Bromelain Restores Glutamatergic Homeostasis via Regulation of NR2A, GLT-1, EAAC1, and xCT in Arsenic-Induced Cerebral Cortex and Hippocampal Neurotoxicity

Ethical approval

The experimental procedure was approved by the Institutional Animal Care and Use Committee of the College of Medicine, University of Nigeria Institutional Animal Care and Use Committee (IACUC) (ethics approval no. COMHREC/2025/01/025), and all efforts were made to avoid unnecessary suffering in animals. Experiments were performed under the guidelines of the U.S. National Research Council Guide for the Care and Use of Laboratory Animals and ARRIVE 2.0 guidelines.

Experimental animals and housing conditions

Eight-week-old Male Wistar rats (Rattus norvegicus) weighing 200-230 g were procured from the animal facility of the University of Nigeria. The rats were kept in a room with controlled temperature (28°C) and humidity (50–60°C) and 12 h/12 h light/dark cycle (8 rats per cage). Animals were allowed free food intake of standard chow and water. The acclimatization period to the facility was 14 days after the start of the experiment.

Sample size and power calculation

The sample size was calculated a priori to achieve sufficient statistical power to identify biologically relevant differences between experimental groups using the G*Power software (version 3.1.9.7, Universitat Dusseldorf, Germany). One-way analysis of variance ANOVA (fixed effects, omnibus, one factor) was selected because the primary comparisons involved multiple independent treatment groups (nine of them). The parameters used were based on the reported effect sizes in other rodent neurotoxicity studies that measured the change in glutamate transporter and receptor expression after pharmacological manipulation (Cohen’s f = 0.5, large effect size).

The analysis showed a minimum of eight animals per group to attain the desired statistical power. This figure is adequate to maintain the balance between scientific sensitivity and ethical minimization of the use of animals in line with the 3Rs principle (Replacement, Reduction, Refinement).

Eight adult male Wistar rats were used in each group, with a total of 72 rats in the nine treatment groups. This sample size is sufficient to provide strong power to detect treatment effects on biochemical and molecular responses (NR2A, GLT-1, EAAC1, and xCT expression) and a reasonable amount of attrition due to unanticipated health problems.

Experimental grouping and treatment regimen

The 72 rats were randomly divided into nine experimental groups (n = 8 per group) following acclimatization as follows:

The dosage regimen was based on previous studies on arsenic neurotoxicity and bromelain efficacy.^37,43 In the first two weeks, sodium arsenite (NaAsO 2; Merck KGaA, Darmstadt, Germany) was administered by oral gavage once daily. The 20 mg/kg dose was selected from pilot studies as the lowest dose that induced significant learning memory deficits without excessive systemic toxicity.

Freshly prepared in distilled water, bromelain (Pure Encapsulations 1000 mg, Sudbury, MA, USA) was orally gavaged daily to the respective groups in the second two weeks (Days 15-28). The doses (5, 10, 15 mg/kg) were selected to encompass a low-to-high therapeutic range based on previous animal investigations in which cognitive enhancement was observed at approximately 10 mg/kg. Donepezil (Aricept 2 mg/kg) was used as a reference neuroprotective agent, which has been shown to improve memory in toxin-induced models.

The control and arsenic-only groups were treated with a similar amount of vehicle (distilled water) throughout the treatment period.

Animal monitoring and welfare

The health status of the rats and their signs of toxicity were monitored daily. Body weight was recorded weekly. No mortalities or major adverse events occurred during the course of the study.

To minimize possible confounding factors, the experiment was conducted in two cohorts, where the representation of the groups was equal. An observer blinded to the group assignments was administered the behavioral testing.

Experimental timeline

Cognitive performance was assessed after arsenic or control treatment (day 15) using the Morris water maze). Bromelain or donepezil treatments were then applied for the next 14 days. Behavioral testing was performed at the endpoint (day 28) prior to euthanasia and tissue collection.

