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. 2025 Mar 12;15(4):86. doi: 10.1007/s13205-025-04239-z

Neuroprotective and antioxidant effects of Urtica dioica extract against chlorpyrifos-induced toxicity: an in vivo study

Ahlem Chira 1,✉, Imen Dridi 2, Fatma Rahmouni 3, Ichrak Ben Amor 4, Bochra Gargouri 4, Choumous Kallel 5, Kamel Jamoussi 6, Abdelfattah El Feki 1, Mongi Saoudi 1
PMCID: PMC11904075  PMID: 40092451

Abstract

This study examines the neuroprotective and antioxidant effects of Urtica dioica ethanolic extract (UDExt) and melatonin (Mel) in mitigating chlorpyrifos (CPF)-induced toxicity in rats, with melatonin serving as a standard antioxidant. Male Wistar rats were divided into six groups: (I) Control, (II) UDExt (200 mg/kg), (III) Mel (20 mg/kg), (IV) CPF (10 mg/kg), (V) UDExt + CPF, and (VI) Mel + CPF. UDExt and Mel were administered for 7 days prior to CPF exposure, followed by a treatment of 14 days. The findings revealed that CPF exposure significantly impaired hematological parameters (WBC, PLT, LYM), increased lipid peroxidation and protein oxidation, and decreased antioxidant enzyme activities (SOD, CAT, GPx) in brain and cerebellar tissues compared to control rats. Behavioral tests, including the open field test, showed that CPF reduced locomotor activity and increased anxiogenic symptoms. The Y-maze test revealed impaired recognition memory, and the tail suspension test indicated increased depressive symptoms. Histological analysis revealed neuronal damage in the brain and cerebellum tissues. Treatment with UDExt or melatonin, following CPF exposure, significantly improved hematological parameters, reduced lipid peroxidation and protein oxidation, normalized antioxidant enzyme activities, and alleviated neurobehavioral alterations. Histopathological analysis also showed that UDExt or melatonin combined with CPF notably reduced neuronal damage in the brain and cerebellum. UDExt and melatonin mitigated the CPF-induced neurobehavioral alterations associated oxidative injury.

Keywords: Anxiety, Locomotor activity, Working memory, Oxidative stress, Urtica dioica L., Chlorpyrifos

Introduction

Historically, approximately 2.5 million tons of pesticides have been applied to crops annually (Zhang 2018). However, less than 0.3% of these pesticides effectively reach the target pests, while 99.7% are dispersed into the environment, contributing to widespread ecological and health issues (Van der Werf 1996; Badraoui et al. 2020; Saoudi et al. 2021). The extensive use of synthetic pesticides, particularly organophosphate pesticides, has been linked to clear signs of toxicity and adverse effects (Karalliedde and Senanayake 1989; Hamdaoui et al. 2020). Acute pesticide poisoning is known to cause neurotoxic effects, including increased prevalence of neurological symptoms and alterations in neurobehavioral performance, which reflect cognitive and psychomotor dysfunction (Jaga and Dharmani 2003; Saoudi et al. 2021). In the past decade, attention has increasingly focused on the effects of prenatal exposure to chlorpyrifos (CPF), an organophosphate insecticide, which has been strongly linked to neurobehavioral deficits in both humans and animal models (Burke et al. 2017). Animal studies have demonstrated that developmental exposure to CPF adversely affects nervous system, brain development and leads to neurobehavioral abnormalities at levels. Such effects have been noticed well below the threshold for systemic toxicity caused by cholinesterase inhibition, which is commonly used to assess exposure and safety (Silva 2020). CPF's neurodevelopmental toxicity is believed to stem not only from cholinesterase inhibition but also from its disruption of cellular mechanisms involved in neuronal replication and differentiation, apoptosis, axon formation, synaptogenesis, and neural circuit formation (Burke et al. 2017).

Moreover, CPF has been implicated in increasing blood–brain barrier permeability (Deepika et al. 2022), and chronic exposure disrupted neurofilaments in the hippocampus and cerebral cortex, which in turn may impairs muscarinic acetylcholine receptors (Naughton and Terry 2018). These disruptions are thought to contribute to neurodegenerative diseases by compromising plasma membrane integrity and triggering key cellular disturbances, including calcium homeostasis imbalance and neurotransmitter receptor dysfunction (Matuz-Mares et al. 2022; Ishak et al. 2024).

In this connection, various herbal plants with diverse biological activities can help mitigate the negative effects of ROS and neurodegenerative diseases (Rehman et al. 2019; Saoudi et al. 2021; Pennisi et al. 2023). Among these, Urtica dioica (U. dioica), commonly known as stinging nettle and belonging to the Urticaceae family, has emerged as a promising candidate in alternative medicine for its neuroprotective effects (Chira et al. 2022; Abi Sleiman et al. 2024). The extract of U. dioica extract effectively normalizes the levels of key neurotransmitters, including dopamine, norepinephrine, and serotonin, which are often disrupted in neurodegenerative diseases, due to the presence of bioactive compounds such as beta-sitosterol, trans-ferulic acid, scopoletin, rutin, quercetin, and other phenolic constituent (Bisht et al. 2017; Ali et al. 2020; Abu Almaaty et al. 2021). Additional compounds identified in UDExt include esculin, which improves the recognition memory in diabetic nephropathy, oxo-octadecadienoic acid, an antioxidant and PPARα activator, and p-coumaric acid, all of which contribute to neuroprotection (Kim et al. 2012; Oh et al. 2021; Song et al. 2021).

