Cannabidiol as a Neuroprotective Agent in Acrylamide‐Induced Neurotoxicity: Effects on Oxidative Stress, Inflammation, and Cholinergic Function in Male Mice
Department of Pharmacy, Laboratory of Molecular Biology and Immunology University of Patras Rio Greece
Department of Biology, Laboratory of Human and Animal Physiology University of Patras Rio Greece
Laboratory of Brain Exosomes & Pathology, Institute of Biosciences & Applications NCSR “Demokritos” Athens Greece
* Correspondence:Korina Atsopardi (kor.atsop@gmail.com)
ABSTRACT
The neuroprotective potential of cannabidiol (CBD) was assessed in a mouse model of acrylamide‐induced neurotoxicity. Acrylamide (AA), an environmental and dietary pollutant, is known to cross the blood–brain barrier and induce oxidative stress, inflammation and neurotoxic effects. Male C57BL/6 mice were randomly assigned to four groups: Control (Con), Acrylamide (AA), Cannabidiol (CBD), and a combination treatment (AA + CBD). The AA group received acrylamide (10 mg/kg, i.p.) daily for 5 days. CBD was administered (10 mg/kg, i.p.) for 10 days in the CBD and AA + CBD groups. In the AA + CBD group, acrylamide (10 mg/kg, i.p.) was co‐administered during the last 5 days of CBD treatment. Behavioral outcomes were analyzed using the open field test, revealing that CBD mitigated anxiety‐like behavior induced by acrylamide, enhancing movement and center exploration. Further, CBD treatment modulated oxidative stress responses, reducing MDA levels and partially restoring antioxidant markers (GSH, SOD, and CAT) in the hippocampus and striatum. Inflammatory markers were also assessed, revealing that acrylamide elevated pro‐inflammatory cytokines TNF‐α and IL‐6. Notably, CBD co‐treatment reduced TNF‐α levels in the hippocampus and cortex and attenuated IL‐6 levels in the cortex and striatum, suggesting an anti‐inflammatory effect. Additionally, CBD modulated neuroplasticity by increasing BDNF levels in the hippocampus, counteracting the reduction caused by acrylamide. CBD also influenced cholinergic activity by restoring Ach levels and altering AChE activity across brain regions. Findings suggest that CBD exhibits neuroprotective properties by reducing oxidative stress, inflammation and cholinergic dysregulation, thereby offering a promising therapeutic approach for mitigating pollutant‐induced neurotoxicity and potentially treating neurodegenerative disorders.
Graphical
Cannabidiol (CBD) protects against acrylamide‐induced neurotoxicity in mice by reducing oxidative stress, inflammation, and cholinergic dysfunction, while restoring BDNF levels. These findings highlight CBD's potential as a therapeutic agent for preventing environmental neurotoxicity and supporting brain health.
Boxed Text
Article notes
Atsopardi, K. , K. Mesiakaris , I. Sotiropoulos , M. Margarity , and K. Poulas . 2025. “Cannabidiol as a Neuroprotective Agent in Acrylamide‐Induced Neurotoxicity: Effects on Oxidative Stress, Inflammation, and Cholinergic Function in Male Mice.” Journal of Neuroscience Research 103, no. 12: e70098. 10.1002/jnr.70098.
Footnote Group
1Untitled section
Boxed Text
- Acrylamide, a widespread environmental and dietary pollutant, induces severe oxidative, inflammatory and cholinergic brain disruptions.
- CBD improves neurobehavioral outcomes, reversing acrylamide‐evoked anxiety‐like behavior in the Open Field Test.
- CBD counters acrylamide‐induced oxidative and inflammatory damage, restoring redox balance across hippocampus, cortex and striatum.
- CBD preserves neuroplastic and cholinergic function, normalizing hippocampal BDNF and partially restoring acetylcholine signaling.
2Introduction
Environmental pollution is widely recognized as one of the most pressing global health threats, with adverse effects extending to nearly all biological systems, including the central nervous system (Grandjean and Landrigan 2014; Landrigan et al. 2018). Acrylamide (AA) represents a particularly relevant example, as it reflects both environmental and dietary exposure routes (Ghasemzadeh Rahbardar et al. 2021; Rifai and Saleh 2020; Rivadeneyra‐Domínguez et al. 2018; Tareke et al. 2002). The capacity of AA to induce oxidative stress, neuroinflammation and neuronal dysfunction highlights its significance as a neurotoxicant (Fang et al. 2022; Gur et al. 2021; Mirzavi et al. 2024; Zhao, Zhang, and Deng 2022). Given the increasing interest in the impact of environmental/food pollution on the brain and its contribution to brain pathologies and based on the reported AA neurotoxic role impact, the identification of neuroprotective strategies against AA brain lesions is of great importance. In this context, Cannabidiol (CBD), a non‐psychoactive phytocannabinoid derived from Cannabis sativa , has recently gained attention for its antioxidant, anti‐inflammatory and neuroprotective properties, making it a promising candidate to counteract environmentally and dietarily relevant neurotoxic insults (Atalay et al. 2020; Hickey et al. 2024).
Over the last decades, CBD has attracted increasing scientific and clinical interest as a promising therapeutic compound for a wide range of pathological conditions. Unlike Δ9‐tetrahydrocannabinol (THC), CBD is a C21 terpeno‐phenolic cannabinoid that lacks the euphoric and addictive effects of THC (Bergamaschi et al. 2011; Calapai et al. 2020; Crippa et al. 2018; Hudson et al. 2019). CBD is considered one of the most promising candidates for clinical application in various pathological conditions due to its lack of cognitive or psychoactive effects and its exceptional tolerability in humans (Larsen and Shahinas 2020). This tolerability has been confirmed in multiple clinical trials, including those involving high doses and extended treatment periods (Larsen and Shahinas 2020). CBD demonstrates a wide range of pharmacological effects, including anxiolytic (Blessing et al. 2015; Campos et al. 2013; Mesiakaris et al. 2024), antioxidant (Borges et al. 2013; Chen et al. 2016), anti‐inflammatory (Chen et al. 2016; Mecha et al. 2013), and neuroprotective properties (Lim et al. 2017). The molecular mechanism of CBD, especially its neuroprotective effect, is associated with its interaction with the human endocannabinoid system (eCB) (García‐Arencibia et al. 2007; Mouslech and Valla 2009). The eCB consists of two primary types of endogenous G protein‐coupled receptors: CB1, mainly expressed in the brain and CB2, which is primarily found in peripheral tissues. While many studies indicate that CBD exhibits low affinity for CB1 and CB2 receptors (Campos et al. 2016; Fouad et al. 2013), some of its effects appear to involve them. This contradiction could be explained by CBD's in vivo interaction as an antagonist/inverse agonist of the cannabinoid receptors or by an indirect elevation of anandamide levels through inhibition of its metabolism/uptake (Bisogno et al. 2001; Campos et al. 2013; Castillo et al. 2010). Furthermore, several of CBD's neuroprotective effects have been associated with mechanisms involving the 5‐HT1A serotonin receptor (Sartim et al. 2016).