The arsenic-only group (Group B) was humanely euthanized on day 15 after completing arsenic exposure to obtain maximum arsenic effects without recovery. The remaining rats were euthanized on day 28. The staggered endpoint was intended to distinguish between the acute effects of arsenic and the post-treatment outcomes. Samples were collected at the respective sacrifice times and analyzed behaviorally and biochemically.

Behavioral assessment: Morris Water Maze Test

The Morris water maze (MWM) method was used to assess spatial learning and memory before the treatments on the 14th study day and then on study day 28 to measure cognitive impairment and recovery. The MWM instrument was a round pool (126 cm diameter and 75 cm height) that contained water (depth 40 cm) controlled at 24 1 C. Water was opacified with non-toxic opaque paint (white tempera) or skim milk powder to conceal a sunken escape platform. Considering the concept of small areas, four quadrants (NE, NW, SE, and SW) were depicted on the walls of the pool and were located visually using specific markings. During training and testing a clear plastic platform (10 cm diameter) was positioned 2 cm beneath the water surface in the target quadrant (SW quadrant, ~10 cm from the wall).

All the rats underwent two stages acquisition (training) and retrieval (testing). The rats were subjected to four training trials three times during the acquisition phase (days 12-14 of the experiment). During each trial, the rat was calmly placed in the water at a pseudo-random start position (switching between NE, NW, and SE start locations) and given a maximum of 60 s to discover the hidden platform. If a rat was unable to locate within 60 s, it was directed to the platform by the experimenter. The duration was 15 seconds, after which the rats were removed from the platform. This training was modified based on Forero et al. (2023)⁴⁵ with slight changes. The aim was to allow rats to learn where the hidden platform was using spatial cues.

On the test day, 24 h after the final training trial, a probe trial was conducted (day 15 for the arsenic-only group and day 28 for all others). During the probe test, the platform was removed from the pool. Each rat was dropped into a new starting point and permitted to swim freely all over the arena for 60 s. As the main indicator of spatial memory, escape latency (the time taken to reach the old platform position) was registered. A smaller escape latency indicates an enhanced memory of the location of the platform. Patterns of swim paths were also examined, so that no significant sensorimotor impairments were present (all rats could swim normally). It was filmed using a video camera above the pool, and tracking was conducted manually using a stopwatch (computer automated tracking software was an option, but resource issues were not considered).

Tissue collection and processing

At the end of the experiment, the rats were anesthetized (using intraperitoneal injection of sodium pentobarbital (100 mg/kg) followed by decapitation, as recommended by the AVMA Guidelines for the Euthanasia of Animals (2020). This method ensures rapid loss of consciousness and is approved under the COMHREC/2025/01/025 protocol. In the arsenic-only group (Group B), euthanasia occurred on day 15, immediately after the final memory test, whereas in all other groups, the process took place on the 28th day. The schedule ensured that the arsenic-alone animals were tested at the peak of impairment, whereas the treatment groups were examined after the recovery/treatment phase.

Immediately after sacrifice, sterile scissors were used to quickly open the skull. The entire brain was removed and washed with ice-cold saline to remove the blood. To maintain RNA and protein integrity in brain tissues, they were processed on a cold surface. The brains were blotted dry and then meticulously dissected to separate the hippocampus and prefrontal cortex on both sides. The protocol for guided freehand dissection was presented by Spijker.⁴⁶ Briefly, the brain was longitudinally cut in the midsagittal plane, the hippocampus of each half-brain was pulled out of the latter on the ventral surface and peeled off, and a mild slice was removed from the prefrontal cortex (~3 mm in front of the frontal pole). This dissection procedure produces tissue samples that have highly represented targeted areas, as well as limited cross-contamination between other regions.