Additionally, UDExt contains a variety of neuroactive phytochemicals, such as choline acetyltransferase, 5-hydroxytryptamine, acetylcholine, gentisic acid, esculetin, scopoletin, and rutin, which collectively contribute to the wide range of pharmacological potentials (Semwal et al. 2023). It was recently reported that U. dioica stimulates the synthesis of trophic factors that enhanced learning, memory, and long-term potentiation (Ghasemi et al. 2019). Furthermore, U. dioica supplementation has demonstrated protective activity against various neurological disorders and exhibited neuropharmacological effects by increasing levels of specific cell survival proteins (Loshali et al. 2021). Despite these promising findings, scientific data validating the neurophysiological benefits of U. dioica are still limited and need to be further documented. To date, the protective effects of UDExt against CPF-induced neurotoxicity in rats have not been comprehensively explored. This study aims to explore the in vivo effects of UDExt, specifically by investigating the potential protective effects of the UDExt in mitigating the damages caused by sub-acute chlorpyrifos exposure, with assessments of oxidative stress biomarkers, behavioral disturbances-including anxiety-like behavior, locomotor activity, and working memory in male Wistar rats-and brain histopathology.

Materials and methods

Chemicals

The pesticide chlorpyrifos [diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate, EC48% formulation, (CAS No. 2921-88-2)] and melatonin (CAS No. 73-31-4) were used in this study. Both chemical products were obtained from Sigma-Aldrich (Aldrich Chemical Company).

Plant material and extraction

The aerial parts of U. dioica were collected from Bir Ali, Sfax, Tunisia, in February 2023 and air-dried for one week. The dried plant material was then extracted with 70% ethanol at 37 °C for three days using maceration. The extract was filtered through Whatman filter paper in a Buchner funnel, and the residues were re-extracted three times. The combined filtrates were evaporated under vacuum at 40 °C with a rotary evaporator.

Animals

Rats farming

Male albino Wistar rats, weighing between 220 and 260 g, were used in the study. These animals were sourced from the Pasteur Institute (Tunisia). They were maintained at a temperature of 22 ± 3 °C with a 12-h light/dark cycle and a minimum relative humidity of 40%. The rats were fed a commercial balanced diet (Nutrisud, Sfax, Tunisia) and had access to drinking water ad libitum. All animal procedures were performed in accordance with the Ethical Committee Guidelines for Laboratory Animal Care and Use of our institution, the Faculty of Sciences of Sfax, Tunisia. Our procedures were in accordance with Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010, on the protection of animals used for scientific purposes.

Experimental condition

After a 2-week acclimatization period, the rats were randomly assigned to six different experimental groups of six animals each.

Group I (C): Control rats received the vehicle (corn oil, 1 mL/kg) via intraperitoneal injection daily for 14 consecutive days, with unrestricted access to standard food and water.

Group II (UDExt): Rats in this group were treated orally with ethanolic extract of U. dioica (UDExt) at a dose of 200 mg/kg/rat/day for 14 days (Ait Haj Said et al. 2016).

Group III (Mel): Rats in this group were treated via intraperitoneal injection with melatonin (Mel) at a dose of 20 mg/kg prepared by dissolving it in a small volume of pure ethanol and then diluting it in drinking water to achieve a final concentration of 0.066% (w/v), equivalent to a dose of 20 mg/kg body weight. Melatonin was administered for 14 days (Pang et al. 2001).

Group IV (CPF): Rats in this group were treated via intraperitoneal injection with CPF at a dose of 10 mg/kg/day in corn oil for 14 days (Mandal and Das 2011; Abolaji et al. 2017).

Group V (UDExt + CPF): Rats in this group were treated orally with UDExt at a dose of 200 mg/kg/rat/day for one week, followed by simultaneous intraperitoneal administration of CPF at a dose of 10 mg/kg/day during the second week.

Group VI (Mel + CPF): Rats in this group were treated via intraperitoneal injection with Mel at a dose of 20 mg/kg for one week, followed by simultaneous intraperitoneal administration of CPF at a dose of 10 mg/kg/day during the second week.

For the combined treatments (UDExt + CPF and Mel + CPF), the animals were administered UDExt or Mel daily for 14 days. During the second week, they received CPF every day, 30 min after the administration of UDExt or Mel. The doses of UDExt (200 mg/kg) and Mel (20 mg/kg) were selected based on their demonstrated significant neuroprotective and antioxidant effects in previous published studies. The corn oil used as vehicle in CPF groups as evidenced by Bebe and Panemangalore (2003) and several other studies. Fresh melatonin and vehicle solutions were prepared weekly, with doses adjusted for body weight throughout the study. Water bottles were wrapped in aluminum foil for light protection, and fluids were replaced twice a week.