Moreover, studies suggest that CBD has anxiolytic (Blessing et al. 2015; Lee et al. 2017; Melas et al. 2021), antidepressant (Melas et al. 2021; Sales et al. 2019; Sartim et al. 2016), and antipsychotic‐like properties (Elsaid et al. 2019; Leweke et al. 2012). Additionally, other studies highlight the significant benefits of CBD in managing neurological disorders, by reducing neuroinflammation and protecting against neurodegeneration, including, Parkinson's (Del‐Bel et al. 2024; Patricio et al. 2020; Santos et al. 2015), Huntington's (Consroe et al. 1991), Alzheimer's disease (Chen et al. 2023; Cheng et al. 2014; Karl et al. 2017; Marques and Campos 2024; Zhang et al. 2022), and epilepsy (Arzimanoglou et al. 2020; Carlini and Cunha 1981; Devinsky et al. 2014; Golub and Reddy 2021), alongside its contribution to neurogenesis (Liput et al. 2013), and cognition enhancement (Ma et al. 2024; Osborne et al. 2017). These effects underscore its potential as a therapeutic option for neuropsychiatric disorders and substance use conditions (Burstein 2015; Watt and Karl 2017). However, determining precise dosing guidelines for CBD remains a challenge at this time.
Acrylamide (AA) is a synthetic chemical, used in the production of polyacrylamide and has wide uses in different industries (Ghasemzadeh Rahbardar et al. 2021; Rivadeneyra‐Domínguez et al. 2018) such as cosmetics, printing, textile, etc. It is also formed naturally when high‐carbohydrate foods are exposed to or cooked at high temperatures (Kunnel et al. 2019; Rawi 2012; Triningsih et al. 2021). AA has been related to different types of toxicity such as hepatotoxicity, nephrotoxicity, neurotoxicity, reproductive toxicity and carcinogenicity in experimental animals (Aboubakr 2019; Aboubakr et al. 2023; Al‐Ghamdi et al. 2023; Bin‐Jumah et al. 2021; Ibrahim 2024; Klaunig 2008; Pyo et al. 2020). Beyond its toxicological profile in animals, AA is of direct human relevance since chronic low‐level exposure occurs mainly through the dietary intake of high‐temperature processed foods and occupational exposure, with cigarette smoke as an additional source (Rivadeneyra‐Domínguez et al. 2018; Tareke et al. 2002). Recent human dietary exposure studies report intakes ranging from 0.165 to 2.81 μg/kg bw/day (Basaran 2024; Kucuksullu et al. 2025; Yu et al. 2023), highlighting that although these levels are much lower than experimental doses, their chronic and long‐term nature raises important health concerns.
AA penetrates the blood–brain barrier, contributing to its neurotoxic effects (Zhao, Zhang, and Deng 2022), inducing oxidative stress, apoptosis, inflammation and alterations in brain function (Edres et al. 2021; Fang et al. 2022; Gur et al. 2021; Mirzavi et al. 2024). Moreover, it induces neurotoxicity through oxidative stress mechanisms in both in vitro and in vivo models (Mehri et al. 2015; Prasad 2012; Yousef and El‐Demerdash 2006; Zhao, Deng, et al. 2022). Previous studies suggest that AA exposure disrupts antioxidant defense mechanisms, such as an increase in reactive oxygen species (ROS) and malondialdehyde (MDA) levels and a decrease of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) (Bicer et al. 2022; Goudarzi et al. 2019; Kopańska et al. 2015; Pan et al. 2018; Uthra et al. 2022). Oxidative stress induced by AA administration was suggested to trigger inflammatory responses by increasing the production of pro‐inflammatory markers, including interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and tumor necrosis factor‐alpha (TNF‐α) (Acaroz et al. 2018; Elblehi et al. 2020; Pan et al. 2018; Zhao et al. 2017; Zong et al. 2019). Additionally, elevated levels of ROS and pro‐inflammatory molecules can reduce the levels of brain‐derived neurotrophic factor (BDNF), a vital neurotrophin essential for regulating brain development, synaptic plasticity and neurogenesis (Lima Giacobbo et al. 2019). Moreover, BDNF is highly expressed in the hippocampus and cortex (Fang et al. 2022; Lima Giacobbo et al. 2019). AA administration downregulates BDNF expression in the hippocampus and cerebral cortex thereby impairing its neuroprotective effects (Erdemli et al. 2018; Yan et al. 2018). This reduction in BDNF levels exacerbates oxidative stress and neuroinflammation, creating a self‐perpetuating cycle of neurotoxicity (Lima Giacobbo et al. 2019). Furthermore, evidence indicates that AA induces oxidative stress through the production of free radicals, which interfere with acetylcholinesterase (AChE) receptors leading to a reduction of AChE activity in the brain (Farouk et al. 2021; Uthra et al. 2017). Other studies also suggest that AA exposure disrupts cholinergic signaling by altering acetylcholine (ACh) synthesis, release, or receptor functionality, contributing to neurobehavioral deficits, including anxiety and cognitive impairments (Elblehi et al. 2020; Kopańska et al. 2022; Zhao, Deng, et al. 2022).
Recent in vitro and in vivo studies have highlighted the potential of natural compounds with anti‐apoptotic, antioxidant and anti‐inflammatory properties to reduce AA‐induced neurotoxicity (Bicer et al. 2022; Edres et al. 2021; Farouk et al. 2021; Ibrahim and Shahen 2023; Shrivastava et al. 2019; Sun et al. 1988; Uthra et al. 2022). However, the understanding of AA's neurotoxic effects on the brain remains limited. While CBD's beneficial action against different neuropsychiatric and neurodegenerative disorders (e.g., depression, Alzheimer's) is characterized by similar brain lesions such as oxidative stress, synaptic malfunction and neurotoxicity (Del‐Bel et al. 2024; Marques and Campos 2024), the potential protective effects of CBD against toxicant‐induced brain damage, particularly acrylamide neurotoxicity, remain largely unexplored. Therefore, this study aims to evaluate the therapeutic potential of cannabidiol in mitigating redox and pro‐inflammatory imbalances, neurotransmitter disruptions and BDNF alterations, alongside behavioral imbalances associated with AA‐induced neurotoxicity.
3Materials and Methods
3.1Chemicals
Acrylamide (AA; C3H5NO), DMSO, Tween‐80 and all necessary reagents were supplied from Sigma‐Aldrich. Cannabidiol (CBD) was supplied by Cayman Chemical (CAS No. 13956‐29‐1). BDNF (MOFI00015), TNF‐α (HUFI00262), and IL‐6 (HUFI00180) ELISA Kits (Assay Genie) were used for our experimental purposes.
3.2Animals
The experiments were performed on male C57BL/6 mice weighing 27 ± 4 g (3–4‐month‐old). Animals were housed and acclimatized under standard husbandry conditions (22°C ± 1°C temp, 50%–60%, relative humidity, 12 h light and dark cycle) in laboratory polyacrylic cages. The experimental protocol was approved by the National Veterinary Administration authorities (License: ΠΔΕ/ΔΚ/923/3, approval date 18 January 2021).