The snipped tissues were immediately placed in RNAlater stabilization solution (Ambion, USA) to inhibit RNA degradation. The samples were stored overnight in RNAlater at 4°C followed by storage at -80°C until molecular analysis. (In preliminary tests, a 2-week fixation in RNAlater did not significantly degrade RNA quality; tissues remained suitable for PCR) Part of each hippocampus was also flash-frozen and stored in a freezer at -80°C in order to perform glutamate assay. For this part of our study, we did not subject the animals to perfusion fixation, because our endpoints were molecular. Every effort was made to ensure that animal suffering and pain was brought to the barest minimum.

RT-qPCR Gene analysis expression

Total RNA was extracted from the hippocampal and prefrontal cortex using a commercial RNA isolation kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Approximately 30 mg of tissue was homogenized in 1 mL of TRIzol reagent in the presence of 0.1 M 8-mercaptoethanol (Life Technologies, Gaithersburg, MD, USA) as a reducing agent. The homogenates were maintained at room temperature and incubated for 5 min to ensure that all nucleoprotein complexes were dissociated. Chloroform (200 μL per mL TRIzol) was added, and the samples were shaken vigorously for 15 s and then centrifuged at 12,000 × g for 15 min at 4°C. The RNA in the aqueous phase was carefully transferred to a new tube, and an equal amount of 70 percent ethanol was added. A silica gel membrane spin column was used to apply the solution. The column that was bound was washed with the supplied buffers and RNA was eluted with RNase-free water. This step was followed by on-column DNase I digestion step that was meant to avoid contamination with genomic DNA. The quality and quantity of RNA were determined based on absorbance measured at 260 nm and 280 nm using the NanoDrop 2000 (ThermoFisher Scientific), with A260/280 between 1.9 and 2.1. DNA integrity was validated through electrophoresis on a 1 percent agarose gel with clear 18 S and 28 S rRNA bands.

cDNA Synthesis

Complementary DNA (cDNA) was synthesized from 2–3 μg of total RNA per sample using a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA). RNA was initially denatured at 65°C for 5 min and cooled rapidly. The reverse transcription reaction (volume of 20 μL) consisted of RNA template, 4 μL volume of 5x RT buffer, 2 μL of dNTP mix (10 mM each), 1 μL of random hexamer primers, 1ul of Moloney murine leukemia virus (MuLV) reverse transcriptase, and RNase free water. The reaction was incubated at 42°C to be incubated with 30 min after which it was incubated at 95°C to destroy the enzyme after 5 min. The obtained cDNA was kept at -20°C until qPCR.

Real-time PCR: Q-PCR was performed using Rotor-Gene Q real-time PCR with SYBR Green chemistry. The target genes, including cystine/glutamate antiporter xCT (Slc7a11), EAAT1 (Slc1a3), GLT1 (Slc1a2), NMDA receptor NR2A subunit (Grin2a), glial fibrillary acidic protein GFAP (Gfap), and housekeeping gene β-actin (Actb) were prepared using specific primer pairs.

Primer sequences are as follows:

Each PCR assay consisted of a 25 μL reaction containing 12.5 μL of 2x SYBR Green Master Mix (Applied Biosystems), 0.5 μM of forward and reverse primer and 2 μL of diluted cDNA template(~100 ng RNA equivalent). The thermal cycling conditions consisted of an initial denaturation at 95°C for 10 min and 35 cycles of 95°C for 30 s, 57–60°C for 40 s and 72°C for 40 s, depending on the primer annealing temperature. An analysis of the melting curve between 60°C and 95°C at cycling completion was also performed to confirm the specificity of amplification (a single product peak per product). Each primer pair contained no-template control (NTC) to rule out possible contamination or primer dimers; the NTCs were negative in each case.

Quantification

Cycle threshold (CT) values were obtained for each sample. The expression levels of the target genes were normalized to β-actin as an internal reference in the same sample (ΔCT = CT (target) – CT(β-actin)). The relative expression of each target gene in the treated groups was calculated using the 2–ΔΔCT method, with the normal control Group A (Group A) as the calibrator. This yielded a fold-change in mRNA expression relative to that in the control. Each PCR was run in duplicate, and the average CT was used for analysis. Data are presented as mean fold-change ± standard error (SE) for n = 5 animals per group (except for the arsenic-only group n = 5 at its time point).