Tissue collection and preparation

At the end of the treatment period, the animals were euthanized by decapitation. Blood samples were collected in heparinized tubes, allowed to clot at room temperature, and then analyzed directly for various hematological parameters. Both the brain and cerebellum of each rat were promptly excised, minced in ice-cold saline, and blotted dry on filter paper. The tissues were then homogenized in 50 mM phosphate buffer (pH 7.4) at a 1:2 (w/v) ratio. The homogenates were centrifuged at 10,000×g for 15 min at 4 °C using an Ultra Turrax T25. The supernatants were collected, stored in aliquots at − 20 °C, until used for the analysis of oxidative stress parameters. Protein content in the supernatants was quantified using the Lowry et al. (1951) method.

Hematological variables

The white blood cells count (WBC) (103/μL), the red blood cells count (RBC) (106/μL), hemoglobin level (Hb) (g/dL), mean corpuscular volume (MCV) (fL), mean corpuscular hemoglobin (MCH) (pg), mean corpuscular hemoglobin concentration (MCHC) (g/dL), platelet count (PLT) (103/µL) and lymphocytes (LYM) (103/µL) were measured using an automatic hematological assay analyzer at CHU Habib Bourguiba, Sfax, Tunisia.

Oxidative stress in brain and cerebellar tissue

Oxidative stress markers

Lipid peroxidation (LPO) was measured as thiobarbituric acid reactive substances (TBARS) following the method of Buege and Aust (1978). Malondialdehyde (MDA), a degradation product of peroxidized lipids, forms a pink chromophore with thiobarbituric acid (TBA), which absorbs maximally at 532 nm. The TBARS values were expressed as nanomoles of MDA/mg protein.

Protein carbonyl (PC) content was measured using the method of Reznick and Packer (1994). Proteins were precipitated with trichloroacetic acid (TCA), reacted with 2,4-dinitrophenylhydrazine (DNPH) in hydrochloric acid, and then treated with guanidine–HCl. The protein carbonyl content was quantified at 370 nm using a guanidine blank.

Antioxidant enzymes activities

In the brain and cerebellum tissues, the activities of antioxidant enzymes were measured as follows: Superoxide dismutase (SOD) activity was assessed using the colorimetric method described by Beyer and Fridovich (1987), involving the oxidation of nitroblue tetrazolium (NBT); Catalase (CAT) activity was determined using the UV colorimetric method outlined by Aebi (1984), with hydrogen peroxide (H₂O₂) as the substrate; and Glutathione peroxidase (GPx) activity was evaluated through a modified colorimetric method based on Flohé and Günzler (1984), utilizing both H₂O₂ and reduced glutathione (GSH) as substrates.

Behavioral tests

Behavioral tasks were conducted under dim red lighting in a secluded and silent room during the dark phase, 1 h after the lights were turned off (i.e., between 9 p.m. and 1 a.m.), for a maximum of 4 h per experimental day. Different groups of rats (6 rats per group) underwent behavioral assessments to measure locomotor activity, anxiety levels, spatial learning abilities, and the psychotropic effects of drugs and medicinal substances. The evaluations included the open field, Y maze, and tail suspension tests as detailed by Dridi et al. (2016).

Rats underwent 7 days of pre-training on all the behavioral tests. Between each test, the cages have been cleaned with 70% ethanol.

Open field for locomotion and anxiety

The open field test (OFT), developed by Hall and Ballachey (1932), is a widely used method for evaluating exploratory behavior and general activity in rats, where both the quality and quantity of the activity can be measured. This test primarily measures "movement," which can be influenced by factors such as motor output, exploratory drive, fear-related behavior, sickness, and the circadian cycle. The apparatus consists of a circular platform with a diameter of 50 cm, divided into 36 squares: 20 squares in the peripheral zone, 12 in the median zone, and 4 in the central zone. The platform is surrounded by 27-cm-high walls with uniform lighting to prevent escape. During the test, rats are individually placed in the peripheral zone of the apparatus, facing the wall, and allowed to explore freely for 6 min. After a 1-min habituation period, the total number of squares crossed by the animal is recorded during the following 5 min to measure horizontal and vertical activity, providing insights into locomotion. The time spent in the peripheral and central zones provides insights into anxiety (Dridi et al. 2016).

Y-maze for working memory

Immediate working memory performance was evaluated by observing spontaneous alternation behavior in a Y-maze. The maze was constructed of black-painted wood, with each arm measuring 30 cm in length, 19 cm in height, and 8 cm in width, positioned at equal angles. Rats were placed at the end of one arm and allowed to explore the maze freely for 3 min. The number of arm entries was recorded, with an entry being counted when all four paws of the rat were fully inside the arm. An alternation was defined as a sequence of three different arms entered consecutively, without revisiting the first arm on the third entry. The percentage of spontaneous alternation was calculated by dividing the number of successful alternations by the total possible alternations (total arm entries minus two) and multiplying by 100 (Dridi et al. 2016).

Tail Suspension for depressive-like behavior

The Tail Suspension Test (TST) was conducted as described by Steru et al. (1985). Rats were suspended 35 cm above a tabletop by their tail using adhesive tape. The test duration was 6 min, with immobility time measured during the final 4 min. Immobility, indicative of behavioral despair, was defined as the absence of movement. This test is utilized to evaluate depressive-like states and assess the effects of antidepressant and sedative substances.

