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. 2023 Dec 21;19(12):2750–2759. doi: 10.4103/1673-5374.391300

Vanillylacetone attenuates cadmium chloride-induced hippocampal damage and memory loss through up-regulation of nuclear factor erythroid 2-related factor 2 gene and protein expression

Fahaid H AL-Hashem 1, Salah O Bashir 1, Amal F Dawood 2, Moutasem S Aboonq 3, Ismaeel Bin-Jaliah 1, Abdulaiziz M Al-Garni 4, Mohamed D Morsy 1,*
PMCID: PMC11168521  PMID: 38595292

graphic file with name NRR-19-2750-g001.jpg

Keywords: hippocampus, neuroprotective, Nrf2 gene, oxidative stress, vanillylacetone

Abstract

Memory loss and dementia are major public health concerns with a substantial economic burden. Oxidative stress has been shown to play a crucial role in the pathophysiology of hippocampal damage-induced memory impairment. To investigate whether the antioxidant and anti-inflammatory compound vanillylacetone (zingerone) can protect against hippocampal damage and memory loss induced by cadmium chloride (CdCl2) administration in rats, we explored the potential involvement of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which is known to modulate oxidative stress and inflammation. Sixty healthy male Wistar rats were divided into five groups: vehicle-treated (control), vanillylacetone, CdCl2, vanillylacetone + CdCl2, vanillylacetone + CdCl2 + brusatol (a selective pharmacological Nrf2 inhibitor) groups. Vanillylacetone effectively attenuated CdCl2-induced damage in the dental gyrus of the hippocampus and improved the memory function assessed by the Morris Water Maze test. Additionally, vanillylacetone markedly decreased the hippocampal tissue levels of inflammatory biomarkers (interleukin-6, tumor necrosis factor-α, intracellular cell adhesive molecules) and apoptosis biomarkers (Bax and cleaved caspase-3). The control and CdCl2-treated groups treated with vanillylacetone showed reduced generation of reactive oxygen species, decreased malondialdehyde levels, and increased superoxide dismutase and glutathione activities, along with significant elevation of nuclear Nrf2 mRNA and protein expression in hippocampal tissue. All the protective effects of vanillylacetone were substantially blocked by the co-administration of brusatol (a selective Nrf2 inhibitor). Vanillylacetone mitigated hippocampal damage and memory loss induced by CdCl2, at least in part, by activating the nuclear transcription factor Nrf2. Additionally, vanillylacetone exerted its potent antioxidant and anti-inflammatory actions.

Introduction

Exposure to environmental toxins is associated with an increased risk factor for developing several neurological disorders (Chin-Chan et al., 2015). Cadmium (Cd) ion is one of the most known toxic heavy metals abundantly found in soil, tobacco smoke, vegetables, fruits, batteries, and other industrial and commercial products, therefore having different routes of entry into the body (Genchi et al., 2020). Once inside the body, Cd ions can accumulate in the liver, kidneys, and brain, leading to severe toxicities and tissue damage, mainly due to the upregulation of oxidative stress, inflammation, and apoptosis (Genchi et al., 2020). In the brain, the toxicities of Cd ions are well-documented in all brain areas, including the cerebral cortex and hippocampus. They are associated with peripheral neuropathy, unsteady gait, cognitive impairment, memory deficits, and poor attention (Saenghong et al., 2012). In addition, intoxication with Cd ions is linked to an increased risk of Parkinson’s and Alzheimer’s disorders (PD & AD, respectively; Branca et al., 2018; Gonçalves et al., 2021).

During the last decades, much interest has been given by researchers to identify the precise mechanisms by which Cd ions induce neural damage and apoptosis. In developing brains, Cd ions can alter the permeability of the blood-brain barrier (BBB), leading to neurodegeneration (Wang and Du, 2013). However, in adults, Cd ions primarily enter the central nervous system (CNS) through the olfactory route, disrupting the BBB and causing neurodegeneration (Branca et al., 2018). Various studies have demonstrated that the overproduction of reactive oxygen species (ROS) by cadmium chloride (CdCl2) exposure is the primary mechanism underlying the activation of neuroinflammatory and various apoptotic pathways (e.g., p38, JNK, and mTOR). This elevated ROS level also disrupts synaptic connections and neurotransmission (Wang and Du, 2013). The nuclear factor-erythroid 2-related factor 2 (Nrf2) is the primary antioxidant transcription factor that binds to the antioxidant responsive element (ARE) to stimulate glutathione (GSH) synthesis and phase II antioxidant enzymes such as heme oxygenase-1 (HO-1), catalase (CAT), and superoxide dismutase (SOD) in response to oxidative damage (Loboda et al., 2016). Moreover, Nrf2 also inhibits apoptosis by upregulating the expression of Bcl2 and suppressing the phosphorylation of the JNK protein (Loboda et al., 2016). Under normal conditions, Nrf2 is inactive in the cytoplasm due to its conjugation with its natural inhibitory protein, kelch-like ECH-associated protein 1 (Keap-1) (Loboda et al., 2016). However, under stress and oxidative damage, Nrf2 dissociates from Keap-1 and is translocated to the nucleus, thereby enhancing its transcriptional activity. The levels and activities of Nrf2 have been observed to be reduced in several animal models of neurodegeneration. In contrast, the activation of this transcription factor protects against oxidative/inflammatory damage and neural apoptosis, mitigating the impairments in memory function (El-Kott et al., 2020). Similarly, prolonged exposure and high doses of Cd ions in adult rats are linked to increased expression of Keap-1, reduced expression and transactivation of Nrf2, diminished levels and expression of GSH and antioxidant enzymes, hippocampal damage, and memory loss (Ali et al., 2021). However, these researchers demonstrated that pharmacological activation of Nrf2 ameliorates Cd ions-induced cortical and hippocampal damage and represents an effective strategy for improving memory function.