Determination of glutamate level

The concentration of brain glutamate was determined using a competitive Enzyme-Linked Immunosorbent Assay (ELISA) kit (Analytik Jena GmbH, Germany) according to the manufacturer’s instructions. Hippocampal tissue samples (~50 mg) were homogenized in ice-cold 0.1 M phosphate-buffered saline (pH 7.4) and centrifuged (10,000 g, 10 min, 4°C) to obtain supernatants. The supernatants were discarded, and the samples were diluted and subjected to ELISA.

The principle of the assay is competitive binding; unused glutamate in the samples competes with a glutamate-enzyme conjugate to combine with glutamate-specific antibodies mounted on the wells of a microplate. The wells of a microtiter plate coated with anti-glutamate antibody were filled with 25 μL of each sample or glutamate standard solution. Then, 50 μL of glutamate-type analog conjugated enzyme was added to each well. Adhesive film was applied on the plates and incubated overnight at 4°C with competition binding. The wells were then washed thrice using the wash buffer provided in the kit to remove unbound material. Next, 100 μL of each well was subjected to TMB substrate solution and incubated at room temperature (20-25) for approximately 20 min under gentle shaking in the dark. The reaction was stopped by adding 100 μL of stop solution (1 N HCl), which yielded a yellow color. A microplate reader was used to measure the absorbance (450 nm) with reference absorbance (600 nm).

A standard curve was developed using known glutamate concentrations (ranging from 0 to 50 μg/mL of glutamate), and sample concentrations of glutamate were calculated using the obtained standard curve. All samples were analyzed in triplicate. In this competitive ELISA, the optical density is proportional to the inverse of the glutamate concentration (glutamate in the sample competes with conjugation of the enzyme); the lower the glutamate, the higher the optical density. We generated a standard inhibition plot and calculated the levels of glutamate in each sample, measured in μmol per gram of tissue.

Statistical analysis

Results are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) using the appropriate multiple comparison tests was performed using behavioral (escape latency) and biochemical data (glutamate level and gene expression fold-changes). In particular, all treatment groups were compared with the arsenic-only group using Dunnett’s test, and multiple bromelain dosages and donepezil were compared using Tukey’s HSD test. To examine learning curves in the water maze escape latency (acquisition trial interval over days), two-way repeated-measures ANOVA (factors: group and training day) was used, and Bonferroni correction of multiple comparisons was obtained on each day. All tests were defined at a significance level of p < 0.05. Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, CA, USA). The sample sizes (n = 5) within each group were selected using power calculations against pilot data, where n = 5 was estimated to be able to detect a 20 per cent reduction in escape latency or a 2-fold degree of change in gene expression in a study with an alpha value of 0.05, with a level of power greater than 80 per cent. To prevent bias, all analyses were performed with the experimenter not knowing who was in the group.