Histopathology examination

Tissues from the brain and cerebellum were collected, rinsed with normal saline, and processed separately for histopathological examination. The brain tissue was preserved in Bouin's solution, dehydrated using a series of graded alcohol concentrations, and embedded in paraffin. Thin sections (4–5 μm) were prepared and stained with hematoxylin and eosin (H&E). These sections were then examined under a microscope to identify any histopathological changes.

Statistical analysis

All results were expressed as mean ± SEM. Comparisons between the groups were performed by one-way ANOVA followed by Tukey’s post hoc test, and statistical analysis was conducted with Prism 9 software. Differences were considered significant at p < 0.05.

Results

Haematological parameters

As shown in Table 1, CPF-induced toxicity resulted in significant alterations in hematological parameters, specifically WBC, PLT, and LYM, when compared to the control group. However, pretreatment with UDExt followed by CPF administration (UDExt + CPF), as well as pretreatment with Mel followed by CPF administration (Mel + CPF), significantly mitigated these hematological disturbances compared to the CPF group. Despite these protective effects, there were no significant changes observed in the red blood cell count (RBC), hemoglobin level (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) across all groups. Additionally, treatment with UDExt or Mel alone did not significantly alter hematological parameters compared to the control group.

Table 1.

Effects of pretreatment with Urtica dioica extract (UDExt) and melatonin (Mel) on chlorpyrifos (CPF)-induced haematological changes in control and experimental rats

Treatment and parameters C UDExt Mel CPF UDExt + CPF Mel + CPF
WBC (103/µL) 14.2 ± 2.91 11.5 ± 2.19 12.33 ± 1.26 21.73 ± 3.65* 15 ± 2.3## 16.8 ± 2.99##
RBC (106/µL) 4 ± 1.41 3.4 ± 0.49 3.5 ± 0.75 5.7 ± 1.36 4.53 ± 0.56 5.13 ± 0.66
HB (g/dL) 11.7 ± 0.98 10 ± 1,3 10.4 ± 0.69 11.65 ± 0.49 11.33 ± 1.34 11.43 ± 1
MCV (fL) 31.5 ± 0.70 28.33 ± 1.52 30.33 ± 2.88 32.66 ± 0.17 32.06 ± 0.90 31.66 ± 0.30
MCH (g/dL) 12.76 ± 2.35 12.33 ± 0.15 12.66 ± 0.35 13.5 ± 0.50 13 ± 1,04 12.99 ± 0.84
MCHC (g/dL) 21.5 ± 2.12 20.4 ± 1.50 20.66 ± 2.08 23.5 ± 1.52 22.66 ± 0.57 23.33 ± 0.57
PLT (103/µL) 875 ± 24 868 ± 30.78 830.3 ± 39.92 1015 ± 32.71*** 929 ± 2.42**# 975 ± 16.50**#
LYM (103/µL) 10.9 ± 1.68 9.36 ± 2.31 8.3 ± 1.65 15.5 ± 0.75** 12.83 ± 2.70*# 11.6 ± 2.76#
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Values are mean ± SEM for six rats in each group. *p < 0.05; **p < 0.01; ***p < 0.001 compared to control group. #p < 0.05; ##p < 0.01 compared to CPF group

Oxidative stress

Markers of oxidative stress

Table 2 presents the levels of TBARS and CP in brain and cerebellum tissues. Exposure to CPF resulted in a significant increase in both TBARS and CP levels in rats compared to the control group. Pretreatment with UDExt followed by CPF exposure (UDExt + CPF), as well as pretreatment with Mel followed by CPF exposure (Mel + CPF), significantly decreased TBARS and CP levels compared to the CPF group (#p < 0.05, ## p < 0.01). Notably, UDExt + CPF or Mel + CPF pretreatment did not significantly affect TBARS levels in the brain compared to the CPF group. Additionally, treatment with UDExt or Mel alone did not significantly alter TBARS and CP levels compared to the control group.

Table 2.

Effects of Urtica dioica extract (UDExt) and melatonin (Mel) pretreatment on TBARS and CP levels in the brain and cerebellum following chlorpyrifos (CPF) exposure

Treatment and parameters C UDExt Mel CPF UDExt + CPF Mel + CPF
Brain
 TBARS (nmol/mg protein) 2.20 ± 0.69 2.98 ± 0.28 2.14 ± 0.41 3.90 ± 0.62* 3.71 ± 0.75 3.30 ± 0.62
 CP (µmol/mg protein) 19.10 ± 3.94 19.51 ± 0.58 18.70 ± 0.18 25.33 ± 0.28** 23.31 ± 0.24* 21.09 ± 0.09#
Cerebellum
 TBARS (nmol MDA/mg protein) 5.48 ± 0.54 4.59 ± 1.73 3.47 ± 0.43 9.56 ± 0.41** 7.67 ± 2.08 5.73 ± 1.12#
 CP (µmol/mg protein) 17.09 ± 2.31 16.77 ± 3.68 16.11 ± 1.73 24.03 ± 0.59** 20.83 ± 1.48*# 19.21 ± 1.36#
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Values are mean ± SEM for three rats in each group. *p < 0.05; **p < 0.01 compared to control group. #p < 0.05 compared to CPF group. #p < 0.05; ##p < 0.01 compared to CPF group

Antioxidant enzyme activities

Table 3 shows the SOD, CAT, and GPx antioxidant enzyme activities in the brain and cerebellum tissues of the control and experimental rats. In the CPF-treated group, SOD, CAT, and GPx activities were significantly decreased compared to the control rats. However, pretreatment with UDExt followed by CPF exposure (UDExt + CPF), as well as pretreatment with Mel followed by CPF exposure (Mel + CPF), showed a significant restoration of SOD and CAT activities to normal levels in brain and cerebellum tissues (#p < 0.05, ##p < 0.01). Notably, UDExt + CPF did not significantly affect GPx levels in the brain. Additionally, UDExt or Mel alone did not significantly affect the concentrations of SOD, CAT, and GPx compared to the control group.

