Evaluation of Urtica dioica Extract as a Protective Agent against Hepatic and Renal Toxicity Induced by Insecticides

Introduction

Pesticides are lipophilic substances known for their neurotoxic effects and relatively low toxicity in mammals [1-5]. Their widespread use in agriculture has led to significant environmental contamination, contributing to various health issues. Exposure to different pesticide classes, including organochlorines (OCs), organophosphorus (OP), carbamates, and pyrethroids, has been associated with a range of adverse effects, including neurological disorders, carcinogenicity, respiratory complications, dermatological conditions, and reproductive abnormalities [6]. Among these, pyrethroids have been reported to cause alterations in hematologic, biochemical, and reproductive parameters [7].

Deltamethrin (DLM), a type II pyrethroid, and chlorpyrifos (CPF), an organophosphorus insecticide, belong to distinct pesticide classes but are both well-documented for their harmful effects [8]. DLM is widely utilized in agriculture as well as in household applications, with studies highlighting its immunotoxic and genotoxic impacts on mammalian species [9]. Meanwhile, CPF has been implicated in oxidative stress, inflammation, and apoptosis [10]. Due to its toxicological concerns, CPF usage was restricted in the United States in 2002 [8].

For centuries, medicinal plants have played a crucial role in traditional medicine, primarily due to their antioxidant properties, which help mitigate oxidative damage [11]. Urtica dioica (U. dioica) is one such plant, known for its therapeutic applications. Research has demonstrated its efficacy in treating nasal and menstrual hemorrhages, nephritis, anemia, and hematuria, as well as its blood-purifying properties. Additionally, it has been investigated for its potential benefits in managing diabetes, atherosclerosis, cardiovascular diseases, and prostate cancer [12]. U. dioica is also widely used in traditional medicine for conditions such as allergies, renal stones, burns, internal bleeding, and diabetes [13]. In Turkey and Iran, it has been employed for alleviating stomachaches, rheumatic pain, colds, and coughs [14], while its application extends to hypertension, allergic rhinitis, and cardiovascular diseases [15]. The plant’s diuretic properties contribute to blood pressure reduction by lowering vascular tension [16]. Moreover, studies have reported that the aqueous extract of U. dioica exhibits hypoglycemic effects, and the hydroalcoholic extract of its leaves has been shown to enhance insulin secretion in rats [17].

Given these pharmacological properties, the present study aims to evaluate the protective potential of U. dioica extract against DLM- and CPF-induced hepatotoxicity, nephrotoxicity, and rats' oxidative stress.

Materials and Methods

Preparation of U. dioica extract

Fresh leaves of U. dioica were air-dried at 20 °C for one week under laboratory conditions. A total of 10 g of the dried leaf powder was then boiled in 100 ml of distilled water for twenty minutes, with intermittent stirring. The resulting decoction and the obtained extract underwent lyophilization for 48 hours. The lyophilized product was subsequently stored at 4 °C for further use.

Quantification of total polyphenolic content

The Folin-Ciocalteu method, as described by Wolfe et al. [18], was employed to determine the total phenolic content, with modifications for microscale analysis. A 10 µl aliquot of the diluted extract solution was mixed with 50 µl of Folin-Ciocalteu reagent and allowed to react for 5 minutes. Subsequently, 150 µl of 20% Na₂CO₃ was introduced, followed by additional shaking for 1 minute. The final volume was adjusted to 790 µl using distilled water. After an incubation period of 90 minutes, the absorbance was measured at 760 nm using a spectrophotometer. A calibration curve was generated with gallic acid as the reference standard, and the total phenolic content was displayed as mg of gallic acid equivalent per gram of dry extract based on the linear regression equation derived from the calibration curve.