The role of plant polyphenols in preventing oxidative stress-induced neurodegeneration has garnered significant attention in recent decades. Vanillylacetone (also known as zingerone) is isolated from the ginger plant (Zingiber officinale) and is commonly used as a food flavoring. Vanillylacetone possesses numerous health benefits, including hypoglycemic, hypolipidemic, insulin-sensitizing, antioxidant, and anti-inflammatory effects (Zhu et al., 2021). In several animal models, the protective roles of vanillylacetone have been demonstrated against neural, hepatic, renal, and cardiac damage. These protective effects are attributed to scavenging ROS, improving mitochondrial structure and function, upregulating antioxidants, suppressing the production of inflammatory cytokines, and activating Nrf2 (Anand and Dhikav, 2012).

According to existing literature, only two studies have examined the neuroprotective effects of vanillylacetone against cerebral and hippocampal damage in animal models of transient ischemia and epilepsy (Vaibhav et al., 2013; Rashid et al., 2021). In both studies, vanillylacetone prevented against neural damage by reducing ROS generation, stimulating antioxidant (GSH and SOD) levels, upregulating Bcl2, and inhibiting Bax/caspase-3-mediated apoptosis. However, the neuroprotective effects of vanillylacetone were poorly investigated. Therefore, further studies examining the neuroprotective effects of vanillylacetone and its mechanism of action are highly necessary.

This study showed that daily dietary administration of vanillylacetone mitigated hippocampal damage and memory loss induced by CdCl2 in adult rats. It can be suggested that the preferred mechanism of action of vanillylacetone involves at least the suppression of oxidative stress and inflammation, mainly through the upregulation of Nrf2.

Methods

Ethics statement

This study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23) and approved by the Animal Ethical Committee of the College of Medicine, King Khalid University, Abha, Saudi Arabia on March 8, 2021 (approval No. REC # 2021-03-08). This study was reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments; Percie du Sert et al., 2020).

Animals

To avoid the interference of female hormones on hippocampal activities, sixty healthy male Wistar rats aged 10 weeks, weighing 260 ± 20 g, were obtained from the animal facility department at King Khalid University (KKU). The animals were maintained at a constant temperature of 22°C, 60–65% humidity, and a 12-hour dark/light cycle throughout the study. All rats had unlimited access to their food and drinking water.

Experimental design

Vanillylacetone (Cat# W312401, Sigma-Aldrich, Gillingham, UK) powder was always freshly prepared in 5% carboxymethylcellulose (CMC). After a 1-week acclimation period, the rats were randomly allocated into five groups (each with 12 rats): 1) control rats: 5% CMC (vehicle); 2) vanillylacetone-treated rats: 5% CMC + vanillylacetone solution (100 mg/kg); 3) CdCl2-treated group rats: 5% CMC + CdCl2 solution (Sigma-Aldrich) (0.5 mg/kg); 4) vanillylacetone + CdCl2: provided vanillylacetone solution (100 mg/kg) and concomitantly administered CdCl2 solution (0.5 mg/kg); and 5) vanillylacetone + CdCl2 + brusatol (a selective Nrf2 inhibitor) (Sigma-Aldrich): were treated as in group 4 but were also intraperitoneally administered brusatol (2 mg/kg twice per week). All other treatments were given orally (by gavage) and daily. The experiment was terminated after four weeks. Body weights and food intake were recorded daily. Our study showed a mortality rate of 10% due to CdCl2 toxicity or hypersensitivity. Three rats from the CdCl2 group, two from the CdCl2 + vanillylacetone + brusatol-treated group, and one from the control group died. The dead rats underwent necropsy and histopathological examination revealed severe hepatitis as the reason behind their death.

Doses of drugs

The CdCl2 dose was selected based on previous research showing that this dosage is capable of causing hippocampal damage and memory loss in rats after 4 weeks of treatment (El-Kott et al., 2020). A recent study has demonstrated the ability of brusatol to inhibit the hepatic activation of Nrf2 after chronic administration at a dose of 2 mg/kg twice per week, therefore, we used the dose and route of administration as outlined in this study (Ahmad et al., 2018). The dose of vanillylacetone was shown to be the most effective therapeutic dose for alleviating renal and hepatic oxidative stress and inflammation and preventing hippocampal damage after induction of focal ischemia in rats (Vaibhav et al., 2013).

Morris Water Maze test

We used the Morris water maze (MWM) test to measure spatial memory on 4 consecutive days (days 32–35) as previously described by Morris (1984). The maze (a circular swimming pool with a diameter of 180 cm) was filled with water (60 cm deep and a temperature of 22 ± 2°C). A circular platform (with a diameter of 10 cm) was submerged 2 cm below the surface of milky water in the middle of the SW quadrant (the target quadrant). All animals were tested to find this hidden platform. Each rat was placed at a different starting position (N, E, or NE) and released to find the hidden platform. This procedure was repeated for 4 days with three trials per day (each trial, which lasted 90 seconds, was separated by an interval of 5 minutes), and conducted from different starting positions (N, E, or SE) each day. The selected starting positions created an even distribution of paths, reducing variability in the results. If a rat was unable to find the platform, it was guided by the investigator and left on the platform for 15 seconds. The examiner recorded the escape time, which was the time taken by each rat to find the hidden platform. Furthermore, an additional probe trial was conducted for all rats one hour later. During this trial, the examiner removed the rescue platform and recorded the total number of times each rat crossed the area where the hidden platform had been. This was used to assess the rats’ long-term memory of the platform’s location.

