Neuroprotection by Nauclea latifolia extract in arsenite & high-fat diet-induced brain stress

Article information

Environ Anal Health Toxicol. 2025;40.e2025016

Publication date (electronic) : 2025 June 30

doi : https://doi.org/10.5620/eaht.2025016

Wusa Makena ¹^,

, Aisha Aminu ²

, Onyinoyi Bethel Onimisi ³

, John Tabakwot Ayuba ¹

, Gidok Kogi Abednego ¹

, Victor Kayode Jerome ⁴, Abel Yashim Solomon ⁵

, Barka Ishaku ⁶

¹Department of Human Anatomy, Kampala International University, Western Campus, Bushenyi, Uganda

²Department of Human Anatomy, Kaduna State University, Kaduna, Nigeria

³Department of Human Anatomy, Usmanu Danfodiyo University, Sokoto, Nigeria

⁴Department of Human Physiology, Ahmadu Bello University, Zaria, Kaduna, Nigeria

⁵Department of Human Anatomy, Nile University of Nigeria, Abuja, FCT, Nigeria

⁶Department of Human Anatomy, University of Maiduguri, Maiduguri, Borno, Nigeria

^*Correspondence: wusa.makena@kiu.ac.ug

Received 2025 February 2; Accepted 2025 June 10.

Abstract

Sodium arsenite (NaAsO₂) and high fat diet (HFD) are already documented to provoke oxidative stress, neuro inflammation and learning and memory deficits. This work aimed to determine the possible neuroprotection of the root extract of Nauclea latifolia (NlREq) against NaAsO₂/HFD induced neurotoxicity in Wistar rats. Twenty-five rats were divided into five groups: groups include control; NaAsO₂/HFD treated; NaAsO₂/HFD + NlREq at 200 mg /kg and 400 mg/kg; and NaAsO₂/HFD treated with silymar in at the dose of 50 mg/kg. The behavioral assessments (elevated plus maze and T-maze), biochemical analysis and histological investigations were performed. As shown in the present study, NaAsO₂/HFD group exhibited enhanced anxiety related behaviour, memory deficit, oxidative stress (MDA, TNF-α, IL-1β) and decreased antioxidant enzymes (SOD, CAT, GSH) activity. The histological examination revealed significant neuronal loss and remarkable architectural alteration in hippocampus, prefrontal cortex and cerebellum. These effects were ameliorated by NlREq administered in a dose-dependent manner, with the 400 mg/kg dose enhancing memory in the affected animals, reducing inflammation, replenishing antioxidant defence systems, and maintaining integrity of neurons. These results indicate that Nauclea latifolia root extract has strong neuroprotective potential and may be used as a phytochemical for managing neurotoxicity and cognitive impairment due to exposure to toxins in the environment and poor diet.

Keywords: Nauclea latifolia; Neuroprotection; Oxidative stress; High-fat diet; Sodium arsenite; Inflammatory markers

Introduction

Public health faces rising challenges from widespread high-fat diet (HFD) use coupled with arsenic toxin exposure because these factors both speed up the development of metabolic disorders and cardiovascular diseases and now demonstrate increased potential to cause neurotoxicity [1]. Studies demonstrate that combined exposure to HFD and arsenic compounds aggravates adverse medical outcomes resulting in evident physiological abnormalities including cognitive impairment. The simultaneous exposure to HFD and arsenic causes synergistic effects which generate intensified neurological deficits and memory impairment according to Alboghobeish et al. [2] and Ashraf et al. [3]. Studies have proven that exposure to both agents in unison results in neurotoxic damage that exceeds results from single-agent exposure by threefold [2]. Oxidative stress has augmented through exposure while information from brain antioxidant defense mechanisms has diminished. Many studies have demonstrated that arsenic exposure could potentially have opposite anti-obesity outcomes to dietary fat consumption while modifying neurotoxicity effects [4].