Behavioral performance in Morris Water Maze

There was a significant decline in spatial learning and memory after exposure to arsenic, but the administration of bromelain restored cognitive performance. The morris water maze test in the arsenic-only group (Group B) demonstrated escape latency that was significantly longer than that of normal rats (Group A), signaling major learning and memory impairment in this group. On the last day of training, arsenic-exposed rats spent significantly more time finding the hidden platform (escape latency 55–60 s) than control rats (20–25 s). These arsenic-only rats generally took a considerably longer time to discover the former position of the platform than they had done earlier or failed within 60 seconds, indicating poor retention of spatial memory. In contrast, rats administered bromelain (Groups C, D, and E) escaped in a similar fashion to the controls; escape latencies were as short and did not vary significantly over the period of training as those of Group A, demonstrating that bromelain alone did not have a negative impact on learning or memory. Interestingly, bromelain therapy following exposure to arsenic (Groups F-H) remarkably enhanced maze retention scores compared with untreated arsenic rats. At the end of treatment, the arsenic + bromelain group exhibited decreased escape latencies (p < 0.05, compared with Group B) and effectively countered the arsenic-induced deficit (Figure 1). Medium-dose bromelain (10 mg/kg, Group G) was remarkable, and the mean escape times were nearly equal to those of the controls. The group administered a high dose of bromelain (15 mg/kg, Group H) showed a shorter escape latency than the donepezil group (Group I) during memory retrieval. Quantitatively, the mean time taken to reach the target was approximately 15 per cent less in the case of high-dose bromelain rats compared to donepezil-treated rats, but this was not significant. Unsurprisingly, donepezil treatment also significantly improved performance compared to arsenic only (p < 0.01), although bromelain at high doses had an even more significant effect on spatial memory. This finding indicates that bromelain has the potential to salvage cognitive dysfunction caused by arsenic and that it is as effective as the conventional medication donepezil. Notably, there were no rats on bromelain-only who demonstrated prolonged latencies or abnormal behavior, indicating that there was no evident adverse effect on cognition with bromelain at the doses used, at least not at inference levels.

Figure 1. Bromelain improves spatial learning and memory in arsenic-exposed rats.

Escape latency (time to find the hidden platform) was significantly longer in arsenic-only rats vs. controls. Bromelain (low, medium, high dose) reduced latency, with high-dose effects comparable to donepezil. Data are mean ± SEM. ^ap < 0.05 vs. control; ^bp < 0.05 vs. arsenic-only; ^cp < 0.05 vs. low-dose bromelain; ^dp < 0.05 vs. medium-dose bromelain; ^ep < 0.05 vs. high-dose bromelain; ^fp < 0.05 vs. donepezil.

NMDA receptor NR2A and glutamate transporter expression

The neurotoxic effects of arsenic were linked to the general downregulation of glutamate transporters and receptor genes in both the hippocampus and prefrontal cortex, which bromelain treatment notably overcame. In our arsenic-only rats (Group B), mRNA levels of the NMDA receptor NR2A subunit, the transporters GLT1, EAAC1, and xCT were significantly reduced in both brain regions compared to those in the normal controls (see Figures 2 and 3). This validates that the transcriptional regulation of major glutamate-handling proteins is affected by arsenic exposure, as previously reported. In quantitative terms, arsenic hippocampal NR2A expression was reduced by ~40% of control, GLT1 by ~50%, EAAC1 by ~45%, and xCT by ~60% of control (all p < 0.01 compared with Group A). The same was true for the prefrontal cortex. These changes support the concept that arsenic aggravates glutamate uptake and receptor operations, resulting in a condition that is susceptible to excitotoxicity.

Figure 2. Bromelain upregulates NMDA receptor subunit NR2A and glutamate transporter genes in the hippocampus.

mRNA levels of NR2A, xCT, EAAC1, and GLT1 were downregulated by arsenic vs. control. Bromelain (low, medium, high dose) significantly restored expression, with the medium dose showing the strongest effect, approaching control values. Data are mean ± SEM. ^ap < 0.05 vs. control; ^bp < 0.05 vs. arsenic-only; ^cp < 0.05 vs. low-dose bromelain; ^dp < 0.05 vs. medium-dose bromelain; ^ep < 0.05 vs. high-dose bromelain; ^fp < 0.05 vs. donepezil.

Figure 3. Bromelain upregulates NR2A and transporter genes in the prefrontal cortex.

Arsenic suppressed mRNA expression of NR2A, xCT, EAAC1, and GLT1, while bromelain (notably high dose) significantly enhanced expression. Effects were dose-dependent, with strong upregulation of xCT and GLT1. Donepezil generally produced higher expression, though bromelain showed stronger effects on EAAC1 and xCT. Data are mean ± SEM. ^ap < 0.05 vs. control; ^bp < 0.05 vs. arsenic-only; ^cp < 0.05 vs. low-dose bromelain; ^dp < 0.05 vs. medium-dose bromelain; ^ep < 0.05 vs. high-dose bromelain; ^fp < 0.05 vs. donepezil.