Table 3.

Effects of Urtica dioica extract (UDExt) and melatonin (Mel) pretreatment on SOD, CAT, and GPx activities in the brain and cerebellum following chlorpyrifos (CPF) exposure

Treatment and parameters C UDExt Mel CPF UDExt + CPF Mel + CPF
Brain
 SOD (Units/mg protein) 11.52 ± 0.91 11.62 ± 2.02 12.22 ± 0.73 6.47 ± 0.22** 8.68 ± 0.99*## 9.44 ± 1.61##
 CAT (μmol H2O2/mg protein) 14.80 ± 1.9 15.04 ± 1.10 18.29 ± 3.41 9.37 ± 0.35*** 11.25 ± 1.58*# 13.13 ± 0.62##
 GPx (μmol GSH/min/mg protein) 0.13 ± 0.05 0.11 ± 0.01 0.15 ± 0.06 0.07 ± 0.012*** 0.08 ± 0.03** 0.10 ± 0.02*
Cerebellum
 SOD (Units /mg protein) 10.71 ± 0.43 9.54 ± 1.82 12.22 ± 1.21 6.99 ± 1.32* 8.05 ± 0.67 8.81 ± 0.76
 CAT (μmol H2O2/mg protein) 39.28 ± 1.79 41.87 ± 4.86 42.75 ± 1.72 31.38 ± 1.47*** 34.04 ± 4.00* 34.83 ± 4.69*
 GPx (μmol GSH/min/mg protein) 0.16 ± 0.07 0.18 ± 0.06 0.17 ± 0.04 0.08 ± 0.01*** 0.11 ± 0.06**## 0.13 ± 0.03*##
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Values are mean ± SEM for three rats in each group

*p < 0.05; **p < 0.01 compared to control group. #p < 0.05; ##p < 0.01 compared to CPF group

Evaluation of behavioral test outcomes

Open field

The results presented in Fig. 1A, B showed that the number of vertical and horizontal locomotions was significantly decreased in the group of rats exposed to CPF, compared to the control rats (p < 0.001). This indicates that CPF causes a marked impairment of motor activity. However, the pre-treatment of rats with UDExt followed by CPF treatment (UDExt + CPF) or pre-treatment with Mel followed by CPF treatment (Mel + CPF) showed an increase in the total number of locomotions compared to the CPF-treated group. This signifies a restoration of the number of locomotions towards a normal state. Although no significant differences were observed between the UDExt, Mel, and control groups.

Fig. 1.

Fig. 1

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Effects of Urtica dioica extract (UDExt), melatonin (Mel), chlorpyrifos (CPF), UDExt + CPF combination, and Mel + CPF combination on locomotor and anxiolytic behaviors in rats, evaluated through the open field test. Rats were monitored for 5 min to assess different types of locomotor and anxiety-related behaviors across treatment groups. A Number of locomotions in the vertical zone; B Number of locomotions in the horizontal zone; C number of locomotions in the peripheral zone; D Number of locomotions in the central zone. Values are expressed as mean ± SEM for six rats in each group. Black dots represent the group means, and red error bars indicate the SEM

The results presented in Fig. 1C, D showed that rats treated with CPF exhibited a significant increase in the number of locomotions in the peripheral zone and a decrease in the number of locomotions in the central zone compared to the control rats. This indicates a state of "anxious" stress accompanied by a sensation of fear. Despite this, the pre-treatment of rats with UDExt followed by CPF treatment (UDExt + CPF) or pre-treatment with Mel followed by CPF treatment (Mel + CPF) resulted in a decrease in the number of locomotions in the peripheral zone and an increase in the number of locomotions in the central zone compared to the CPF-treated groups. This demonstrates the relaxing effect of UDExt and a restoration of a sense of well-being. Although no significant differences were observed between the UDExt, Mel, and control groups. These results demonstrate the neuroprotective effects of UDExt by inducing significant locomotor activity and a significant decrease in anxiolytic activity for the groups of rats (UDExt + CPF and Mel + CPF) compared to the control group of rats.

Y-maze

The second test conducted was to evaluate the spontaneous alternation behavior in the Y-maze to measure working memory. The principle of this test is based on the natural tendency of rodents to spontaneously and alternately explore the different arms of the maze, with a successful alternation rate of about 60% to 70% (Belzung 1999). Our results, presented in Fig. 2, showed that the percentage of spontaneous alternation was significantly reduced in the group of rats treated with CPF (41.89 ± 3.25%) compared to the control rats (71.40 ± 6.49%). However, this alternation percentage was restored in rats pre-treated with UDExt followed by CPF treatment (UDExt + CPF) or pre-treated with Mel followed by CPF treatment (Mel + CPF) compared to the groups of rats intoxicated with CPF only (CPF). However, no significant differences was observed in the rats treated with UDExt or Mel alone. These results demonstrate that pre-treatment with UDExt significantly attenuates neurodegenerative disorders by correcting the memory center of the rats.