Ferric reducing antioxidant power (FRAP) assay

The reducing power of a compound serves as an essential marker of its potential antioxidant activity [19]. To assess this, 0.5 ml of the extract at varying concentrations, along with the reference antioxidant ascorbic acid, was combined with one ml of a 1% potassium ferricyanide [K₃Fe(CN)₆] solution and 1 ml of phosphate buffer (0.2 M, pH = 6.6). The mixture was then incubated at 50 °C for 20 minutes. Following this, the reaction was terminated by adding 1 ml of 10% trichloroacetic acid (TCA), and the solution was centrifuged at 3000 rpm for 10 minutes.

After centrifugation, 1.5 ml of the supernatant was mixed with 1.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride (FeCl₃). The solution was incubated for 10 minutes, after which the absorbance was recorded at 700 nm. To standardize the comparison we used ascorbic acid.

DPPH free radical scavenging activity

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay is one of the earliest methods used to examine the antioxidant structure-activity relationships of phenolic compounds [20]. DPPH (2,2-diphenyl-1-picrylhydrazyl) undergoes a color change from deep violet to yellow, signifying its reduction to 2,2-diphenyl-1-picrylhydrazine.

To evaluate this effect, 0.5 ml of the extract at different concentrations, along with ascorbic acid as the reference antioxidant, was mixed with 0.5 ml of DPPH dissolved in methanol. The reaction mixture was then incubated in the dark for 30 minutes. Absorbance readings were taken at 517 nm, with antioxidant activity assessed about a control solution containing absolute methanol and DPPH.

Animal selection and experimental conditions

This study utilized adult male albino Wistar rats, which were procured from the Central Pharmacy of Tunisia (SIPHAT, Tunisia). The rats were housed under controlled laboratory conditions, maintaining a 12-hour light/dark cycle, with regulated temperature and humidity levels. Throughout the study, the rats had free access to food and water (ad libitum). All experimental procedures were reviewed and approved by the Institutional Animal Care Committee, ensuring that the rats were handled in compliance with established institutional ethical guidelines.

Experimental design and grouping

Following a two-week acclimatization period, the rats were randomly assigned to five distinct experimental groups:

1. Control group (Group 1): Rats were provided with distilled water and a standard diet ad libitum throughout the study.
2. DLM/CPF group (Group 2): This group received a daily gavage administration of a DLM/CPF mixture (1 mg/kg/day DLM + 5 mg/kg/day CPF) for 20 consecutive days [21].
3. Vitamin CE + DLM/CPF group (Group 3): Rats were treated with vitamins C and E (100 mg/kg/day for each vitamin) [22] over 20 days. Additionally, during the final 10 days, they simultaneously received the DLM/CPF mixture via gavage.
4. U. dioica group (Group 4): This group was administered an aqueous extract of U. dioica (200 mg/kg/day) via gavage for 20 days.
5. U. dioica + DLM/CPF group (Group 5): Rats were treated with the aqueous extract of U. dioica (200 mg/kg/day) for 20 days, while simultaneously receiving the DLM/CPF mixture (1 mg/kg/day DLM + 5 mg/kg/day CPF) during the final 10 days via gavage.

Body weight was recorded throughout the treatment period. On day 20, the animals were sacrificed by decapitation. After collecting the blood samples, we left them to clot at 20 °C. After clotting, the samples were centrifuged at 2700 g for fifteen minutes to separate the serum, which was then stored for various biochemical analyses.

The liver and kidneys were promptly removed, cut into small pieces, and washed with ice-cold saline before blotting them on filter paper. Tissue homogenates were prepared by mixing the organs in 50 mM phosphate buffer (pH = 7.4) at a 1:2 weight-to-volume ratio. The homogenates were then centrifuged at 10,000 g for 15 minutes at 4 °C using an Ultra Turrax T25 (Germany). The resulting supernatant and serum samples were aliquoted and stored at -30 °C until further analysis.

Hematological measurements

Hematological parameters were assessed, including red blood cell (RBC) count,  hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin (Hb), mean corpuscular hemoglobin concentration (MCHC), and white blood cell (WBC) count. These parameters were measured using an automated Sysmex Kx-21N system at CHU Habib Bourguiba Sfax.