3.3Experimental Design
Cannabidiol (CBD) was dissolved in saline containing 5% DMSO and 2% Tween‐80 and administered sub‐chronically via intraperitoneal (i.p.) injection at a dose of 10 mg/kg/day for 10 consecutive days. Acrylamide (AA) was dissolved in saline and delivered intraperitoneally (i.p.) at a dose of 10 mg/kg for 5 consecutive days. Both treatments were administered using a volume of less than 10 μL/g. Mice were randomly divided into four groups (8 mice in each group). Group I (Control; Con) received saline with 5% DMSO and 2% Tween‐80; Group II (AA) received acrylamide (10 mg/kg/day, i.p.) for 5 days; Group III (CBD) received cannabidiol (10 mg/kg/day, i.p.) for 10 days; Group IV (AA + CBD) received cannabidiol (10 mg/kg/day, i.p.) for 10 days and acrylamide (10 mg/kg/day, i.p.) for the last 5 days of CBD treatment (Figure 1). Six animals per group were subsequently euthanized by cervical dislocation. Blood samples were collected through cardiac puncture into tubes containing anticoagulants (K3 EDTA, Sarstedt). These samples were then centrifuged at 1500 g for 10 min and the plasma was isolated and stored at −80°C. Three brain regions, the hippocampus, cerebral cortex and striatum, were isolated and stored at −80°C until further homogenization. This experimental design was selected to evaluate the overall protective effects of CBD based on previously reported results with rutin in AA neurotoxicity (Motamedshariaty et al. 2014). The acrylamide dose (10 mg/kg/day, i.p. for 5 days) was chosen within a range commonly reported in rodent studies (Aboubakr 2019; Aboubakr et al. 2023; Acaroz et al. 2018; Motamedshariaty et al. 2014) as an intermediate regimen that induces neurotoxicity while maintaining tolerability.
3.4Open Field Test
To evaluate the impact of CBD and acrylamide treatments on anxiety‐like behavior, the Open Field Test (OFT) was used. The OFT relies on the mice's inherent fear of open spaces and their natural tendency to explore new environments. The apparatus (42 × 42 × 42 cm) used in the test was first introduced by Simon (Simon et al. 1994) and the test was performed 24 h after the final injections. Prior to the test, mice were placed in a dimly lit room for 1 h to allow habituation. Each mouse was individually placed in the center of the apparatus and allowed to explore it for 10 min. Animal movement was quantified automatically using Any‐Maze video tracking software (Kraeuter et al. 2019), which recorded total distance traveled (m), number of entries into the center and the time spent in the center (s).
3.5Blood Test Analysis
The Samsung PT10V Compact Blood Analyzer, which utilizes a combination of optical and electrochemical detection methods, was used for metabolic analysis of the plasma samples.
Specifically, the determination of metabolic markers in the plasma included glucose (GLU), blood urea nitrogen (BUN), creatinine (CREA), calcium (CA), total protein (TP), globulin (Glob), albumin (ALB), alanine transaminase (ALT), alkaline phosphatase (ALP), bilirubin (TBIL), cholesterol (CHOL), amylase (AMYL) and lipase (LIP).
3.6Tissue Homogenization
Tissue samples were prepared for analysis by homogenizing them in phosphate‐buffered saline (PBS) supplemented with an EDTA‐free Protease Inhibitor Cocktail (ROCHE/04693159001). Homogenization was carried out using a glass/Teflon homogenizer under cold conditions on ice. The resulting homogenate was then centrifuged at 15,000 g for 20 min at 4°C. The supernatants, containing the salt‐soluble (SS) fraction with the target components, were aliquoted and stored at −80°C. The pellets were resuspended in an equal volume of 1% Triton X‐100 and centrifuged again at 15,000 g for 20 min at 4°C to isolate the detergent‐soluble (DS) fraction, which was used for acetylcholinesterase activity determination. Total protein concentration was quantified using the Bradford assay (Olson and Markwell 2007).
3.7Redox Markers
Redox markers were evaluated in the brain regions, including the hippocampus, cerebral cortex and striatum, to assess oxidative and antioxidative status. The markers measured included malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), each representing different aspects of redox homeostasis.
3.7.1Malondialdehyde
Malondialdehyde (MDA), a byproduct of lipid peroxidation and a well‐established marker of lipid oxidative stress, was quantified using the thiobarbituric acid reactive substances (TBARS) assay (Afonso et al. 2013; Ritter et al. 2003). In this method, 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid were added to homogenized brain samples. The mixture was heated in a water bath at 100°C for 15 min to induce the acid‐heating reaction. The resulting chromogenic product was measured at 535 nm using a spectrophotometer. MDA levels were calculated and expressed as nanomoles of MDA equivalents per milligram of protein.
3.7.2Reduced Glutathione
Reduced glutathione (GSH), a critical non‐enzymatic antioxidant responsible for neutralizing free radicals and maintaining cellular redox balance, was measured using the GSH Assay Kit (Abcam, ab239727). The assay was performed according to the manufacturer's protocol, which quantifies the thiol group reactivity of GSH. GSH levels were normalized to total protein content and reported as μmol/mg of protein.
3.7.3Superoxide Dismutase
Superoxide dismutase (SOD), an enzymatic antioxidant that catalyzes the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen, was assessed using the SOD Assay Kit (Cayman Chemicals, Item No. 706002). This assay measured total SOD activity, including both cytosolic (Cu/Zn‐SOD) and mitochondrial (Mn‐SOD) isoforms, following the manufacturer's instructions. SOD activity was expressed in units (U) per milligram of protein, providing insights into the brain's capacity to mitigate superoxide‐induced oxidative damage.
3.7.4Catalase
Catalase (CAT), an essential antioxidant enzyme that decomposes hydrogen peroxide into water and oxygen, was evaluated using the Mouse Catalase Assay Kit (Assay Genie, Item No. MOFI00210). The assay involved measuring the change in absorbance at 450 nm, corresponding to CAT‐mediated reactions. The procedure was carried out in accordance with the manufacturer's protocol. CAT activity was normalized to protein concentration and expressed as units (U) per milligram of protein.
3.8Pro‐Inflammatory Cytokines
The pro‐inflammatory cytokines tumor necrosis factor‐alpha (TNF‐α) and interleukin‐6 (IL‐6) were quantified using ELISA kits (Assay Genie; TNF‐α: Item No. HUFI00262, IL‐6: Item No. HUFI00180) following the manufacturer's protocols. Absorbance for both assays was measured at 450 nm and cytokine concentrations were normalized to protein content and reported as pg/mg of protein.
3.9Brain‐Derived Neurotrophic Factor
Brain‐derived neurotrophic factor (BDNF) protein levels were quantified using the Mouse BDNF ELISA Kit (Assay Genie, Item No. MOFI00015), following the manufacturer's protocol. To ensure precise quantification, BDNF levels were normalized to total protein content. Sample dilutions were optimized and verified to fall within the kit's dynamic detection range.
3.10Cholinergic Analysis
3.10.1Acetylcholinesterase Activity
Acetylcholinesterase (AChE) activity was assessed in both the SS and DS fractions of all tissue homogenates using the Ellman's colorimetric method (Ellman et al. 1961). The SS fraction primarily contains the G1 (cytosolic) isoform of AChE, while the G4 (membrane‐bound) isoform predominates in the DS fraction (Das et al. 2005). In this assay, the production rate of thiocholine, resulting from the hydrolysis of acetylthiocholine iodide by AChE, was measured. Results are expressed as micromoles of thiocholine produced per minute per gram of wet tissue (μmol/min/g wet tissue).