When bromelain was used alone (Groups C, D, and E), minor upregulation of genes related to glutamate was observed relative to the control experiments. When arsenic was absent, low-dose bromelain tended to elevate NR2A, GLT1, EAAC1, and xCT expression by ~10-20% compared to controls, whereas medium- and high-dose bromelain led to minor but statistically significant alterations in the expression of some genes (p < 0.05, vs. Group A subset of targets). Although these modifications in br-only rats were small, they indicate that bromelain has regulatory effects on the expression of neurotransmitter genes, even under normal conditions. Remarkably, this level of upregulation did not result in any cognitive changes, as the bromelain-only rats exhibited normal behavior in the MWM, suggesting that the gene modulatory effects of bromelain are positive but not deleterious.

Most importantly, mRNA levels of NR2A, GLT1, EAAC1, and xCT were strongly enhanced by bromelain therapy after arsenic exposure (Groups F, G, and H) compared to those in the arsenic alone group (Figures 2 and 3). Bromelain at any dose significantly increased the expression of these genes in the hippocampus (p < 0.01 vs. Group B). The medium dose (10 mg/kg) proved especially effective, as most of the mRNA levels were close to or exceeded the control levels. For example, hippocampal NR2A expression in the medium-dose group was ~95% of the control (a 2.3-fold increase compared to the presence of arsenic-only), whereas GLT1 was ~110% of the control (2-fold increase compared to arsenic-only). Small and large doses of bromelain also brought about considerable changes, but the medium dose resulted in the greatest overall performance in the hippocampus. Surprisingly, the dose-response was slightly different in the prefrontal cortex: high-dose bromelain demonstrated the highest upregulation of xCT and GLT1 (even surpassing the same effect produced by the medium dose), but the upregulation of NR2A and EAAC1 did not increase further with the high dose. This indicated a localized, dose-dependent effect of bromelain on gene regulation. While moderate dosing could best target hippocampal neurons (where NR2A plays a significant role in synaptic plasticity), higher doses could also be effective in enhancing cortical astrocytic function (reported by an increase in xCT and GLT1 in the cortex). Regardless of dose, bromelain-treated arsenic groups demonstrated a significant upregulation in all measured genes compared to the corresponding untreated arsenic group, demonstrating a definite reversal of the suppressive effects of arsenic.

For comparison, these glutamate-related genes were also upregulated by donepezil treatment (Group I) administered after arsenic. Among all groups, donepezil showed the greatest expression of NR2A, GLT1, EAAC1, and xCT in both the hippocampus and cortex, and it was stronger than the effect of bromelain in most drug conditions (p < 0.05 among donepezil and bromelain high-dose conditions when tested on NR2A and GLT1). For example, the hippocampal NR2A level in the donepezil group was approximately 1.3-fold higher than that in the medium bromelain group. This was not surprising since the positive effect of donepezil on glutamatergic neurotransmission was achieved indirectly by increasing cholinergic neurotransmission and neurotrophic support. Nonetheless, some interesting findings were that bromelain performed better than donepezil in some of the measures. In the prefrontal cortex, the high-dose bromelain group showed greater EAAC1 and xCT than donepezil. Cortical xCT mRNA in Group H was approximately 20 percent greater than Group I, and EAAC1 mRNA was approximately 15 percent greater, indicating that bromelain might have an exclusive benefit in regulating glial antioxidant transport systems (xCT) and cortical neuronal glutamate uptake. These minuscule disparities allude to a possibly different condition of bromelain that may supplement or surpass the outcomes of typical treatment in particular brain areas. Collectively, the gene expression data show that bromelain significantly prevents arsenic-induced repression of essential excitatory signaling components, and can therefore restore the potential of glutamate clearance and the normal functioning of NMDARs.