Serum biochemical analysis

For serum biochemical parameters, levels of aspartate aminotransferase (ASAT), creatinine, alanine aminotransferase (ALAT), urea, and uric acid were determined using commercial kits from France. The tests were performed on an automated system at CHU Hedi Chaker, Sfax, Tunisia. The results for ASAT and ALAT were expressed in U/L, while creatinine and urea were measured in mmol/L and uric acid in µmol/L.

Protein content measurement

The protein levels in both liver and kidney tissues were quantified using the method described by Lowry et al. [23], with bovine serum albumin serving as the reference standard.

Assessment of advanced oxidation protein products (AOPP)

The concentration of AOPP was measured based on the procedure outlined by Kayali et al. [24]. In this method, 0.4 mL of tissue homogenate supernatant was mixed with 0.8 mL of Tris-buffered saline (TBS, 0.1 mol/L, pH 7.4). After two minutes, 0.1 mL of 1.16 mol/L potassium iodide and 0.2 mL of acetic acid were added. The reaction mixture’s absorbance was recorded at 340 nm immediately. The AOPP concentration was obtained using the extinction coefficient (261 cm⁻¹mM⁻¹) and was expressed as µmol of AOPP per milligram of protein.

Protein carbonyl content measurement

The assessment of protein carbonyl content was performed following the protocol described by Ardestani and Yazdanparast [25]. For this, 1 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) dissolved in 2 M HCl was added to 1 mg of the sample. The mixture was incubated for 30 minutes at room temperature. Afterward, 1 mL of cold 10% trichloroacetic acid (TCA) was introduced, and the samples were centrifuged at 3000 g for 10 minutes. We washed The resulting protein pellet 3 times with a 1:1 ethanol/ethyl acetate mixture (2 mL each), then dissolved in 1 mL of 6 M guanidine hydrochloride (pH 2.3). The absorbance of the solution was measured at 370 nm. We determined  The carbonyl content using the molar extinction coefficient of DNPH (e = 2.2 × 10⁴ cm⁻¹M⁻¹) and expressed as nmol per mg of protein.

Lipid peroxidation (TBARS Assay)

To estimate lipid peroxidation, TBARS levels were measured, which reflect the concentration of malondialdehyde (MDA), the end product of lipid degradation, based on Yagi’s method [26]. In brief, 125 µL of the supernatant was sonicated with 50 µL of Tris-buffered saline (TBS), followed by the addition of 125 µL of TCA-BHT to precipitate proteins. The mixture was centrifuged at 1000 × g for 10 minutes at 4 °C. To 200 µL of the supernatant, 40 µL of 0.6 M HCl and 160 µL of TBA dissolved in Tris were added. The mixture was incubated at 80 °C for 10 minutes, and the absorbance was recorded at 530 nm. We calculated The concentration of TBARS using the extinction coefficient of 156 × 10⁵ mM⁻¹ cm⁻¹.

Conjugated diene assay

The conjugated diene levels were determined by taking 25 µL of the sample, followed by the addition of 3 mL of chloroform/methanol (2:1 v/v). After centrifugation at 3000 rpm for 5 minutes, the supernatant was removed, and the extract was dried in an oven at 45 °C overnight. The residue was re-dissolved in 2 mL of methanol and the optical density (OD) was measured at 190 nm using a quartz cuvette, as described by [27].

Histopathological analysis

Tissues from the liver and kidneys were collected and preserved in a 10% formalin solution. The specimens underwent dehydration through a stepwise ethanol treatment and were then cleared with xylene. Following this, they were stained in paraffin. Sections of 5 µm thickness were cut and embedded with hematoxylin and eosin. The stained slides were observed under a microscope for any histological changes.

Statistical analysis

Results were expressed as means ± standard error of the mean (SEM). Statistical comparisons were made using one-way ANOVA, followed by the Tukey post hoc test. A p-value of less than 0.05 was considered significant.