3.10.2Acetylcholine Levels
Acetylcholine (ACh) levels were determined using a modified version of Hestrin's method to enable rapid and sensitive determination in small sample volumes. Briefly, brain homogenate aliquots were mixed with distilled water, 1.5 mM eserine and 1.84 M trichloroacetic acid and the mixture was thoroughly blended. After centrifugation at 10,000 rpm for 15 s at 4°C, the supernatant was collected and the sample was transferred to a microplate. To each sample, basic hydroxylamine solution (equal volumes of 2 M hydroxylamine hydrochloride and 3.5 M NaOH) was added and the mixture was incubated at 25°C for 20 min. Following incubation, 3.75 M HCl and 0.75 M FeCl3 were added. Absorbance was measured at 540 nm using a spectrophotometer. Results were expressed as micromoles of acetylcholine per milligram of protein (μmol/mg protein). Both AChE activity and ACh levels were determined from the same brain tissue homogenates of each animal, with separate aliquots processed for each measurement.
3.11Statistical Analysis and Graphs
Statistical analysis was performed using GraphPad Prism version 8.0.2. The two‐way analysis of variance (ANOVA) followed by Post Hoc Tukey's test has been used in order to analyze the differences among mean scores. Two‐way ANOVA was used for comparisons across the four subgroups (Control, AA, CBD, AA + CBD). For behavioral analysis, 8 per group were used while molecular/biochemical analyses were based on 6 animals per group. Tukey's post hoc test has been used to determine specific differences between groups, with a 95% confidence interval. Statistical significance was denoted as p < 0.05, p < 0.01 and p < 0.001. Data distribution is depicted with scatterplots. All graphs were generated using GraphPad Prism 8.0.2. and every value is expressed as Mean ± SEM.
4Results
4.1Behavioral Analysis
4.1.1Pretreatment With CBD Reduces AA Effects on Behavioral Parameters
For monitoring the role of CBD against AA‐induced brain lesions and malfunction, we used mice that we administered AA or vehicle for 5 days while two other groups received CBD for 10 days as shown in Figure 1A. One day after the last injection, animals were tested using the Open Field Test (OFT) to evaluate locomotor activity and anxious behavior (Figure 1B). Distance traveled was measured revealing an overall effect of AA (two‐way ANOVA, F 1,28 = 17.97, p < 0.0002) and an overall effect of CBD (two‐way ANOVA, F 1,28 = 17.97, p < 0.0002). As seen in Figure 1, AA administration reduced behavioral markers, while CBD coadministration led to a significant increase.
Specifically, AA intoxication resulted in reduced total distance traveled (25.500 ± 1.225, p < 0.032), entries in the center (17.655 ± 0.804, p < 0.0452) and time in the center (39.063 ± 6.886, p < 0.0452) when compared to the control group (38.250 ± 2.161, 21.984 ± 0.894 and 82.01 ± 6.56). On the contrary, CBD treatment led to increased total distance traveled (51.63 ± 5.134, p < 0.0023), entries in the center (29.81 ± 1.76, p < 0.0001) and time in the center (125.8 ± 11.23, p < 0.0001) when compared to the control group. CBD and AA coadministration (AA + CBD) resulted in an increase in the total distance traveled compared to the AA group (38.25 ± 2.34, p < 0.032). Similar to total distance traveled, CBD coadministration (AA + CBD) resulted in increased entries in the center (27.07 ± 0.57, p < 0.0001) and increased time in the center (74.44 ± 8,10, p < 0.0001) when compared to AA administration. Moreover, the AA + CBD group had reduced total distance traveled and time in the center compared to the CBD group (p < 0.0023, p < 0.001).
4.2Biochemical Blood Analysis
In order to clarify the general effect of AA on the overall health of animals including liver function, we performed biochemical blood analysis of different metabolic parameters. Table 1 presents the results of the biochemical analysis of blood samples from all four groups of animals.
| Con | AA | CBD | AA + CBD | |
|---|---|---|---|---|
| GLU (mg/dL) | 197.833 ± 19.992 | 207.333 ± 18.270 | 197.667 ± 15.121 | 194.833 ± 19.939 |
| BUN (mg/dL) | 20.800 ± 1.464 | 21.200 ± 1.468 | 19.917 ± 1.304 | 19.633 ± 1.309 |
| CREA (mg/dL) | 0.300 ± 0.037 | 0.250 ± 0.034 | 0.317 ± 0.031 | 0.300 ± 0.037 |
| CA (mg/dL) | 9.933 ± 0.226 | 9.767 ± 0.112 | 9.533 ± 0.143 | 9.517 ± 0.122 |
| TP (g/dL) | 5.833 ± 0.136 | 6.133 ± 0.092 | 5.867 ± 0.105 | 5.950 ± 0.134 |
| ALB (g/dL) | 2.983 ± 0.111 | 3.133 ± 0.067 | 3.250 ± 0.067 | 3.133 ± 0.095 |
| GLOB (g/dL) | 2.817 ± 0.114 | 2.917 ± 0.079 | 2.683 ± 0.117 | 2.783 ± 0.060 |
| ALT (U/L) | 60.667 ± 6.081 | 118.00 ± 13.974 b | 56.500 ± 1.945 | 73.833 ± 4.512 c |
| ALP (U/L) | 95.167 ± 4.377 | 128.33 ± 8.488 a | 96.333 ± 5.777 | 89.833 ± 4.799 c |
| TBIL (mg/dL) | 0.182 ± 0.028 | 0.518 ± 0.036 b | 0.195 ± 0.029 | 0.247 ± 0.027 d |
| CHOL (mg/dL) | 91.167 ± 6.426 | 82.333 ± 5.812 | 85.667 ± 4.566 | 95.500 ± 4.710 |
| AMYL (U/L) | 1324.424 ± 73.521 | 1267.284 ± 37.240 | 1386.075 ± 50.533 | 1282.723 ± 43.720 |
| LIP (U/L) | 73.167 ± 6.690 | 84.333 ± 4.759 | 69.500 ± 7.384 | 67.000 ± 4.008 |
Following acrylamide exposure, significant alterations were observed in metabolic markers primarily associated with liver function. Specifically, statistical analysis exhibited a CBD × AA interaction effect in levels of alanine aminotransferase (ALT) (two‐way ANOVA: F 1,20 = 6.241, p < 0.0213), alkaline phosphatase (ALP) (F 1,20 = 10.66, p < 0.004) and total bilirubin (TBIL) (F 1,20 = 22.55, p < 0.0001). Quantification of ALT, ALP and TBIL levels revealed a significant overall effect of AA (two‐way ANOVA: ALT F 1,20 = 21.74, p < 0.0001; ALP F 1,20 = 4.817, p < 0.040; TBIL F 1,20 = 41.87, p < 0.0001). Similarly, a significant overall effect of CBD was observed on ALT (F 1,20 = 9.112, p < 0.0001), ALP (F 1,20 = 9.442, p < 0.006) and TBIL (F 1,20 = 18.53, p < 0.0003) levels. Specifically, AA exposure led to elevated ALT (p < 0.0003), ALP (p < 0.005) and TBIL (p < 0.0001) levels compared to the control group. However, CBD treatment demonstrated a protective effect when administered prior to and together with AA. In the AA + CBD group, CBD significantly reduced ALT (p < 0.0045), ALP (p < 0.0012) and TBIL (p < 0.0001) levels compared to the AA group. Overall, the above findings suggest an AA‐driven liver malfunction offering a validation of the AA animal model while, importantly, CBD treatment in AA animals exhibited a protective effect.