Isolation of the hippocampus and drug administration

After the MWM test, the animals were anesthetized by intramuscular injection of 60 mg/kg ketamine hydrochloride (Pfizer, New York, NY, USA). Following the completion of the MWM test, the animals were euthanized by neck dislocation, and their skulls were opened. The brains were immediately removed and placed on ice. Some brains (n = 4 per group) were placed in a 10% buffered formalin solution and used within 24 hours for the study of the hippocampal structure. The remaining eight brains in each group were used to harvest the hippocampi under a dissecting microscope. These hippocampi were then stored at -80°C for future analysis.

Analysis of Cd levels in the hippocampus

Freshly harvested hippocampal samples were homogenized in phosphate-buffered saline (pH 7.4) and then 5 mL of 70% nitric acid (HNO3) was added to each sample. The mixture was heated at 85°C for 30 minutes, and then allowed to cool. 1.5 mL of 70% perchloric acid was then added to each sample and heated at 220°C. The clear solution was isolated and cooled, and the volume was made up to 10 mL with deionized distilled water. The solution was then filtered and analyzed using graphite furnace (electrothermal) atomic absorption spectrometry (Model AA-6800, Shimadzu, Korea) (http://www.a-a.co.kr/pdf/AA-6800.pdf). Cd ions were measured using standard samples.

Biochemical analysis in the hippocampus

Parts of the frozen hippocampal tissues (40 mg) from each sample were homogenized in ice-cold PBS. After centrifugation at 4°C for 10 minutes at 11,000 × g, the supernatant homogenates were collected and stored at –80°C. In addition, the Celllytik isolation kit (NXTRACT, Sigma, St. Louis, MO, USA) (https://www.sigmaaldrich.com/IN/en/product/sigma/nxtract) was used to isolate nuclear proteins from various areas of the hippocampi. SIRT1 and Nrf2 levels in nuclear extracts were determined using rat-specific enzyme-linked immunosorbent assay (ELISA) kits (Cat# 2600246 and Cat# 752046, respectively, from MyBioSource, Waltham, MA, USA) (https://www.mybiosource.com). The total homogenate levels of glutathione (GSH), malondialdehyde (MDA), interleukin-6 (IL-6), intracellular cell adhesive molecules (ICAMs), tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD) were measured using special rat kits (Cat# 265966, 268427, 175908, 267983, 2881838, MyBioSource, San Diego, CA, USA). A rat-specific fluorometric kit (Cat# 186027, Abcam, Cambridge, MA, USA) was used to quantify the levels of ROS and reactive nitrogen species (RNS). All measurements were performed in triplicate and in accordance with the suppliers’ specifications.

Real-time PCR

Primers were synthesized and purchased from Thermo Fisher Scientific, Bremen, Germany, to study the transcription of Nrf2, Bax, Bcl2, and β-actin. The sequences of the primers for Nrf2 were as follows: CAC ATC CAG ACA GAC ACC AGT and R: CTA CAA ATG GGA ATG TCT CTG C (103 bp). For Bcl2, the sequences were F: TGG GAT GCC TTT GTG GAA CT and R: TCT TCA GAG ACT GCC AGG AGA AA (73 bp). For Bax, the sequences were F: ATG GAG CTG CAG AGG ATG ATT and R: TGA AGT TGC CAT CAG CAA ACA (97 bp). For β-actin, the sequences were F: TAC CCA GGC ATT GCT GAC AG and R: AGC CAC CAA TCC ACA CAG AG (115 bp). Total RNA was isolated from the frozen hippocampal samples using a commercial kit (Cat# 12183018A, Thermo Fisher Scientific, https://www.thermofisher.com/de/en/home/life-science/sequencing.html). The purity of the DNA was determined as the absorption at 260/280 nm in a nanodrop machine (LightMachinery, Ottawa, ON, Canad). A commercially available cDNA synthesis kit (Cat# K1621, Thermo Fisher Scientific; https://www.thermofisher.com/de/en/home/life-science/sequencing.html) was used for the synthesis of the first-strand cDNA. The real-time PCR reaction was performed in a CFX96 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA; https://www.bio-rad.com/en-us/product/cfx96-touch-real-time-pcr-detection-system?ID=LJB1YU15). The Ssofast Evergreen Supermix was used for the amplification reaction. The quantities of all included ingredients and amplification steps were based on the supplier’s instructions (Cat# 172-5200, Bio-Rad Laboratories; https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10014647A.pdf). In brief, the amplification reaction (20 µL) contained the following: 500 ng/µL template cDNA (2 μL), 0.4 μL of the stock 10 μM forward primers (200 nm/well); 0.4 μL of the stock 10 μM reverse primers (200 nm/well); 10 μL of the Supermix, and 7.2 μL of nuclease-free water. The following were the steps for the amplification: an initial heating at 95°C for 30 seconds (1 cycle), denaturation at 95°C for 5 seconds (40 cycles), annealing at 60°C for 60 seconds (40 cycles), and one melting cycle at 95°C for 30 seconds. All target gene expression levels were measured using the associated software (https://imagelab 6-0.software.informer.com/ Bio-Rad Laboratories). The expression levels were presented relative to the expression of the reference gene, β-actin, using the ΔΔCT method (El-Kott et al., 2020). Finally, the Bax/Bcl2 ratio was calculated.