Results and Discussion

Total polyphenolic content and antioxidant activity of U. dioica

The U. dioica extract was found to have a total polyphenolic content of 712.375 µg GAE per mg of dry plant extract. The antioxidant potential of U. dioica was assessed through its FRAP reduction power and antiradical activity, using the DPPH free radical assay. The results showed an IC50 value of 0.09 ± 1.3 for the extract and 0.07 ± 0.005 for ascorbic acid, which served as a reference (IC50 = 0.07 mg/ml for ascorbic acid and 0.02 ± 0.004 mg/ml for U. dioica) (Table 1).

Table 1. Antioxidant tests of U. dioica by FRAP and DPPH

Growth curve analysis of control and treated rats

Growth assessment is an essential indicator of overall health and nutritional status. The data indicated that U. dioica had a protective effect in the DLM/CPF group, showing improvement compared to the group receiving only the DLM/CPF mixture. Additionally, the inclusion of vitamin CE appeared to support the restoration of body growth, in contrast to the rats receiving the DLM/CPF mix alone (Figure 1).

After the treatment period, the final body weight measurements revealed a significant reduction in the body weight of the DLM/CPF group compared to the control rats. The weight gain in this group was notably lower. However, the rats that were pretreated with U. dioica extract and vitamin CE showed a significant improvement in weight gain and body weight recovery, reversing the disturbances caused by the DLM/CPF mixture.

Hematological parameters

As shown in Table 2, significant reductions in red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and white blood cell count (WBC) were observed in the DLM/CPF group comparing to the control group, with decreases of -26.27%, -21.9%, -26.42%, -25.31%, 1.34%, -0.17%, and 35.66%, respectively. The groups treated with U. dioica+DLM/CPF or VitCE+DLM/CPF showed improvements in these parameters comparing to the DLM/CPF group, with increases of +34.36%, +32.37%, +1.43%, +3.49%, +25.39%, +16.36%, and +25.06% for U. dioica+DLM/CPF, and +33%, +37.14%, +5.45%, +37.1%, +26.68%, +29.23%, and +25.15% for VitCE+DLM/CPF. Importantly, no significant difference was observed between the control group and the group treated with U. dioica alone.

Table 2. Effects of DLM/CPF, U.dioica, Vit CE, and their combination (Vit CE + DLM/CPF, U. dioica + DLM/CPF) on some hematology parameters in blood.

Values were expressed as means ± SEM. The number of determinations was n = 5 ; * P ≤ 0.05 vs control U. dioica + DLM/CPF ; Vit CE + DLM/CPF vs DLM/CPF : # P ≤ 0.05

Biochemical parameters

The biochemical analysis of liver and kidney markers is presented in Table 3. A marked increase in serum ASAT (+11.06%), ALAT (+32.57%), ALP (+16.14%), and LDH (+13.16%) was observed in the DLM/CPF treated rats, indicating hepatotoxicity. However, pre-treatment with U. dioica extract significantly attenuated the rise in these liver enzymes, indicating a protective effect. In contrast, U. dioica alone did not result in any significant changes compared to the control group.

For renal markers, including urea, creatinine, and uric acid, no notable changes were found in the U. dioica only group compared to controls. The DLM/CPF group showed a significant increase in creatinine (+13.25%), urea (+9.89%), and uric acid (+24.91%). However, the groups pretreated with U. dioica + DLM/CPF and VitCE + DLM/CPF exhibited significant reductions in these markers, with decreases of -26%, -2%, and -14.16% for U. dioica+DLM/CPF, and -4%, -9.71%, and -41.61% for VitCE+DLM/CPF, respectively.

Notably, the antioxidant properties of U. dioica extract played a role in mitigating the toxicity removed by the DLM/CPF mixture.