Glutamate levels in brain tissue

In line with the results of gene and behavioral analyses, the use of bromelain as a treatment reversed the arsenic-induced imbalance in glutamate homeostasis. Exposure to arsenic results in a high accumulation of glutamate in the brain tissue. In the hippocampal homogenates (and cortical homogenates), the arsenic-only group (Group B) had an increased concentration of glutamate compared to the control group (Figures 2 and 3). Arsenic-only rats exhibited ~1.8-fold greater levels of glutamate than controls on average (p < 0.01), indicative of impaired clearance and excitotoxic saturation. This is consistent with the idea that arsenic produces glutamate dyshomeostasis, likely as a result of impaired transporter activity and hyperrelease. The glutamate ELISA, In feasible terms, unraveled concentrations of approximately 12-15 micromole/gram in arsenic brains in comparison to ~7-8 micromole/gram controls.

Glutamate levels were not significantly different between the bromelain-only groups (C, D, E) and the control group. Indeed, glutamate levels in these rats were low (similar to Group A), which means that bromelain alone does not exhaust glutamate below normal levels or leads to its abnormal accumulation. This is a vital safety assurance that bromelain has no effect on destabilizing baseline neurotransmitters.

Glutamate concentration was also significantly decreased in arsenic-treated rats administered bromelain (Groups F and G) compared to arsenic only in Group D. A trend of reduced glutamate with low-dose bromelain was observed (Figure 4); however, the difference was not significant (glutamate = 12 (micro sign) m/g in Group B vs. 15 (micro sign) m/g in Group A, p = 0.09). The middle dose produced a strong adjustment (p < 0.05, compared with Group B) and re-approached the norm of glutamate concentration (~9-10 mU/g). The strongest effect was achieved with high-dose bromelain, whose Glu content was close to that of the control (~8 μmol/g, not significantly different from the control) and significantly lower than that in the medium-dose and arsenic-only groups. Indeed, high-dose bromelain decreased brain glutamate levels even more than donepezil (Group I glutamate ~9 μmol/g compared with Group H ~8 μmol/g). Regardless of the fact that both treatments reduced the influence of glutamate accumulation, the large amount of bromelain was more effective in relation to donepezil. This indicates the effectiveness of bromelain in increasing glutamate clearance or decreasing the release of glutamate in toxic states. Statistical comparisons revealed that the glutamate level in Group H was significantly lower than that in Group I (p < 0.05), proving that bromelain has a strong effect when administered at high doses.

Figure 4. Bromelain reduces arsenic-elevated glutamate in hippocampal tissue.

Arsenic significantly increased glutamate vs. control, indicating excitotoxicity. Bromelain lowered glutamate dose-dependently, with high dose normalizing levels and outperforming donepezil. Data are mean ± SEM. ^ap < 0.05 vs. control; ^bp < 0.05 vs. arsenic-only; ^cp < 0.05 vs. low-dose bromelain; ^dp < 0.05 vs. medium-dose bromelain; ^ep < 0.05 vs. high-dose bromelain; ^fp < 0.05 vs. donepezil.

Taken together, bromelain can normalize the neurochemical imbalance caused by arsenic. Bromelain probably prevents neuronal over-excitation by preventing glutamate accumulation with excitotoxic effects. The glutamate results are consistent with the increases in transporter expression; when bromelain increased the transporter expression of GLT1, EAC, and xCT, the ability of the brain to remove or recycle glutamate was enhanced, causing reduced steady-state levels of glutamate tissue. This biochemical finding supports the behavioral findings, suggesting that there is a mechanistic relationship between the improved cognitive agent bromelain and restored glutamate homoeostasis.

Declarations

Clinical trial number: Not applicable.

Ethics approval and consent to participate

The experimental protocol was approved by the College of Medicine, University of Nigeria Institutional Animal Care and Use Committee (IACUC) (ethics approval no. COMHREC/2025/01/025). All procedures were conducted in compliance with the ethical standards.