Fig. 2.

Fig. 2

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Effects of Urtica dioica extract (UDExt), melatonin (Mel), chlorpyrifos (CPF), UDExt + CPF combination, and Mel + CPF combination on the percentage of spontaneous alternation in rats, assessed through the Y-maze test. Values are expressed as mean ± SEM for six rats in each group. Black dots represent the group means, and red error bars indicate the SEM

Tail suspension

Our results showed that the group of rats exposed to CPF exhibited a highly significant increase in immobility time compared to the control group. This suggests the presence of a state of despair leading to "depressive-like" immobility. However, the pre-treatment of rats with UDExt followed by CPF treatment (UDExt + CPF) or pre-treatment with Mel followed by CPF treatment (Mel + CPF) resulted in a highly significant reduction in immobility time compared to the CPF group, which is considered a correction of depressive-like behavior. No significant changes in these levels were observed for the group of rats treated with UDExt or Mel alone compared to the control group (Fig. 3).

Fig. 3.

Fig. 3

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Effects of Urtica dioica extract (UDExt), melatonin (Mel), chlorpyrifos (CPF), UDExt + CPF combination, and Mel + CPF combination on immobilization time in the tail suspension test. Values are expressed as mean ± SEM for six rats in each group. Black dots represent the group means, and red error bars indicate the SEM

Brain histopathology

For the histological examination of the brain and cerebellum, the control rats and those treated with UDExt or Mel displayed normal histology, showing various types of normal Purkinje neurons and well-developed molecular and granular layers (Figs. 4A–C and 5A–C, respectively). The rats receiving CPF exhibited abnormal cellular morphology with severe vacuolization and necrosis of cerebral and cerebellar neurons (Figs. 4D and 5D). However, the pre-treatment of rats with UDExt followed by CPF treatment (UDExt + CPF) or pre-treatment with Mel followed by CPF treatment (Mel + CPF) demonstrated an improvement in the histopathological alterations in CPF-treated rats (Figs. 4E, F and 5E, F, respectively).

Fig. 4.

Fig. 4

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Histological study of brain tissue in control and treated rats. Representative images of brain sections from A control rats, B Urtica dioica extract (UDExt)-treated group, C melatonin (Mel)-treated group, D chlorpyrifos (CPF)-treated group, E UDExt + CPF-treated group, and F Mel + CPF-treated group. Sections were stained with hematoxylin and eosin and examined under a microscope with original magnification of × 400 (scale bar = 50 µm) to assess cellular morphology and treatment-induced alterations. ML molecular layer ML, GL granular layer, N neurons, + : necrosis, *neuronal vacuolation in the molecular layer

Fig. 5.

Fig. 5

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Histological study of cerebellum in control and treated rats. Representative images of brain sections from A control rats, B Urtica dioica extract (UDExt)-treated group, C melatonin (Mel)-treated group, D chlorpyrifos (CPF)-treated group, E UDExt + CPF-treated group, and F Mel + CPF-treated group. Sections were stained with hematoxylin and eosin and examined under a microscope with original magnification of × 400 (scale bar = 50 µm) to assess cellular morphology and treatment-induced alterations. CM molecular layer, GL granular layer, P Purkinje neurons, +: necrosis, *: neuronal vacuolation

Discussion

Throughout this investigation, we observed that CPF exposure was closely linked to neurotoxicity, oxidative stress, depressive and anxiogenic behaviors, as well as impairments in locomotor and memory functions in rats. The protective potential of UDExt in counteracting CPF-induced toxicity was further explored, particularly on oxidative stress and neurobehavioral impairments. Our results show that CPF-induced toxicity significantly alters hematological parameters, likely due to ROS activity. The increased leukocyte count can be attributed to heightened ROS production and inflammation, consistent with Umapathy et al. (2010), who observed similar changes in CPF-treated rats. Duerschmied et al. (2013) also reported increased leukocyte count and platelet reactivity. These disruptions have been linked to cytokine-driven neutrophil recruitment and platelet-leukocyte interactions. Additionally, Yadala et al. (2021) found substantial lymphocyte apoptosis in CPF-treated mice. Our study shows that UDExt has a notable effect on mitigating these alterations. The protective effects of UDExt align with Insaf Fatima Zohra et al. (2015), who reported hemoprotective properties of UDExt. Wambui et al. (2024) also highlight the anti-inflammatory potential of flavonoid content of the UDExt. Hence, further research is needed to clarify and decipher the underlying mechanisms.