Western blot assay

Radio-immunoprecipitation (RIPA) buffer with a protease inhibitor cocktail was used to homogenize portions of the frozen hippocampus tissues (Cat# 201111, Abcam). To separate the supernatants containing the total proteins, all samples were centrifuged for 15 minutes at 11,500 × g at 4°C. The quantities of protein in all supernatants were determined using an assay kit (Cat# 23225, Thermo Fisher Scientific) and then diluted in the loading buffer (130 mM sodium carbonate pH 7.0) to a final concentration of 2 µg/L. After boiling for 5 minutes, equal amounts of each sample were loaded onto the SDS-PAGE and separated for 2 hours at 100 V. After that, all proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) for 2 hours at 100 V. The membranes were then blocked for 1 hour with 5% skim milk, diluted with washing buffer (1× TBST), and then washed three times for 10 minutes with this washing buffer. The membranes were then incubated for 2 hours at room temperature (23 ± 2°C) with primary antibodies against Nrf2 (61 kDa, 1:1000, Cat# 365949, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and cleaved caspase-3 (Cat# 9661, 17/19 kDa, 1:500, Cell Signaling Technology, Danvers, MA, USA). All the primary antibodies were specific for rats. All membranes were washed with the TBST buffer three times and then incubated with the HRP-secondary antibody (Sigma) for 2 hours at room temperature (23 ± 2°C). The interaction between the antibodies (bands) was detected after incubating the membranes with the enhanced chemiluminescent (ECL) (Cat# 32106, Thermo Fisher Scientific). All bands were photographed using iBright Western Blot Imaging Systems with the high-resolution 9.1 MP camera (Thermo Fisher Scientific, Bremen, Germany). The intensities of all bands were analyzed using the C-Di Git blot scanner (Biosciences, Lincoln, NEB).

Morphological analysis

All formalin-fixed hippocampal tissues were deparaffinized and rehydrated in 100%, 90%, and 70% ethanol solutions. All tissues were then embedded in wax and cut into 3–5 µm sections. Staining was then performed using Haematoxylin and glacial acetic acid solution. The slides were then washed with deionized distilled water. Distaining was then performed using the HCL/70% ethanol solution (1:400 v/v). Slides were then stained with Eosin and dehydrated with 95% and 100% ethanol (Haematoxylin and Eosin and glacial acetic acid were purchased from Abcam). A mounting media was added, and all slides were visualized under a light microscope (Olympus, Tokyo, Japan) the next day at 200×.

Statistical analysis

No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to those reported in previous publications (El-Kott et al., 2020). All analysis and graphing were performed on the GraphPad prism analysis software (version 8). Normality was tested using the Kolmogorov-Smirnov test. Comparison between all data was made using the one-way analysis of variance followed by Tukey’s post hoc test. A level of P < 0.05 was considered statistically significant. All data were presented as the mean ± standard deviation (mean ± SD).

Results

Effects on the dental gyrus structure

Typical dental gyrus areas of the hippocampi, characterized by intact three layers [i.e., the polymorphic (P), pyramidal or glandular (GL), and molecular layers], were detected in both the control and vanillylacetone-treated rats (Figure 1A and B). In both groups, abundant microglial cells with multiple layers forming the GL were seen. In addition, few glial cells were seen in both groups of rats’ P and M layers (Figure 1A and B). An increased number of microglial cells in both the P and M layers, with an evident decrease in the number of layers forming the GL layers, was observed in the CdCl2-treated rats, and many pyknotic cells were also seen in this group of rats (Figure 1C). The number of layers and cells forming the GL and microglial cells in the P and M layers were significantly reduced in rats treated by CdCl2 and vanillylacetone, with a reduction in the number of pyknotic cells in the GL (Figure 1D and E). However, inhibition of Nrf2 by brusatol (CdCl2 + vanillylacetone + brusatol) induced similar pathological changes in the hippocampi observed in the model group (CdCl2-treated rats; Figure 1F).

Figure 1.

Figure 1

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Photomicrographs of the dental gyrus area of the hippocampus of all groups of rats.

(A and B) The normal three layers named the polymorphic (P), pyramidal or glandular (GL), and molecular (M) layers in rats in the control and vanillylacetone groups. The GL layers contained numerous cell layers that have intact nuclei (long arrow). Besides, microglial cells were seen in both M and P layers (short and thick arrow). (C) CdCl2-treated rats showed an obvious reduction in the number of layers forming the GL with the appearance of pyknotic cells (short, thin arrow). (D and E) CdCl2 + vanillylacetone-treated rats showed an increased number of cell layers of the GL and disappearance of the pyknotic cells (short and thick arrow). (F) CdCl2 + vanillylacetone + brusatol-treated rats showed almost similar pathological changes to those seen in the CdCl2-treated rats. Hematoxylin and eosin staining (200×, magnification). Scale bars: 50 μm.

Alterations in rat memory

The administration of vanillylacetone to control rats did not change the escape latencies and their AUC compared with the control rats. However, there were no significant changes in the escape latencies between days 2, 3, and 4 in the CdCl2-model and CdCl2 + vanillylacetone + brusatol-treated rats compared to their measured latencies on day one (Figure 2A). A significant and progressive decline in the escape latencies was seen between days 1, 2, 3, and 4 in control, vanillylacetone, and CdCl2 + vanillylacetone-treated rats (Figure 2A). The escape latencies measured on days 1 and 4 and their presentative AUC were all significantly higher in CdCl2-treated rats than the latencies measured on the corresponding days in the control rats (Figure 2A and B).

Figure 2.

Figure 2

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Vanillylacetone improves memory function in rats without affecting the hippocampal Cd levels.

(A) The times required to find the hidden platform during the 4 days of the training in the MWM test. (B) Area under the curve of all graphs obtained in A. (C) Number of times each rat crossed over the removed rescue platform during the probe trial of the MWM. (D) Cd levels in the hippocampi of all groups of rats. *P < 0.05, vs. control; #P < 0.05, vs. vanillylacetone (zingerone)-treated rats; †P < 0.05, vs. CdCl2-treated rats; ‡P < 0.05, vs. CdCl2 + vanillylacetone-treated rats. All data are presented as the mean (±SD) of three independent experiments. Cd: Cadmium; MWM: Morris Water Maze.