4.3Oxidative Stress
As AA is known to induce oxidative stress in the brain, we next monitor markers of oxidative stress such as superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and catalase (CAT) in three brain regions: hippocampus, cerebral cortex and striatum. Results show that Acrylamide markedly elevated oxidative stress by impacting these markers across all examined brain regions (Table 2).
| Con | AA | CBD | AA + CBD | |
|---|---|---|---|---|
| MDA (pmol/mg protein) | ||||
| Hippocampus | 0.023 ± 0.001 | 0.035 ± 0.002 c | 0.024 ± 0.002 | 0.026 ± 0.001 e |
| Cerebral Cortex | 0.005 ± 0.001 | 0.012 ± 0.001 c | 0.003 ± 0.001 | 0.004 ± 0.001 f |
| Striatum | 0.022 ± 0.002 | 0.038 ± 0.002 c | 0.021 ± 0.002 | 0.020 ± 0.001 f |
| GSH (μmol/mg protein) | ||||
| Hippocampus | 1.369 ± 0.034 | 1.172 ± 0.056 a | 1.284 ± 0.032 | 1.350 ± 0.043 d |
| Cerebral Cortex | 0.402 ± 0.023 | 0.277 ± 0.017 a | 0.341 ± 0.029 | 0.331 ± 0.034 |
| Striatum | 1.933 ± 0.191 | 1.359 ± 0.080 a | 1.987 ± 0.126 | 2.073 ± 0.105 d |
| SOD (U/mg protein) | ||||
| Hippocampus | 5.802 ± 0.407 | 3.517 ± 0.317 a | 6.295 ± 0.736 | 5.071 ± 0.572 |
| Cerebral Cortex | 4.085 ± 0.377 | 1.692 ± 0.356 b | 4.606 ± 0.450 | 3.065 ± 0.595 |
| Striatum | 7.476 ± 0.733 | 3.836 ± 0.381 a | 8.868 ± 0.894 | 7.864 ± 1.288 d |
| CAT (U/mg protein) | ||||
| Hippocampus | 13.192 ± 1.274 | 7.499 ± 0.746 a | 10.628 ± 1.192 | 10.281 ± 1.415 |
| Cerebral Cortex | 15.800 ± 1.327 | 7.378 ± 0.846 c | 13.102 ± 0.759 | 13.238 ± 1.188 e |
| Striatum | 24.537 ± 1.950 | 15.623 ± 1.088 a | 24.815 ± 2.063 | 21.903 ± 2.718 |
4.3.1Malondialdehyde
Analysis of malondialdehyde (MDA) revealed an interaction between treatment and MDA levels across brain regions (two‐way ANOVA: hippocampus F 1,20 = 9.79, p < 0.005; cortex F 1,20 = 8.776, p < 0.0077; striatum F 1,20 = 20.56, p < 0.0002) (Figure 2). Results demonstrated an overall effect of AA in all brain regions studied (two‐way ANOVA: hippocampus F 1,20 = 15.09, p < 0.0009; cortex F 1,20 = 16.17, p < 0.0007; striatum F 1,20 = 15.86, p < 0.0007), as well as an overall effect of CBD (two‐way ANOVA: hippocampus F 1,20 = 4.838, p < 0.0398; cortex F 1,20 = 30.90, p < 0.0001; striatum F 1,20 = 22.91, p < 0.0001). Post hoc analysis showed a significant increase in MDA levels in the hippocampus (p < 0.0004), cortex (p < 0.0004) and striatum (p < 0.0001), after AA administration, compared to the control group (Figure 2A). Conversely, CBD coadministration in the AA + CBD group significantly lowered MDA levels, in all brain regions studied compared to the AA group, particularly in the hippocampus (p < 0.0061), cortex (p < 0.0001) and striatum (p < 0.0001). Additionally, CBD alone had no effects on MDA levels (Figure 2A).
4.3.2Glutathione
Two‐way ANOVA analysis of glutathione (GSH) levels showed a CBD × AA interaction in all brain regions studied (two‐way ANOVA: hippocampus F 1,20 = 9.654, p < 0.0056; cortex F 1,20 = 4.667, p < 0.0431; striatum F 1,20 = 9.654, p < 0.0056). AA treatment revealed an overall AA effect only in the cerebral cortex (two‐way ANOVA: F 1,20 = 6.513, p < 0.0190). Additionally, acrylamide significantly reduced GSH levels in all brain regions compared to the control group (hippocampus, p < 0.0173; cortex, p < 0.0161; striatum, p < 0.0173) (Figure 2B). CBD alone had no effects on GSH levels but CBD pretreatment in the AA + CBD group effectively reversed this reduction in the hippocampus (p < 0.0340) and striatum (p < 0.0340).
4.3.3Superoxide Dismutase
Statistical analysis of superoxide dismutase (SOD) data revealed an overall AA effect in the hippocampus, cerebral cortex and striatum (two‐way ANOVA, F 1,20 = 10.85, p < 0.0036; F 1,20 = 18.75, p < 0.0003; F 1,20 = 6.897, p < 0.0164). In addition, CBD administration showed an overall CBD effect only in the striatum (two‐way ANOVA, F 1,20 = 9.348, p < 0.0062). Post hoc analysis revealed a significant decrease in SOD levels following acrylamide exposure in the hippocampus (p < 0.0307), cerebral cortex (p < 0.0067) and striatum (p < 0.0402) compared to the Con group (Figure 2C). However, CBD pretreatment in the AA + CBD group significantly reversed the reduction in SOD levels in the striatum (p < 0.0209 compared to the AA group) while CBD alone had no effects on SOD levels (Figure 2C).
4.3.4Catalase
Catalase (CAT) data analysis revealed a CBD × AA interaction in the hippocampus and cerebral cortex (two‐way ANOVA F 1,20 = 5.100, p < 0.0352; F 1,20 = 16.40, p < 0.0006) (Figure 2D) as well as an overall AA effect across the brain regions (two‐way ANOVA, hippocampus: F 1,20 = 6.510, p < 0.0190; cerebral cortex: F 1,20 = 15.37, p < 0.0008; striatum F 1,20 = 8.411, p < 0.0089). Specifically, AA exposure significantly reduced CAT levels in the hippocampus (p < 0.0139), cerebral cortex (p < 0.0001) and striatum (p < 0.0271) compared to the Con group. No significant effect was observed in the CBD group; however, CBD pretreatment in the AA + CBD group increased CAT levels in the cerebral cortex (p < 0.0043) compared to the AA group. Overall, the above findings suggest that AA exposure triggers oxidative stress while CBD in AA‐treated animals blocks this AA effect.
4.4Pro‐Inflammatory Analysis
As oxidative stress is related to inflammatory response the pro‐inflammatory cytokines tumor necrosis factor‐alpha (TNF‐α) and Interleukin 6 (IL‐6) were quantified after exposure to acrylamide, CBD and their combined administration in three brain regions: the hippocampus, cerebral cortex and striatum (Figure 3A,B). Two‐way ANOVA analysis of the pro‐inflammatory cytokine TNF‐α revealed a CBD × AA interaction in the cerebral cortex (F 1,20 = 16.82, p < 0.0006) and striatum (F 1,20 = 14.19, p < 0.0012). Post hoc analysis revealed that AA significantly increased TNF‐α levels compared to controls in all regions (hippocampus, p < 0.0001; cortex, p < 0.0001; striatum, p < 0.0002), whereas CBD pretreatment minimized these AA‐induced increases (AA + CBD vs. AA: hippocampus, p < 0.0114; cortex, p < 0.0004; striatum, p < 0.0007). In the hippocampus, TNF‐α levels in the AA + CBD group remained higher than in the CBD group (p < 0.0039).