CPF-induced neurotoxicity is driven by oxidative stress, which arises from an imbalance between pro-oxidants and antioxidants, leading to biomolecular damage (Dubey et al. 2013; Saoudi et al. 2021). In our study, CPF exposure significantly increased MDA and PCO levels in the brain and cerebellum, indicating oxidative damage and potential proteasomal impairment (Barouki 2006). The protein oxidation disrupts the neuronal lipid matrix, leading to intracellular ATP loss, reduced neuronal viability, hippocampal degeneration, brain damage, and neurobehavioral impairments (Ekaluo et al. 2013; Saoudi et al. 2021). Our results demonstrate that both UDExt and melatonin effectively counteract CPF-induced oxidative stress by normalizing elevated MDA and PCO levels in the brain and cerebellum. UDExt enhances BDNF expression, promoting neuronal survival and synaptic plasticity, while regulating genes to inhibit pro-apoptotic factors (Ahmed et al. 2020; Mhalhel et al. 2024). In contrast, melatonin increases Bcl-2 levels, inhibits Bax, prevents apoptosis, and protects mitochondrial function by regulating mitochondrial permeability transition pore (mPTP) and calcium homeostasis (Tiong et al. 2019; Fang et al. 2020).

The brain is highly vulnerable to oxidative stress, with antioxidant enzymes like CAT, SOD, and GPx play a crucial role in its defense (Lee et al. 2020). Reduced levels of antioxidant enzymes are associated with neurodegenerative conditions, including Alzheimer's and Parkinson's diseases (Pathak et al. 2022). CPF exposure significantly reduces the activity of these antioxidant enzymes in the brain tissues of rats, suggesting an indirect increase in mitochondrial and cytosolic H2O2 (Ghahramani et al. 2018; Saoudi et al. 2021). This disruption of redox homeostasis aligns with the findings by Ekaluo et al. (2013), linking CPF exposure to neuronal damage and neurodegenerative diseases. The reduced GPx and CAT activity may lead to H2O2 accumulation, triggering OH° radical production via the Fenton reaction (Icenogle et al. 2004; Cory-Slechta et al. 2005).

Thus, UDExt significantly mitigates oxidative stress by preserving enzymatic antioxidant activity (Saoudi et al. 2021; Chira et al. 2024). Behboodi et al. (2021) reported reduced neuronal oxidative stress with U. dioica treatment. Furthermore, several studies have highlighted the neuroprotective action of kaempferol, a key component of U. dioica, which protects vulnerable neurons by improving their current functioning or promoting their regeneration (Hfaiedh et al. 2013). Moreover, flavonoids have a remarkable ability to chelate transition metals, preventing them from the free radical-generating reactions (Rahmouni et al. 2018; Malacaria et al. 2021; Sulieman et al. 2024). This metal chelation can effectively reduce oxidation (Tian et al. 2021). In another study, Calderaro et al. (2022) examined the effect of several flavonoids on beta-amyloid peptide-induced cytotoxicity in cortical neurons. Pre-incubation of these cells with 50 μM of kaempferol, apigenin, luteolin, and quercetin significantly reduced the increase in ROS production resulting from senile plaque aggregation (Tian et al. 2021).

Neurobehavioral studies in animal models are crucial for detecting subtle forms of toxicity (Dubovický et al. 2008; Saoudi et al. 2021). Organophosphorus compounds like CPF primarily affect the peripheral nervous system, but their liposolubility allows them to cross the blood–brain-barrier and cause neurotoxic effects (Braquenier et al. 2010), leading to impaired memory, anxiety, and depression (Braquenier 2009). To assess these effects, we conducted the Y-maze, tail suspension test, and open field test, which examine memory, cognitive function, depressive-like behaviors, as well as locomotor activity and anxiety levels in CPF-exposed rats. In our study, CPF exposure was found to significantly reduces motor activity and induce depressive symptoms, as evidenced by a preference for peripheral zones in the open field test (Hühne et al. 2020). This test is effective in assessing basal activity in rodents and their response to new or anxiety-inducing environments, genetic modifications, lesions, or pharmacological interventions (Pentkowski et al. 2021). Anxiety, often manifesting as inner turmoil, is characterized by feelings of unease, nervousness, or worry (Azizah et al. 2024). It is one of the most prevalent psychiatric disorders and can lead to behavioral disorders such as pacing, physical complaints, and excessive worrying. As anticipated, CPF treatment induced significant anxiogenic effects in rats, consistent with previous reports that link CPF exposure to increased anxiety behaviors (Umapathy et al. 2010). Our findings complement this by demonstrating that pre-treatment with UDExt significantly reduced anxiety-like behaviors, as evidenced by increased exploration in the open field test. This outcome is consistent with earlier studies highlighting the anxiolytic effects of both aqueous and alcoholic U. dioica extracts (Doukkali et al. 2016; Izunwanne et al. 2024), with various species offering distinct mechanisms to mitigate neuronal damage. For instance, Mucuna pruriens (Mp) demonstrated neuroprotective properties in mice intoxicated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a Parkinson’s disease model, by alleviating behavioral abnormalities, reducing neuroinflammation, and modulating key pathways like NF-κB and pAkt (Rai et al. 2017a). It helps mitigate the neuroinflammatory cascade by reducing proinflammatory cytokines and oxidative stress, with compounds like L-DOPA and ursolic acid. Studies show it lowers iNOS expression, nitrite levels, and lipid peroxidation, protecting dopaminergic neurons in the substantia nigra (Yadav et al. 2017; Rai et al. 2017b). Similarly, Withania somnifera (Ws) reduces oxidative stress and inflammation in a Parkinson's disease mouse model induced by maneb and paraquat. Ws treatment improves locomotor activity, restores dopamine levels, and reduces oxidative damage by lowering iNOS expression and balancing apoptosis (Prakash et al. 2014).