However, a significant reduction in the escape time of all test days was observed in vanillylacetone-treated rats compared with CdCl2-treated rats, which was associated with a significantly lower AUC (Figure 2A and B). The escape latencies measured on all test days of the study and their AUC were significantly higher in the CdCl2 + vanillylacetone + brusatol-treated rats (P < 0.01) compared with the corresponding values observed in CdCl2 + vanillylacetone-treated rats. These values significantly decreased compared with CdCl2-treated rats (P < 0.01; Figure 2A and B).

Additionally, the number of crossing times over the removed escape platform did not significantly vary in the vanillylacetone-treated rats. At the same time, it was significantly reduced in the group of rats treated with CdCl2 compared with the control rats (P < 0.001; Figure 2C). CdCl2 + vanillylacetone-treated animals showed a significant increase in the number of crossing times (P < 0.001) compared with CdCl2-treated rats. On the other hand, the number of crossing times did not show any significant changes when comparing the CdCl2 treated with CdCl2 + vanillylacetone + brusatol-treated rats (Figure 2C).

Changes in hippocampal levels of Cd ions

The levels of Cd ions in the hippocampi of control and control + vanillylacetone-treated rats did not differ significantly (Figure 2D). The levels of Cd ions in the hippocampi of CdCl2, CdCl2 + vanillylacetone, and CdCl2 + vanillylacetone + brusatol-treated rats were significantly higher (P < 0.001) when compared with the control rats (Figure 2D). When comparing the Cd ions levels in the hippocampi of the last three groups, they did not show significant changes between each other (Figure 2D).

Changes in the hippocampal transcription and nuclear localization of Nrf2

The mRNA and nuclear protein levels of Nrf2 were significantly reduced in the hippocampi of rats treated with CdCl2 compared with the control group (P < 0.001; Figure 3A–C). However, mRNA total and nuclear protein levels of Nrf2 significantly increased in the hippocampi of vanillylacetone-treated rats (P < 0.001) and CdCl2 + vanillylacetone-treated rats (P < 0.05) compared with either the control non-treated or CdCl2-treated rats, respectively (Figure 3A–C). However, with no significant alterations in the mRNA and total levels of Nrf2, the nuclear protein levels of Nrf2 significantly reduced in the hippocampi of CdCl2 + vanillylacetone + brusatol-treated rats (P < 0.001) compared with CdCl2 + vanillylacetone-treated rats (Figure 3A–C).

Figure 3.

Figure 3

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Vanillylacetone stimulates the transcription and nuclear transactivation of Nrf2 in the hippocampi of control and CdCl2-treated rats.

(A) The mRNA level of Nrf2 quantified by quantitative polymerase chain reaction. (B) Total protein level of Nrf2 measured by enzyme-linked immunosorbent assay. (C) The nuclear protein levels of Nrf2 detected by western blotting in the hippocampi of all groups of rats. *P < 0.05, vs. control rats (lane I), #P < 0.05, vs. vanillylacetone (zingerone)-treated rats (lane II), †P < 0.05, vs. CdCl2-treated rats (lane III), and ‡P < 0.05, vs. CdCl2 + vanillylacetone-treated rats (lane IV). Lane V: CdCl2 + vanillylacetone + brusatol. All data are presented as the mean (±SD) of three independent experiments. CdCl2: Cadmium chloride; Nrf2: nuclear factor erythroid 2-related factor 2.

Changes in the hippocampal markers of oxidative stress and inflammation

The effects of all treatments on the markers of oxidative stress and antioxidant power in the hippocampi are illustrated in Figure 4A–D. By contrast, those related to neuroinflammation are presented in Figure 5A–C. With stable levels of all measured inflammatory markers, ROS and MDA levels were significantly decreased (P < 0.05), while GSH and SOD levels were significantly increased (P < 0.01) in the hippocampi of control rats treated with vanillylacetone compared with control nontreated rats (Figure 4A–D and Figure 5A–C).

Figure 4.

Figure 4

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Vanillylacetone suppresses ROS and lipid peroxidation and stimulates the cellular antioxidant systems in the hippocampi of control and CdCl2-treated rats in an Nrf2-dependent manner.

(A–D) The total levels of ROS and RNS (A), MDA (B), SOD (C) and GSH (D) in the hippocampi of rats in all groups detected by enzyme-linked immunosorbent assay. *P < 0.05, vs. control rats; #P < 0.05, vs. vanillylacetone (zingerone)-treated rats; †P < 0.05, vs. CdCl2-treated rats; ‡P < 0.05, vs. CdCl2 + vanillylacetone-treated rats. All data are presented as the mean (±SD) of three independent experiments. GSH: Reduced glutathione; MDA: malondialdehyde; RNS: reactive nitrogen species; ROS: reactive oxygen species; SOD: superoxide dismutase.

Figure 5.

Figure 5

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Vanillylacetone suppresses neuroinflammation in the hippocampi of CdCl2-treated rats in an Nrf2-dependent manner.

Total level of (A) IL-6, (B) TNF-α, (C) ICAM-1 in the hippocampi of all groups of rats detected by ELISA. *P < 0.05, vs. control rats; #P < 0.05, vs. vanillylacetone (zingerone)-treated rats; †P < 0.05, vs. CdCl2-treated rats; ‡P < 0.05, vs. CdCl2 + vanillylacetone-treated rats. All data are presented as the mean (±SD) of three independent experiments. ELISA: Enzyme-linked immunosorbent assay; ICAM-1: intracellular adhesive molecule-1; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α.