Regarding IL‐6, the statistical analysis showed a significant CBD × AA interaction effect in the cortex (F 1,20 = 8.411, p < 0.0089) and striatum (F 1,20 = 5.791, p < 0.0259). Moreover, a significant overall AA effect on IL‐6 levels was found in all brain regions (hippocampus F 1,20 = 8.529, p < 0.0085; cortex F 1,20 = 9.202, p < 0.0066; striatum F 1,20 = 55.18, p < 0.0001). However, the overall effect of CBD on IL‐6 levels was significant only in the striatum (F 1,20 = 6.447, p < 0.0195). Post hoc analysis revealed that AA exposure significantly elevated IL‐6 levels compared to control in all three regions (hippocampus, p < 0.0111; cortex, p < 0.0023; striatum, p < 0.0001). CBD pretreatment in the AA + CBD group significantly reduced IL‐6 levels in the cerebral cortex and striatum, compared to the AA group (p < 0.0122, p < 0.0112, respectively). Nevertheless, in the striatum, IL‐6 levels significantly increased in the AA + CBD group compared to the CBD group (p < 0.0099).
4.5Brain‐Derived Neurotrophic Factor Analysis
As previous studies casually related elevated oxidative stress, inflammatory responses and reduced neurotrophic levels, we next analyzed brain‐derived neurotrophic factor (BDNF) levels in the three brain areas (Figure 3C). Statistical analysis of BDNF levels showed an overall AA effect in the hippocampus (two‐way ANOVA, F 1,20 = 23.57, p < 0.0001), cerebral cortex (F 1,20 = 11.43, p < 0.0030) and striatum (F 1,20 = 14.93, p < 0.0010) and an overall CBD effect in the hippocampus (F 1,20 = 72.61, p < 0.0001) and in the striatum (F 1,20 = 5.006, p < 0.0368). Post hoc analysis revealed a significant decrease in BDNF levels in all three brain regions after AA exposure (hippocampus 251.463 ± 26.002 pg/mg of protein, cerebral cortex 169.672 ± 15.364 pg/mg of protein and striatum 316.577 ± 29.662 pg/mg of protein) compared to the control group (hippocampus 365.000 ± 4838 pg/mg of protein, p < 0.0111; cerebral cortex 319.143 ± 35,020 pg/mg of protein, p < 0.0191; striatum 514.547 ± 41.885 pg/mg of protein, p < 0.0049). CBD administration significantly increased BDNF levels in the hippocampus (565.174 ± 24.249 pg/mg of protein) compared to the control group (p < 0.0001). However, this BDNF‐increasing effect of CBD was not found in the cortex or striatum. Additionally, the reduction in BDNF levels observed after AA exposure was reversed with CBD pretreatment in the AA + CBD group in the hippocampus (450.976 ± 30.211 pg/mg of protein, p < 0.0001). However, the AA + CBD group still showed lower BDNF levels compared to the CBD‐only group (p < 0.0126) suggesting that CBD minimized the BDNF‐reducing effect of AA. No significant effect on BDNF levels was found between AA + CBD and AA groups in both cortex and striatum.
4.6Cholinergic Analysis
4.6.1Acetylcholinesterase Activity
Determination of AChE activity in both SS and DS fraction indicated a CBD × AA interaction in all brain regions analyzed (SS fraction: two‐way ANOVA, hippocampus F 1,20 = 20.85, p < 0.0002; cortex F 1,20 = 10.65, p < 0.0039; striatum F 1,20 = 7.889, p < 0.0108; and DS fraction: two‐way ANOVA, hippocampus F 1,20 = 21.62, p < 0.0002; cortex F 1,20 = 10.77, p < 0.0037; striatum F 1,20 = 29.33, p < 0.0001) (Figure 4A,B).
Also, AChE activity in the SS fraction results showed an overall AA effect in the hippocampus (two‐way ANOVA, F 1,20 = 16.13, p < 0.0007), in the cerebral cortex (F 1,20 = 9.737, p < 0.0054) and in the striatum (F 1,20 = 6.534, p < 0.0188) and an overall CBD effect only in the striatum (F 1,20 = 7.200, p < 0.0143). Specifically, AA intoxication significantly decreased AChE activity in the SS fraction in all brain regions: hippocampus (2.263 ± 0.307 μmol/min/g tissue), cerebral cortex (3.127 ± 0.270 μmol/min/g tissue) and striatum (1.872 ± 0.175 μmol/min/g tissue) compared to the control group (5.204 ± 0.558 μmol/min/g tissue, p < 0.0001; 11.140 ± 1.688 μmol/min/g tissue, p < 0.0011; 4.670 ± 0.918 μmol/min/g tissue, p < 0.0058, respectively). Similarly, CBD treatment significantly decreased AChE activity in the SS fraction of the hippocampus (3.338 ± 0.239 μmol/min/g tissue) and striatum (1.805 ± 0.436 μmol/min/g tissue) compared to the control group (p < 0.005 and p < 0.0047). For the hippocampus and striatum, any further reduction of AChE activity in the AA + CBD group remained at similar levels to the AA or CBD groups. However, AChE activity was increased in the cerebral cortex (8.321 ± 1.543 μmol/min/g tissue) compared to the AA group (p < 0.0384).
Similar to SS fraction, in DS fraction results also showed an overall AA effect in hippocampus (two‐way ANOVA, F 1,20 = 16.39, p < 0.0006), in cerebral cortex (F 1,20 = 14.32, p < 0.0012) and in striatum (F 1,20 = 8.726, p < 0.0078) and an overall CBD effect only in hippocampus (F 1,20 = 17.66, p < 0.0004) and striatum (F 1,20 = 8.020, p < 0.0103). Post hoc analysis showed that AA exposure significantly decreased AChE activity in DS fraction in all brain regions, hippocampus (13.977 ± 0.251 μmol/min/g tissue), cerebral cortex (15.123 ± 1.715 μmol/min/g tissue) and striatum (41.908 ± 1.729 μmol/min/g tissue) compared to control group (20.992 ± 1.330 μmol/min/g tissue, p < 0.0001; 42.961 ± 3.856 μmol/min/g tissue, p < 0.0004; 63.321 ± 3.656 μmol/min/g tissue, p < 0.0001, respectively). CBD treatment also significantly decreased AChE activity in the DS fraction of hippocampus (13.853 ± 0.396 μmol/min/g tissue), cerebral cortex (26.475 ± 3.636 μmol/min/g tissue) and striatum (42.221 ± 2.426 μmol/min/g tissue) compared to the control group (p < 0.0001, p < 0.0359 and p < 0.0001). Additionally, AChE activity in the SS fraction of the AA + CBD group was reduced across the regions, hippocampus (14.338 ± 0.783 μmol/min/g tissue), cerebral cortex (24.498 ± 5.573 μmol/min/g tissue) and striatum (48.518 ± 1.985 μmol/min/g tissue) compared to the control group (p < 0.0001, p < 0.0168 and p < 0.0029).
