Introduction
Neurodegenerative diseases represent a heterogeneous group of disorders that affect millions of people worldwide. These diseases are characterized by the progressive and irreversible loss of function or death of neurons in specific regions of the brain, leading to severe cognitive and functional decline. The most well-known neurodegenerative diseases include Alzheimer disease, Parkinson disease, Huntington disease, and amyotrophic lateral sclerosis, with oxidative stress and apoptosis being the primary shared cellular and molecular mechanisms involved in these neurological disorders [1-3]. Evidence suggests that one of the major contributors to the exacerbation and increased risk of neurodegenerative diseases is environmental neurotoxins, which, in modern societies, are increasingly and uncontrollably threatening the health of living organisms directly or indirectly, thereby raising the risk of disease development [4].
Pesticides are environmental neurotoxins that, upon exposure, increase the risk of neurodegenerative diseases [5، 6]. Rotenone, a naturally occurring insecticide and pesticide derived from leguminous plants, is a potent environmental neurotoxin. Due to its highly lipophilic nature, it readily crosses all biological membranes, including the mitochondrial membrane and the blood-brain barrier, without requiring specific transporters [7-9]. The primary neurotoxic mechanism of rotenone involves inhibiting complex I of the mitochondrial electron transport chain. This inhibition leads to oxidative stress, apoptosis, and impaired autophagy. Rotenone disrupts electron transfer from Fe-S centers in complex I to ubiquinone within the electron transport chain, subsequently inhibiting oxidative phosphorylation, reducing adenosine triphosphate levels, and simultaneously increasing reactive oxygen species production, thereby inducing oxidative stress. Elevated reactive oxygen species levels and oxidative damage to DNA, lipids, and proteins contribute to neuronal cell degeneration [10].
Given the rising incidence of neurodegenerative diseases and the association of various environmental neurotoxins, including rotenone, with neurodegenerative disorders, understanding the inflammatory, apoptotic, and oxidative stress pathways involved in rotenone-induced toxicity is crucial. Moreover, protective mechanisms, such as the neuroprotective role of estrogen and the modulatory effects of resveratrol—a well-known polyphenol with anti-apoptotic, anti-inflammatory, and antioxidant properties—require further exploration. The present study investigates these processes by inducing estrogen depletion (ovariectomy in Wistar rats) and exposing the animals to rotenone, a pesticide commonly used in agriculture, to assess its impact on consumers and agricultural workers. Molecular assessments focused on gene expression changes in Bcl2, BAX, and the antioxidant factor superoxide dismutase (SOD) in the hippocampus of the treated rats.

Methods
This study utilized 30 female Wistar rats (weighing approximately 170±20 g) obtained from the Laboratory Animal Center of Babol University of Medical Sciences (Babol City, Iran). The animals were housed under standard conditions (12-h light/dark cycle, ad libitum access to food and water, and an ambient temperature of 20-25 °C). All experimental procedures were performed following the ethical guidelines set by the Animal Ethics Committee of the Shahid Chamran University of Ahvaz.
The rats were randomly assigned to five groups (n=6 per group). Group 1 was the rotenone group (intraperitoneal injection of 5 mg/kg rotenone; Sigma-Aldrich), group 2 was ovariectomy (OVX) (bilateral ovariectomy), group 3 was OVX + resveratrol group (40 mg/kg resveratrol via gavage), group 4 were OVX + rotenone group, and group 5 were OVX + rotenone + resveratrol group. All treatments were administered daily for three weeks. At the end of the experiment, the animals were anesthetized with intraperitoneal injections of ketamine (80 mg/kg) and xylazine (5 mg/kg) (Alfasan, Netherlands). The hippocampal tissue samples were then collected for analysis.

Statistical analysis
Biochemical assay data were analyzed using IBM SPSS Statistics software, version 20, employing a one-way analysis of variance followed by the Tukey post hoc test. A P<0.05 was considered statistically significant.