Table 3. Effects of DLM/CPF, U. dioica, Vit CE and their combination (Vit CE + DLM/CPF, U. dioica + DLM/CPF) on serum liver and kidney of control and treated rats

Values were expressed as means ± SEM. The number of determinations was n = 5 ; * P ≤ 0.05 ; ** P ≤ 0.01 vs control U. dioica + LM/CPF ; Vit CE + DLM/CPF vs DLM/CPF : # P ≤ 0.05 ; ## P ≤ 0.01

Assessment of protein oxidation: AOPP and CP levels

Administration of DLM/CPF led to a notable increase in the levels of AOPP and CP in both the liver and kidneys compared to the control group. However, these levels were significantly reduced in rats pretreated with U. dioica + DLM/CPF or VitCE + DLM/CPF when compared to the DLM/CPF only group. No significant difference was observed between the U. dioica treatment group and the control group, as illustrated in Figure 2.

Lipid oxidation estimation: TBARS and CD levels

A significant rise in the levels of TBARS and CD was observed in the liver and kidney of rats treated with the DLM/CPF mixture, indicating that the pesticides induced lipid oxidation at both membrane and plasma levels. Supplementation with U. dioica extract or VitCE led to a reduction in TBARS and CD content when compared to the DLM/CPF only group. Interestingly, the U. dioica extract alone did not affect the TBARS and CD levels, as shown in Figure 3.

Histopathological observations

Histological examination of the liver revealed a healthy liver structure in the control group. In contrast, significant alterations, including inflammation and leukocyte infiltration, were observed in the liver of rats treated with the DLM/CPF mixture. Pretreatment with U. dioica helped restore the liver’s structure, mitigating the damage caused by the pesticide mixture. These findings suggest that the DLM/CPF combination has a severe hepatotoxic effect, leading to noticeable changes in liver architecture (Figure 4).

In the kidney, severe nephrotoxicity, including inflammation and epithelial detachment, was observed in rats treated with the DLM/CPF mixture. Pretreatment with U. dioica significantly reduced the nephrotoxic effects induced by the pesticide combination (Figure 5).

Our findings revealed that U. dioica is rich in phenolic compounds and exhibits potent antioxidant activity, as demonstrated by both the FRAP and DPPH assays. Treatment with DLM/CPF led to significant reductions in hematological parameters, increases in biochemical markers, and heightened oxidative stress in both the liver and kidneys. These results were further supported by histopathological examination. The antioxidant potential of U. dioica would attributed to its flavonoid content, particularly chrysoeriol, along with other flavonoids and phenolic compounds [28]. These findings are consistent with the study by Vajic et al. [29], which reported that nettle leaves possess considerable antioxidant activity, capable of modulating blood pressure and oxidative damage in hypertensive rats. Similarly, Sadegh et al. [30] reported comparable levels of phenolic compounds.

In our investigation, we examined the effects of subchronic exposure to a mixture of DLM/CPF pesticides, with U. dioica extract used as a pretreatment. DLM/CPF administration caused a high reduction in hematological parameters, such as RBC, Hb, Ht, MCV, MCH, MCHC, and WBC, when compared to the control group. However, pretreatment with U. dioica extract and Vit CE led to significant improvements in these parameters compared to the DLM/CPF group. The decrease in RBC, Hb, and Ht levels may be attributed to inhibited erythropoiesis and chemosynthesis. Furthermore, the reduction in WBC count in the DLM/CPF group could be a result of immune system suppression. Notably, U. dioica extracts significantly mitigated these disturbances. These findings align with Abdel-Daim et al. [31], which reported similar reductions in RBC and Hb levels following exposure to DLM.