4.6.2Acetylcholine
Analysis of the neurotransmitter, acetylcholine, demonstrated an overall AA effect in all brain regions studied (two‐way ANOVA: hippocampus F 1,20 = 25.31, p < 0.0001; cortex F 1,20 = 24.86, p < 0.0001; striatum F 1,20 = 23.52, p < 0.0001), as well as an overall effect of CBD (two‐way ANOVA: hippocampus F 1,20 = 22.58, p < 0.0001; cortex F 1,20 = 16.62, p < 0.0006; striatum F 1,20 = 15.31, p < 0.0009). Post hoc analysis showed that ACh levels of the AA group were significantly lower in all regions examined: hippocampus (0.337 ± 0.036 nmol/mg of protein), cerebral cortex (0.471 ± 0.043 nmol/mg of protein) and striatum (0.460 ± 0.039 nmol/mg of protein), compared to the control group (0.593 ± 0.033 nmol/mg of protein, p < 0.0088; 0.698 ± 0.034 nmol/mg of protein, p < 0.0464; 0.835 ± 0.046 nmol/mg of protein, p < 0.0424, respectively) (Figure 4C).
On the contrary, ACh levels in the CBD group were significantly higher in all regions studied: hippocampus (0.829 ± 0.042 nmol/mg of protein), cerebral cortex (0.983 ± 0.084 nmol/mg of protein) and striatum (1.268 ± 0.164 nmol/mg of protein), compared to the control group (p < 0.0169; p < 0.0095; p < 0.0166, respectively) suggesting that CBD alone enhanced neurotransmission. ACh levels in the AA + CBD group were significantly lower in the hippocampus (0.579 ± 0.077 nmol/mg of protein), cerebral cortex (0.646 ± 0.052 nmol/mg of protein) and striatum (0.749 ± 0.060 nmol/mg of protein) compared to the CBD group (p < 0.0109, p < 0.0022 and p < 0.0038, respectively). Moreover, it is clear in the hippocampus that CBD in AA‐treated animals minimized the AA‐reducing effect as AA + CBD levels of ACh were significantly higher compared to the AA group (p < 0.0136).
5Discussion
The global environmental pollution, its effects on brain health and their relation to age‐related neurodegenerative disorders are among the first priorities of the worldwide research efforts. Acrylamide (AA) is considered an environmental and dietary pollutant while the AA exposure to the human organism and brain can be detrimental. Indeed, AA is a known neurotoxic agent, induces oxidative stress, inflammation and cholinergic dysregulation, contributing to neurodegenerative‐like conditions, to which humans may be exposed primarily through dietary intake (Bin‐Jumah et al. 2021; Edres et al. 2021; Fang et al. 2022; Gur et al. 2021; Ibrahim and Shahen 2023; Mirzavi et al. 2024). However, the neurotoxic effects of AA on brain tissue are not yet fully investigated while the testing of potent protective compounds and strategies is of paramount importance. Hereby, we test for the first time the potentially beneficial effect of CBD against AA‐induced brain lesions on different brain areas and different mechanisms including inflammation (e.g., TNF‐α, IL6), neurotrophic markers (e.g., BDNF) as well as neuronal communication (e.g., cholinergic markers) all related to AA neurotoxicity. This study has been conducted using male mice, in line with previous research; however, this limitation is acknowledged and future studies should include female mice to expand the scope of findings (Data S1).
AA administration led to an imbalance in hepatic enzymes (ALT, ALP, and TBIL), indicating toxicity from AA, which aligns with relevant research (Shrivastava et al. 2019; Uthra et al. 2022). CBD, administered at a dose of 10 mg/kg/day for 10 consecutive days, was well‐tolerated and did not affect hepatic health. Notably, in the AA + CBD group, CBD significantly mitigated the effects of AA on these enzymes.
The Open Field Test results indicate that AA exposure induces anxiety‐like behavior, reducing total distance traveled, center entries and time spent in the center of the field. These findings are consistent with previous studies that highlight AA's neurobehavioral toxicity, which may be linked to disrupted neurotransmitter signaling and increased oxidative stress (Elblehi et al. 2020; Kunnel et al. 2019). CBD treatment significantly attenuated these behavioral changes, improving exploratory activity and reducing anxiety‐like behavior. The anxiolytic effects of CBD, on its own, were clearly demonstrated in our experiments. This is in line with previous findings showing that CBD modulates serotonergic signaling, particularly via 5‐HT1A receptors, which are implicated in anxiety regulation (Blessing et al. 2015; Sartim et al. 2016). Interestingly, in agreement with previous research in mice with nicotine intoxication (Mesiakaris et al. 2024), with chronic unpredictable stress (Campos et al. 2013), with stress‐induced hyperthermia (Shu et al. 2024) and in middle‐aged female rats exposed to social isolation (Saad et al. 2023), the recovery observed in the AA + CBD group indicates that CBD does not fully block AA effects but rather minimizes AA‐induced behavioral impairments, leading to a behavioral profile that approximates that of control animals.
The study's findings demonstrated that CBD could mitigate oxidative damage in brain tissues caused by AA. This protective effect was reflected in increased glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels and a reduction in malondialdehyde (MDA) levels in CBD + AA‐treated mice. In particular, the coadministration of CBD fully reversed the elevated MDA levels caused by AA intoxication in all brain regions. Interestingly, CBD coadministration restored GSH to control values in the striatum and hippocampus, fully reversed SOD imbalances in the striatum, and normalized CAT activity in the cerebral cortex. These results suggest that CBD exhibits tissue‐specific antioxidant capabilities. Moreover, following the AA exposure, the decrease in GSH, SOD, and CAT levels suggests a decrease in antioxidant defense. Additionally, the increased MDA levels indicate an elevation in lipid peroxidation, which may pose a threat to cellular stability. This aligns with prior research showing that AA induces reactive oxygen species (ROS) production, leading to lipid peroxidation and cellular damage (Prasad 2012). In a rat model of ischemic stroke, CBD reduced brain infarction volume, decreased malondialdehyde (MDA) levels and increased antioxidant enzyme activity (Khaksar et al. 2022). Similarly, in a mouse model of multiple sclerosis CBD reduced oxidative stress (Sajjadian et al. 2017). CBD also demonstrated protective effects in a sepsis model in rats, reducing oxidative stress parameters in peripheral organs and the brain (Cassol‐Jr et al. 2010) CBD administration effectively reduced MDA levels and partially restored antioxidant activity, suggesting its neuroprotective role through free radical scavenging and enhancement of endogenous antioxidant mechanisms (Borges et al. 2013; García‐Arencibia et al. 2007; Khaksar et al. 2022). Taken together, the observed biochemical improvements occurred in parallel with behavioral recovery in the OFT, indicating a likely association between redox balance and anxiety‐like behavior. Similar associations have been described in other models of neurotoxicity and neurodegeneration (Fedoce et al. 2018; Ferlemi et al. 2014; Mancini et al. 2025).
Oxidative stress is a central mechanism in tissue damage, arising from an imbalance between excessive reactive oxygen species (ROS) production and the endogenous antioxidant defense systems. Elevated ROS levels trigger lipid peroxidation, protein oxidation and DNA damage, which in turn compromise cellular integrity and promote inflammation (Aboubakr 2019; Aboubakr et al. 2023). Such mechanisms have been well described in models of acrylamide toxicity and other toxic insults, underscoring the importance of targeting redox balance to prevent neurodegeneration (Acaroz et al. 2018).