Results 
Following ovariectomy, the highest malondialdehyde (MDA) levels were observed in the OVX + rotenone group (40.06±2.22 µM/g), which showed a significant increase compared to the OVX group (32.92±1.12 µM/g; P<0.001). MDA levels in the OVX + resveratrol group (21.42±0.97 µM/g) were significantly lower compared to those in the OVX group (P<0.001). Resveratrol administration significantly reduced MDA levels in the OVX + rotenone + resveratrol group (22.35±1.91 µM/g) compared to the OVX + rotenone group (P<0.001).
SOD activity (16.36±0.61 U/mg) significantly decreased in the OVX + rotenone group compared to the OVX group (70.69±1.58 U/mg; P≤0.001). However, resveratrol administration in the OVX + resveratrol group did not significantly increase SOD activity (77.02±2.2 U/mg) compared to the OVX group. In the OVX + rotenone + resveratrol group, SOD activity (57.9±1.94 U/mg) was significantly higher than in the OVX + rotenone group (P<0.001).
The highest BAX gene expression levels were observed in the OVX + rotenone group (5.9±0.05), which was significantly higher than in the OVX group (1.3±0.11; P<0.001). BAX expression in the OVX + resveratrol group (1.2±0.11) did not significantly differ from the OVX group. However, resveratrol administration significantly reduced BAX expression in the OVX + rotenone + resveratrol group (4.5±0.11) compared to the OVX + rotenone group (P<0.001).
The lowest Bcl2 gene expression levels were recorded in the OVX + rotenone group (0.93±0.05), which was significantly lower than in the OVX group (5.9±0.05; P<0.001). Bcl2 expression in the OVX + resveratrol group (1.13±0.03) did not significantly increase compared to the OVX group. However, resveratrol administration significantly elevated Bcl2 expression in the OVX + rotenone + resveratrol group (0.78±0.05) compared to the OVX + rotenone group (P<0.001).

Conclusion
In this study, rotenone was used as a pesticide-induced neurotoxicant, while ovariectomy (estrogen depletion) was used as an experimental model of menopause. The protective effects of resveratrol against rotenone-induced hippocampal damage were evaluated. The findings demonstrated that rotenone significantly reduced SOD activity and Bcl2 gene expression while increasing MDA levels and BAX gene expression in the hippocampus.
Overall, resveratrol mitigated the neurotoxic effects of rotenone and estrogen depletion on apoptosis and oxidative stress markers. This study supports previous findings that resveratrol supplementation may be beneficial in improving key biomarkers associated with neurodegenerative diseases, particularly in high-risk populations, such as agricultural workers and postmenopausal individuals. Future research should investigate the role of proteins, such as Nrf2, SIRT, and NF-κB, along with their signaling pathways, as well as the effects of estrogen replacement therapy at different concentrations on oxidative stress and apoptosis indices.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the Ethics Committee of Shahid Chamran University of Ahvaz, Ahvaz, Iran (Code: EE.1400.3.02.10568).

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions 
Conceptualization and study design: Hossein Najafzadehvarzi, AhmadAli Moazedi and Mahboobeh Aghagolzadeh; Data analysis, interpretation, and statistical analysis: Mahboobeh Aghagolzadeh and Hossein Najafzadehvarzi; Financial resources and project administration, technical or material support: AhmadAli Moazedi and Hussain Naj Fazadeh Warzi; Supervision: Hossein Najafzadehvarzi, Ahmad Ali Moazdi, and Hadi Parsian; Writing the original draft: Mahboobeh Aghagolzadeh; Review and editing: All authors.

Conflicts of interest 
The authors declared no conflict of interest.

Acknowledgements
The authors would like to acknowledge the support of the Department of Biology, Shahid Chamran University of Ahvaz, and the Vice Chancellor for Research, Shahid Chamran University of Ahvaz, Ahvaz, Iran and Babol University of Medical Sciences, Babol, Iran during the implementation of this research.

References