In the Y-maze test, CPF exposure impaired memory, evidenced by decreased motivation and lower alternation percentages in successive tasks (Costa et al. 2015). This suggests potential damage to central memory structures (Lee et al. 2023). These effects may be linked to alterations in serotoninergic, dopaminergic, glutamatergic, and GABAergic systems, which are essential for mood and anxiety regulation (Judge et al. 2016). As observed in the Y-maze test, CPF exposure also impaired cognitive performance, likely due to its neurotoxic effects on central memory structures. However, pre-treatment with UDExt restored cognitive function, aligning with findings from previous studies that demonstrated enhanced learning, cognitive function, and neurogenesis with U. dioica ethanolic extract co-administration in zebrafish model (Mhalhel et al. 2024). In a Parkinsonian model, U. dioica treatment exhibited anti-apoptotic activity, enhancing midbrain dopaminergic neuron recovery and improving motor and cognitive functions in rats (Albadawi et al. 2024). This effect is linked to U. dioica’s regulation of apoptotic markers, including the modulation of Bcl-2 and Bax expression, which may help restore cellular homeostasis and protect against neurodegeneration (Abi Sleiman et al. 2024; Prakash et al. 2014; Mohammadi et al. 2016).

In the tail suspension test, CPF exposure increased immobility time, suggesting depressive-like effects (Ommati et al. 2024). This observation is consistent with studies where CPF exposure led to behavioral changes and symptoms resembling depression in rats (Costa et al. 2015). Depression, marked by profound sadness, persistent low mood, reduced motivation, and disruptions in eating and sleep patterns, is often associated with impaired amine-dependent synaptic transmission (Sanda et al. 2022). The immobility displayed by rats in this test reflects a state of despair or lower mood, which characterizes depressive disorders in humans (Demin et al. 2020). Treatment with UDExt reversed the depressive-like symptoms induced by CPF, indicating that UDExt may contain bioactive components that protect amine-dependent neurons involved in the depressive process (Du et al. 2023). This supports findings that U. dioica has antioxidant, anti-inflammatory, and neuroprotective properties, which may help mitigate oxidative stress, a common factor in neurological disorders linked to CPF exposure (Sajad et al. 2024). Overall, behavioral studies show that pre-treatment with UDExt or melatonin alleviates CPF-induced neurotoxicity, improves behavioral traits, and prevents cognitive deficits. UDExt and melatonin both influence behavioral deficits, depressive symptoms, and anxiety through different mechanisms. U. dioica enhances cognitive function and reduces anxiety by its bioactive compounds, such as flavonoids and amino acids, improving memory and neuroprotection (Keshvari et al. 2020). In contrast, melatonin regulates circadian rhythms and improves sleep quality, indirectly reducing anxiety and irritability, while also exerting antidepressant effects by modulating neurotransmitter systems and reducing oxidative stress (Kholghi et al. 2022; Repova et al. 2022).

Histopathologically, CPF administration caused marked alterations in the brain and cerebellum. Our results are consistent with those of Hernandez et al. (2015), who found that CPF causes structural abnormalities in brain tissue characterized by abnormal cellular morphology. In contrast, treatment of UDExt led to significant histological improvements in brain and cerebellum health following treatment. In a study involving male rabbits treated with ethylene glycol, U. dioica extract (100 mg/kg) resulted in slight improvements in the histological examination of the cerebrum and cerebellum, reducing degeneration and necrosis of neuronal cells (Alsaidya et al. 2022).

Conclusion

Urtica dioica ethanolic extract (UDExt) demonstrates significant neuroprotective and antioxidant properties, effectively mitigating the cerebral toxicity induced by chlorpyrifos (CPF) in rats. The ability of UDExt to enhance hematological parameters, reduce oxidative stress, and improve neurobehavioral functions-particularly locomotor activity, anxiety levels, and working memory-were notably observed in intoxicated rats.

While these enhancements did not fully restore the conditions to those of the control group, they were markedly superior to the CPF group, suggesting that UDExt may serve as a potent therapeutic agent against CPF-induced neurotoxicity. Extrapolating from these results, we also propose that UDExt could be a promising alternative or complementary treatment to standard antioxidants like melatonin for addressing CPF-related health risks. Additional clinical studies are crucial to confirm these findings, and future research should focus on isolating the active compounds in U. dioica, prolonging the treatment period, and investigating different extraction techniques and mechanistic pathways.

Author contributions

A.C: conceptualization, methodology, investigation, formal analysis, visualization, writing—original draft, and writing. I.D, F.R, Ch.K, K.J, A.E.F.: Investigation. M.S: supervision, conceptualization. All authors gave their individual critical revision and final approval of the version to be submitted.

Data availability

Data are available upon request.

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest in the manuscript.

Research involving animals

All procedures involving animals were conducted in accordance with international, national, and/or institutional guidelines for the care and use of animals in research.

Informed consent

This study did not involve human participants.

Consent for publication

All authors have approved the final article and consented to publication.

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