ROS, MDA, TNF-α, ICAM-1, and IL-6 levels were significantly increased (P < 0.01), while GSH and SOD levels were significantly reduced (P < 0.001) in the hippocampi of rats treated with CdCl2 compared with control rats. However, these changes were significantly reversed in the hippocampi of rats treated with CdCl2 and vanillylacetone (Figure 4A–D and Figure 5A–C). Significantly enhanced levels of ROS, MDA, TNF-α, ICAM-1, and IL-6 (P < 0.01) and significantly reduced levels of GSH and SOD (P < 0.001) were observed in the hippocampi of CdCl2 + vanillylacetone + brusatol-treated rats compared with CdCl2 + vanillylacetone-treated rats (Figure 4A–D and Figure 5A–C).

Changes in the hippocampal markers of intrinsic cell apoptosis

While the vanillylacetone-treated control group showed no significant changes in the mRNA levels of Bax and the protein levels of cleaved caspase-3, the mRNA levels of Bcl2 were significantly increased (P < 0.01), along with a significant reduction (P < 0.05) in the Bax/Bcl2 ratio compared to control non-treated rats (Figure 6A–D). The mRNA levels of Bcl2 were significantly decreased (P < 0.01). While the mRNA levels of Bax, protein, cleaved caspase-3, and the ratio of Bax/Bcl2 were significantly increased (P < 0.0001) in the hippocampi of CdCl2-treated rats compared to control non-treated rats (Figure 6A–D). In contrast, the mRNA levels of Bcl2 were significantly increased (P < 0.05), whereas the mRNA levels of Bax, protein, cleaved caspase-3, and the ratio of Bax/Bcl2 were significantly decreased (P < 0.01) in the hippocampi of CdCl2 + vanillylacetone-treated rats compared with CdCl2-treated rats or compared to CdCl2 + vanillylacetone + brusatol-treated rats (Figure 6A–D).

Figure 6.

Figure 6

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Vanillylacetone suppresses intrinsic cell apoptosis in the hippocampi of CdCl2-treated rats in an Nrf2-dependent manner.

(A and B) The mRNA levels of Bax and Bcl2, as quantified by qPCR. (C) The mRNA ratio of Bax/Bcl2 and (D) the protein levels of cleaved caspase-3, as measured by western blotting, in the hippocampi of all groups of rats. *P < 0.05, vs. control rats (lane I); #P < 0.05, vs. vanillylacetone (zingerone)-treated group (lane II); †P < 0.05, vs. CdCl2-treated rats (lane III); ‡P < 0.05, vs. CdCl2 + vanillylacetone-treated rats (lane IV). Lane V: CdCl2 + vanillylacetone + brusatol. All data are presented as the mean (±SD) of three independent experiments. CdCl2: Cadmium chloride.

Discussion

The findings of this study demonstrate that daily dietary supplementation with vanillylacetone is highly effective in mitigating CdCl2-induced hippocampal damage and memory loss in rats. This is achieved by suppressing hippocampal oxidative stress, inflammation, and intrinsic cell apoptosis. Additionally, these effects are mediated by the activation of Nrf2, downregulation of Bax and caspase-3, and upregulation of Bcl2. However, the concomitant administration of brusatol, a selective Nrf2 inhibitor, was able to reverse the neuroprotective structural, biochemical, and learning effects afforded by vanillylacetone. This provides evidence that the neuroprotection of vanillylacetone in this animal model is mediated, at least in part, by upregulation and activation of Nrf2.

The hippocampus, located in the center of the brain, is responsible for regulating mammals’ memory function and learning abilities (Broadbent et al., 2004). Various factors, such as age, sex, diet, and race, can significantly impact the structure and functions of the hippocampus (Jack et al., 2015). In this study, we selected young adult aged-matched male rats to eliminate any variation in the results due to these factors and are supported by many previous studies. In this context, it is well-documented that oxidative stress and neuroinflammation are increased in aged brains and are associated with impaired memory function (Gomar et al., 2015). Furthermore, the structure and memory function of the hippocampus vary between males and females. Indeed, younger rats have more intact spatial and working memories and even cholinergic function compared with old rats (Haider et al., 2014).

Moreover, the differences in the dendritic densities of the Cornu Ammonis (CA), CA1, and C3A regions of the hippocampus, as well as the proliferation rates of the dentate gyrus region, memory-related signaling pathways, and performance in behavioral tests are well-documented between males and females. For example: (i) neurons in males are typically longer than neurons in females (Barrera et al., 2001); (ii) sex hormone receptors in the hippocampus affect depressive-like behaviors in rats (Selakovic et al., 2019); the diverse actions of the female hormone estrogen are well-documented (Spencer et al., 2008); (iii) spatial learning, synaptic activity, and long-term potentiation significantly vary between males and females (Monfort et al., 2015); and (vi) differences in neurotransmitter uptake mechanisms and cholinesterase activity were shown between males and females (Monfort et al., 2015).