Numerous epidemiological studies have suggested a link between pesticide exposure and various health issues in affected populations, with evidence of carcinogenic and neurotoxic effects in animals. Our study focused on assessing the protective and antioxidant properties of U. dioica extract in rats treated with a pesticide mixture, evaluating the effects on liver biomarkers (e.g., ASAT, ALAT, ALP, and LDH) and kidney markers (e.g., creatinine, urea, and uric acid). The results showed a significant increase in hepatic and renal biochemical markers in the DLM/CPF group, which is indicative of tissue damage due to poisoning. However, pretreatment with the aqueous extract of U. dioica notably restored these markers, demonstrating its protective effect against DLM/CPF toxicity. Similarly, pretreatment with Vit CE also mitigated the toxic effects of DLM/CPF. It is noteworthy that the U. dioica extract contains antioxidant compounds that help reduce the overall toxicity. These results are in line with Ncir et al. [32], which found that deltamethrin exposure increased the activity of liver enzymes such as ASAT, ALAT, ALP, and LDH.

The present research demonstrated that the mixture of DLM/CPF caused oxidative damage in both kidney and liver tissues, as evidenced by the notable increase in AOPP and CP levels in rats exposed to these pesticides compared to controls. This increase indicates that the DLM/CPF mixture induces liver and kidney damage. Several studies have highlighted that pesticide exposure leads to alterations in protein levels. Our findings align with Hamdaoui et al. [33], which showed that exposure to Kalach 360 SL (an herbicide) caused ovarian protein damage, indicated by an increase in AOPP levels. Similarly, Ben Amara et al. [34] demonstrated that methyl thiophanate fungicide raised AOPP levels and triggered cellular toxicity in kidney and liver tissues. Additionally, Feriani et al. [35] found that DLM exposure caused an increase in CP levels, while Yazdinezhad et al. [36] reported that CPF overproduces reactive oxygen species (ROS), contributing to protein damage in liver cells. In contrast, pretreatment with either U. dioica extract or a Vit CE mixture significantly reduced both AOPP and CP levels, confirming the antioxidant-rich content of U. dioica and highlighting the protective role of Vit CE in countering the harmful effects of free radicals induced by pesticide exposure.

The current study also showed that DLM/CPF administration resulted in increased TBARS and CD levels in the liver and kidney, signaling lipid oxidation in membrane and plasma levels. These findings are consistent with previous studies [37]. Additionally, the administration of U. dioica extract led to a significant reduction in TBARS formation. Notably, the combination of Vit CE also decreased TBARS levels in both the liver and kidneys. These results are in line with the findings of Niki et al. [38], who recognized Vit CE as a potent natural antioxidant, capable of neutralizing ERO intracellularly and extracellularly [39]. The CD is a byproduct of lipid peroxidation, and its measurement serves as an indicator of oxidative stress. Our findings corroborate the study by Ncir et al. [32], who demonstrated that DLM exposure caused cellular damage in both the brain and kidneys. Treatment with U. dioica extract lowered DC levels, and the application of the Vit CE complex significantly reduced DC levels in comparison to the group exposed only to the pesticide mixture. These results echo Giray et al. [40], who found that vitamin E reduced CD and TBARS levels following cypermethrin exposure.

Histological evaluations further confirmed the protective effects of U. dioica extract and the Vit CE mixture. Our findings suggest that U. dioica could serve as both a preventive and therapeutic agent for oxidative liver and kidney diseases. The DLM/CPF mixture caused noticeable alterations in liver and kidney structure, including inflammation and leukocyte infiltration, compared to the control group. However, pretreatment with U. dioica extract or the Vit CE complex helped reverse the hepatotoxicity and nephrotoxicity induced by the pesticide mixture. These findings align with Abdou et al. [41], who reported that DLM exposure led to significant hepatotoxicity, characterized by necrotic changes and inflammatory cell infiltration.

Conclusion

To summarize, our findings indicate that U. dioica demonstrates therapeutic potential for various health issues, exhibiting significant antioxidant and antiradical properties through its polyphenol content, along with DPPH and FRAP activity. Both U. dioica and the combination of Vit CE contributed to a reduction in biochemical markers, protein oxidation, and lipid peroxidation. These results support the beneficial use of U. dioica for health-related purposes.

Acknowledgments: None

Conflict of Interest: None

Financial Support: None

Ethics Statement: None