Previous research has examined the potential of CBD in mitigating inflammation and its associated effects (Chen et al. 2016; Florensa‐Zanuy et al. 2021; Karimi‐Haghighi et al. 2020; Mecha et al. 2013; Pellati et al. 2018). Recent studies show that CBD reduces TNF‐α and IL‐6 levels in mice following LPS stimulation and nicotine intoxication (Fouda et al. 2022; Mesiakaris et al. 2024). The significant increase in pro‐inflammatory cytokines TNF‐α and IL‐6 in AA‐treated mice confirms AA's role in triggering neuroinflammation, as previously reported (Acaroz et al. 2018; Pan et al. 2018). In a methamphetamine reinstatement model, CBD modulated the expression of pro‐inflammatory cytokines, including IL‐1β, IL‐6, IL‐10, and TNF‐α, in the prefrontal cortex and hippocampus (Karimi‐Haghighi et al. 2020). These effects are likely achieved through CBD's ability to inhibit NF‐κB activation and modulate microglial activity, thereby reducing neuroinflammatory responses (Dos‐Santos‐Pereira et al. 2020). Similarly, in a lipopolysaccharide (LPS)‐induced neuroinflammatory model, CBD decreased IL‐6 levels in both plasma and the brain while simultaneously reducing NF‐κB activation (Florensa‐Zanuy et al. 2021). Our study found that AA intoxication increases the release of TNF‐α and IL‐6 in all brain regions studied. Notably, coadministration of CBD significantly reduces these proinflammatory cytokines, suggesting a protective effect of CBD in the early phase of cytokine production. These results align with previous research, supporting the role of CBD in modulating TNF‐α and IL‐6 levels in response to AA‐induced neuroinflammation. In our study, TNF‐α reduction was observed across all brain regions following CBD coadministration, whereas IL‐6 reduction was significant only in the cortex and striatum. This indicates a region‐specific anti‐inflammatory action of CBD, which may relate to differences in microglial activity and NF‐κB signaling between brain regions, consistent with prior evidence that cytokine modulation can vary between hippocampus, cortex and other areas (Karimi‐Haghighi et al. 2020).
BDNF is a crucial neurotrophic factor for synaptic plasticity that promotes neuron growth and supports the development of noradrenergic and serotonergic neurons and shields them from neurotoxic damage (Fang et al. 2022; Lima Giacobbo et al. 2019; Zaid et al. 2017). Current studies report a decrease in BDNF levels in the brains of rats, attributed to oxidative stress induced by AA in brain tissues (Mansour et al. 2017; Zaid et al. 2017). Our findings align with previous studies demonstrating that AA reduces BDNF expression in key brain regions, impairing neuroplasticity (Erdemli et al. 2018; Lima Giacobbo et al. 2019). CBD treatment increased BDNF levels, consistent with its reported role in promoting neurogenesis (Sartim et al. 2016). In middle‐aged female rats, CBD restored social isolation‐induced decreases in BDNF levels in the nucleus accumbens and increased BDNF expression in the medial prefrontal cortex and basolateral amygdala (Saad et al. 2023). Additionally, repeated CBD treatment in cocaine‐consuming mice increased BDNF expression and neural cell proliferation in the hippocampus, while also reducing cocaine intake (Luján et al. 2018). Our findings show that CBD alone significantly increased BDNF levels only in the hippocampus, a brain region where BDNF is highly expressed, demonstrating a tissue‐specific effect. In the AA + CBD group, CBD counteracted the acrylamide‐induced reduction, restoring hippocampal BDNF to control levels. The improvements observed in the hippocampus suggest that CBD could be beneficial in counteracting AA‐induced neuroplasticity deficits.
Beyond its effects on BDNF, CBD has also been reported to act through upstream molecular pathways, including activation of the Nrf2/HO‐1 antioxidant response (Atalay et al. 2020), inhibition of NF‐κB signaling (Dos‐Santos‐Pereira et al. 2020) and modulation of CREB‐BDNF signaling (Sartim et al. 2016). In addition to these pathways, CBD may exert its protective actions through interactions with the endocannabinoid system and related receptor targets. For instance, CBD indirectly influences CB1 and CB2 receptors by elevating anandamide levels, modulates TRPV1 channels and acts as a partial agonist at 5‐HT1A receptors (Campos et al. 2016; Castillo et al. 2010; Sartim et al. 2016). These receptor‐ and pathway‐related mechanisms may therefore complement the antioxidant and anti‐inflammatory effects observed in our study, providing a broader framework for understanding CBD's neuroprotective actions and pointing to potential relevance beyond acrylamide toxicity. Indeed, similar mechanisms are implicated in neurodegenerative diseases such as Parkinson's and Alzheimer's, suggesting broader translational potential (Chen et al. 2023; Marques and Campos 2024; Patricio et al. 2020).
In our research, AA exposure significantly decreases cholinergic function by reducing ACh levels and AChE activity. AA induced oxidative stress by generating free radicals, which block AChE receptors and lead to a decrease in AChE activity in the rats' brains (Farouk et al. 2021; Shrivastava et al. 2019). These findings support previous research showing that AA disrupts cholinergic signaling, contributing to cognitive dysfunction (Farouk et al. 2021; Kopańska et al. 2022; Shrivastava et al. 2019). In CBD + AA‐treated mice, CBD minimized the AA‐induced decrease in ACh levels, but AChE activity remained low. This reduced AChE activity is due to CBD displaying competitive inhibition on AChE (Puopolo et al. 2022; Suttithumsatid et al. 2023). Furthermore, it has been demonstrated that cannabinoid‐rich extracts exhibit AChE inhibitory activity, with CBD potentially being the active compound (Zhang et al. 2024). These results suggest that CBD has the potential to preserve cholinergic neurotransmission. Its effect on AChE may be mediated both directly, through enzyme inhibition and indirectly, by lowering AA‐induced ROS. Notably, sustaining acetylcholine signaling via AChE inhibition is a recognized therapeutic approach in Alzheimer's disease and CBD's modulatory actions on this pathway may therefore represent a neuroprotective mechanism with translational potential (Chen et al. 2022; Hickey et al. 2024; Mello‐Hortega et al. 2025).
In line with these mechanistic findings, the improvement in behavioral outcomes observed in the OF test appears to reflect, at the functional level, the attenuation of AA‐induced neurochemical disturbances across redox, neurotrophic and cholinergic pathways, further supporting the integrated neuroprotective profile of CBD.
6Conclusion
By improving behavioral outcomes, reducing oxidative stress, modulating inflammation, enhancing neuroplasticity and preserving cholinergic function, CBD shows promise as a potential therapeutic approach for neurotoxic and neurodegenerative conditions. However, further research is necessary to determine combination therapies to fully harness CBD's therapeutic potential in mitigating AA‐induced neurotoxicity. Finally, this study provides valuable insights into the health risks associated with acrylamide exposure, a pollutant of both environmental and dietary relevance and highlights the potential of natural antioxidants such as CBD in mitigating these risks.
Funding
The authors have nothing to report.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Acknowledgments
The publication of this article in OA mode was financially supported by HEAL‐Link.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.