Additionally, exposure to Cd ions has been shown to lead to a progressive reduction in body weight, which On the other hand, research has shown that exposure to cadmium ions can lead to a gradual reduction in body weight, a phenomenon that has been attributed by numerous authors to the changes in the appetite of rodents or the systemic effects of Cd ions (Anand and Dhikav, 2012). An interesting observation in our study is that the administration of vanillylacetone significantly improved weight gain and food intake without affecting the levels of cadmium in the hippocampus of CdCl2-treated rats. This effect was also not observed in control rats. These data indicate that all the observed effects of vanillylacetone in this study are not related to regulating the appetite of rats or chelating Cd ions. However, the improvement in rats’ final body weights after vanillylacetone treatment might be attributed to the restoration of systemic organ function, which could be due to the suppression of oxidative damage and apoptosis induced by CdCl2 in these organs. Indeed, oxidative stress, inflammation, and intrinsic cell death are the primary hallmarks of CdCl2-induced damage in the hippocampus of rodents (Gomar et al., 2015; Branca et al., 2018). Furthermore, an excess of ROS is believed to be the primary mechanism underlying the neurotoxic, inflammatory, and apoptotic effects of CdCl2. In this context, Cd ions can reach the CNS through the olfactory pathway or by disrupting the BBB in adult animals (Gomar et al., 2015).

Several mechanisms contribute to the generation of large amounts of ROS in neurons by Cd ions, including competition with binding sites of essential metals like zinc (Zn2+) and copper (Cu2+), the activation of ROS-generating enzymes such as NADPH oxidase (NOX), interaction with sulfhydryl group-containing proteins, the depletion of glutathione stores, the scavenging of antioxidant enzymes, the suppression of Nrf2, the impairment of mitochondrial function, and the increase in intracellular calcium (Ca2+) levels (Wang and Du, 2013; Wu et al., 2022).

ROS generated by cadmium ions not only promote neural lipid peroxidation and DNA damage but also activate microglial cells and increase the expression of various adhesive molecules. Additionally, ROS stimulate the generation and synthesis of inflammatory cytokines and induce cell apoptosis in cholinergic and non-cholinergic neural cells. However, the best-characterized oxidative stress-induced cell death mode after exposure to CdCl2 in rodents is the intrinsic mitochondria-mediated cell death, which is due to the increase in expression of Bax, release of cytochrome c, downregulation of Bcl2, and activation of caspase-3 (Anand and Dhikav, 2012). This is in agreement with our findings shown in this study.

In this study, administration of CdCl2 to the rats resulted in obvious damage to the dental gyrus area of the hippocampus, significant increases in oxidative stress, induced neuroinflammation, and promoted intrinsic cell death. Additionally, CdCl2 significantly reduced the mRNA and nuclear levels of Nrf2 and concomitantly decreased the levels of GSH, CAT, and SOD in the hippocampi of the model rats. This supports the findings of other authors who have suggested that suppression of Nrf2 is a central mechanism by which CdCl2 exaggerates the production of ROS and hippocampal damage (Gomar et al., 2015; Anand and Dhikav, 2012). Vanillylacetone was able to attenuate these damaging effects in the CdCl2-treated rats and improved the structure of the dental gyrus of the hippocampus. This indicates that vanillylacetone has neuroprotective properties, which are mediated by its potent antioxidant, anti-inflammatory, and anti-apoptotic effects.

Interestingly, the inhibitory effects of vanillylacetone on ROS, inflammatory cytokines, and cell apoptosis markers in the hippocampi of CdCl2-treated rats were prevented after suppressing Nrf2 by brusatol, even in the presence of high total protein and mRNA levels of Nrf2, which were not affected by this inhibitor. The suppression of Nrf2 by brusatol, despite the high total protein and mRNA levels of Nrf2, indicates that the brusatol mechanism of action is mediated by suppressing the nuclear localization of Nrf2 rather than affecting its transcription or levels. In addition, vanillylacetone also affects the mRNA and nuclear activities of Nrf2, as well as levels of GSH, CAT, and SOD in the hippocampi of control rats, without altering the transcription of Bax and the levels of cleaved caspase-3, nor the level of inflammatory markers. Based on these observations, we concluded that the neuroprotective effect of vanillylacetone is mainly mediated through the activation of the Nrf2/antioxidant axis, which is upstream of its anti-inflammatory and anti-apoptotic effects. However, the increased expression of Bcl-2 in the control rats treated with vanillylacetone could also be secondary to the activation of Nrf2, which is a known stimulator of Bcl-2 (Maurer and Williams, 2017). Nonetheless, it is possible that vanillylacetone may directly stimulate Bcl-2 or modulate other pathways to do so, which requires further investigation.

Although the protective effects of vanillylacetone in the hippocampi of CdCl2-treated rats are the first to be shown, many other studies in the hippocampus or other tissues of different animal models may support our findings. For example, Zingiber officinale mitigated hippocampal damage after focal ischemia in rats by upregulating GSH, SOD, and CAT and suppressing lipid peroxidation (Maurer and Williams, 2017). It also prevented epileptic hippocampal damage and apoptosis in mice by attenuating ROS generation, decreasing inflammatory cytokine production, downregulating Bax, and upregulating Bcl-2. In addition, several in vivo and in vitro studies have shown very powerful ROS scavenging and antioxidant abilities of vanillylacetone and distinguished potentials to inhibit mitochondria damage and downregulate Bax and caspases-3 and -9 (Wattanathorn et al., 2010). Furthermore, vanillylacetone prevented hepatic and cardiac damage in diabetic and cisplatin-treated rats by upregulating GSH, CAT, and SOD and reducing the activation of NF-κB, TNF-α, and IL-6 (Wattanathorn et al., 2010). Additionally, vanillylacetone prevented the development of asthma in rodents by upregulating Nrf2, stimulating endogenous antioxidants, and suppressing inflammatory cytokine production (Zhu et al., 2021).

Despite these findings, our study may still have some limitations. Importantly, these findings are still observational and require further experiments to confirm our results, possibly at the level of neural culture and cells or in rats with Nrf2 deficiencies. Additionally, cadmium ions induce peripheral toxicities by promoting oxidative stress and inflammation. The CNS may also be affected by the efflux of inflammatory cytokines from the periphery. Therefore, additional experiments are needed to determine if the effect of vanillylacetone is central or peripherally mediated. Furthermore, Nrf2 is regulated by many other upstream mechanisms that control its transcription and interaction with Keap-1. Thus, further studies are needed to identify the main upstream mechanism by which vanillylacetone stimulates the transactivation of Nrf2.

