prostaglandins and thromboxane [
57]. Furthermore, the PAF (platelet activating factor) system, inflammatory response, and antioxidant reaction are inhibited. The NF-κB system, which is implicated in immunity, is also affected [
19,
58]. In addition, a number of studies have demonstrated that leaf extracts block the release of interleukins IL-2 and IL-1, interferon (IFN), and the tumor necrosis factors (TNF) [
59,
60].
The ability of AEUD to combat oxidative damage and inflammation may be responsible for its protective action against liver injury related to oxidative damage [
18]. Despite this, ROS are known to be beneficial species; however, excessive generation alters the redox balance and results in an oxidative stress state. In this context, it has been noted that an excessive amount of ROS is produced in subjects with UC. Hence, chronic oxidative stress has been demonstrated to have a significant impact on the persistence and etiology of ulcerative colitis [
61]. In our investigation, exposure to DSS was accompanied by colonic oxidative damage that showed up as an increase in the H
2O
2 levels compared to the control group. Treatment with MESA and AEUD has proven to be useful in preventing colonic ROS excess caused by DSS intoxication. In fact, free radicals are molecules created by cellular metabolism, which can be destructive to biological tissues and cause injury to DNA, lipids, cell membranes, and proteins. It is commonly recognized that reactive oxygen species, especially the hydroxyl radical, contribute significantly to inflammation by causing membrane lipid peroxidation, which causes severe cellular damage [
62]. Several studies have demonstrated that leaf extracts exhibit an antioxidant action by scavenging the DPPH radical (1,1-diphenyl-2-picrylhydrazyl). The majority of this antioxidant activity is caused by the presence of phenolic compounds [
63,
64].
One of the main causes of tissue lipid peroxidation is the excessive generation of ROS [
65]. Our findings demonstrate that the colons of DSS control animals had higher MDA levels, whereas the administration of AEUD or MESA considerably decreased them. This shows that AEUD’s active ingredient has a protective impact that is remarkably similar to MESA’s protective effect in reducing oxidative damage. Our data are consistent with a number of previous reports that show that the antioxidant effect of AEUD is mostly due to the presence of phenolic compounds [
63,
64].
The activities of the main antioxidant enzymes SOD, CAT, and GPx were evaluated, since the occurrence of UC is significantly influenced by oxidative stress and inflammatory reactions [
66]. SOD levels, GPx levels, and CAT activities dropped in the group treated with DSS, which could access mucosal cells via pinocytosis, causing cellular oxidation and disruption of the enzymatic antioxidant defense mechanism [
67]. These levels increased and were comparable to those of the control group in the groups treated with AEUD and MESA. These outcomes are in accordance with the findings confirming that GPx, SOD, and CAT activities decreased in the colitis group under the action of the
Urtica dioica antioxidant compounds [
68,
69,
70,
71].