In mammals, memory function is primarily regulated by the hippocampus, and neurodegeneration in this area of the brain is associated with a significant impairment in memory and learning (Wang and Du, 2013). The neurotoxicity and hippocampal damage induced by Cd ions are also associated with impaired memory functions (Anand and Dhikav, 2012; El-Kott et al., 2020). The cholinergic system is an important part of the hippocampus that regulates hippocampal plasticity, neurogenesis, and memory function via its interaction with non-neural cells. In the brain, normal levels of acetylcholine (ACh) are required to stimulate synaptic neuroplasticity and suppress inflammation in non-neural cells, such as microglia (Ahmad et al., 2015). Acetylcholinesterase (AChE), an enzyme, terminates the action of ACh by promoting its hydrolysis (Haam and Yakel, 2017). However, BDNF is the most-known factor required for both ACh synthesis and neural survival (Alshammari et al., 2021).

Some scholars mentioned that reduced levels and expression of BDNF parallel with low levels of Ach, ChAT, and hyper-activated AChE in the cortical and hippocampal tissues after exposure to Cd ions (Oboh et al., 2020; Yao et al., 2021). Indeed, Cd ions directly downregulate the transcription of BDNF. Also, Cd ions inhibit ChAT by occupying its active site (Gonçalves et al., 2021). In addition, Cd ions reduced levels and activities of AChE by suppressing its transcription and blocking the Ca2+ efflux at the nerve terminal, respectively (Oboh et al., 2020). Nonetheless, the reduction in BDNF, Ach, and the activities of ChAT could be related to Cd ion-induced apoptosis in the cholinergic and non-cholinergic neurons.

In line with these studies, which were related to damaged dental gyrus area in CdCl2-treated rats, Cd ions also impaired the spatial and avoidance memory of the treated rats. Additionally, it was associated with a decrease in the hippocampal BDNF and Ach levels, reduced activation of ChAT, and over-activation of AChE (Yao et al., 2021). On the other hand, our study demonstrated that vanillylacetone improved memory function in CdCl2-treated rats and reversed the alterations in BDNF levels and all markers of the cholinergic system in the hippocampi of CdCl2-treated rats, effects of which were blocked by brusatol.

Interestingly, our study found that vanillylacetone increased Ach levels in the hippocampi of control rats without altering the activities of both ChAT and AChE. Nrf2 is a potent inducer of BDNF, and it plays a significant role in Ach synthesis and release (Yao et al., 2021). Thus, we suggest that vanillylacetone may stimulate memory function and Ach levels in control and CdCl2-treated rats through an Nrf2-induced upregulation of BDNF. In addition, this could be a secondary effect due to vanillylacetone-mediated stimulation of cell survival and inhibition of cell apoptosis in the cholinergic neurons.

The enhanced working memory function observed in middle-aged women after dietary supplementation with a fresh extract of Zingiber officinale supports our findings (Saenghong et al., 2012). Similarly, an improvement in memory function was observed in Hyoscine-treated male rats after treatment with an ethanolic extract of Zingiber officinale (Gomar et al., 2015). Additionally, daily treatment with fresh and dried ginger and 6-gingerol extract improves the performance of working memory and avoidance memories in scopolamine-treated rats by increasing BDNF levels (Gomar et al., 2015).

Moreover, a fresh extract of Zingiber officinale alleviated memory loss and improved the forced swim test and elevated plus maze behavioral tests in diabetic rats by increasing the transcription of BDNF and enhancing antioxidant levels (Alshammari et al., 2021). Similarly, it prevented hippocampal damage and improved learning abilities during the MWM in rats exposed to focal ischemia by stimulating GSH and antioxidant levels, including SOD and CAT (Maurer and Williams, 2017). The reduction in escape latency of the MWM in epileptic mice after treatment with vanillylacetone was attributed to the reduction in oxidative stress, inflammation, and neural cell apoptosis (Rashid et al., 2021).

In conclusion, our data demonstrated the neuroprotective potential of vanillylacetone against CdCl2-induced hippocampal damage and memory loss in mammals. Besides, these data open a horizon for future pre-clinical and translational studies to investigate the possibility of using vanillylacetone to manage neurodegenerative disorders related to oxidative stress and reduced levels of Nrf2.

Acknowledgments:

The authors acknowledge the funding and technical support provided by the Research Deanship of King Khalid University and Princess Nourah bint Abdulrahman University. The authors thank Professor Bahjat Al-Ani (Department of Physiology College of Medicine, King Khalid University, Abha, Saudi Arabia) for his input and support. The authors would like to thank Dr. Waiel S Bashir (Department of Medicine, Medical Sciences and Technology, Khartoum University, Sudan) and Ahmed M Darwesh (Department of Orthopedic Surgery, Kasr El Aini Hospital, Cairo University) for their assistance in the practical part of this work. The authors are grateful to Dr. Mariam Al-Ani (Face Studio Clinic, UK) for proofreading the manuscript.

Funding Statement

Funding: This work was funded by the Research Deanship of King Khalid University, No. GRP-215-43 (to FHA); Princess Nourah bint Abdulrahman University Researchers Supporting Project, No. PNURSP2023R110 (to AFD).

Footnotes

Conflicts of interest: Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Data availability statement: No additional data are available.

C-Editor: Zhao M; S-Editor: Li CH; L-Editor: Song LP; T-Editor: Jia Y

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