The environmental pollutant NaAsO₂ generates major neurological damage and impaired mental functions [5]. Repeated absorption of arsenic by the brain leads to concentration-dependent accumulation, initially affecting the hippocampus before spreading to the cerebellum and ultimately the cerebrum [3]. Research shows that the spatial memory process along with learning ability become impaired as a result of these changes observed in hippocampal neurons [6,7]. Stimulated by NaAsO₂ the activation of MMP-2 and MMP-9 degrades the blood-brain barrier (BBB) thus permitting toxic compounds to enter the brain which results in multiple forms of brain damage and dementia development [5]. The compound causes oxidative stress through its action of lowering antioxidants and boosting lipid damage which activates inflammatory pathways NF-κB and Nrf2 thus accelerating neurodegeneration [8]. The neuronal function becomes impaired because of diminished norepinephrine, dopamine and acetylcholinesterase activity [9]. Evidence shows that HFD create adverse effects on brain function through the mechanisms of neuroinflammation combined with hippocampal microRNA alterations and diminished neuroplasticity as well as cognitive impairment [10]. HFD causes dissipation of BBB integrity while producing sex-dependent modifications of neuronal firing and it leads to cognitive plus motor dysfunction [11, 12]. Neurotoxic effects trigger by a combination of High-fat diet and NaAsO₂ impact memory and cognitive function severely along with generating oxidative stress and creating mitochondrial dysfunction [13, 14]. This highlights the need for effective protective strategies against such combined toxicities.

The medicinal plant N. latifolia which goes by its common name African peach stands out for its traditional medical applications and therapeutic abilities. This plant serves multiple cultural purposes for the treatment of diabetes and hypertension and inflammatory diseases [15,16]. The plant extracts demonstrate strong antioxidant capabilities and anti-inflammatory effects that establish it as a useful natural medicine component. African peach received traditional approval for treating diabetes patients alongside those dealing with malaria and hypertension problems [15, 17]. The presence of flavonoids, phenolics, and saponins provides antioxidant properties to the plant [18].

Given the serious health risks linked to combined exposure to a HFD and NaAsO₂, there’s a growing need for effective protective agents that can help counter these harmful effects. N. latifolia, a plant long valued in traditional medicine, has shown promising antioxidant and anti-inflammatory properties. However, its potential to combat the combined impact of a HFD and NaAsO₂ exposure remains largely unexplored. This study aimed to evaluate whether the root extract of N. latifolia could protect against HFD/NaAsO₂-induced anxiety, memory impairment, oxidative stress, elevated pro-inflammatory cytokines (TNF-α, IL-1β), and brain regions (hippocampus, prefrontal cortex, and cerebellum) tissue damage in rats. We hypothesized that the extract would significantly reduce these toxic effects, via its antioxidant and cell-protective actions.

Materials and Methods

Chemicals and reagents

The chemicals used were NaAsO₂, ketamine hydrochloride (PVT Ltd., India,) rat loading activities of the antioxidant enzymes (CAT, SOD, GSH), concentration of MDA, and chemokines (TNF-alpha and IL-1ꞵ rat ELISA kit). The silymarin used in this study was silymarin capsules 70 mg/tablet, purchased from the Micro Labs Ltd, India. All other chemicals and reagents used in the studies were of analytical grade and obtained from standard commercial sources.

Plant collection, identification and extraction

N. latifolia root extracts were collected from a community garden in Shika, Sabo, Kaduna State, Nigeria immediately after the flowering period. Characterization and authentication of the plant root was done by a Taxonomist of Ahmadu Bello University. The voucher specimen number for the plant is 3276, which is deposited in the herbarium of the department. Air-drying of N. latifolia roots was done at 25 °C until they reached a constant mass and then mechanically pounded using an automated mortar and pestle. Subsequently, each dry powder of the sample was dissolved in 600 ml of 95% ethanol and maintained at the same condition for 48 hours. The tested samples, weighing 500 g, were put into the mortar. The samples were filtered through Whatman filter paper no. 1 and then concentrated at 55 °C using rotary evaporator namely Rotavapor R-300, Buchi. The extract was stored on ice and at a temperature of 4 °C was used in this experiment. The rats under experimentation were administered with N. latifolia extract in a daily dose in distilled water.

Experimental animals and ethical compliance

The rats used in this experimental study were 25 healthy six-week-old Wistar albino rats with body weight of 140 to 160g. When housed in these chambers it is important to note that the rats were caged in clear plastic cages with a mesh roof and provisions for the feeding and watering of the animals. The rats were maintained under standard housing condition, temperature of 25 ± 2 °C and light-dark cycle of 12 hours in a vermiculated portion of the animal house of the faculty of pharmaceutical science, ABU, Zaria, Nigeria. The rats also received standard rodent diet and water ad libitum in good hygienic conditions as needed by any standard rodent. These rats were allowed to acclimatized for two weeks before the experiment. The follow-up of the study was enhanced with the permit of the ethic committee of the utilisation of animals in the process of ABU Zaria Animal Use and Care Committee (ABUCAUC/2023/033). All animal studies were carried out in compliance with the European Union guidelines for the use of animals for scientific research (Directive 2010/63/ EU).

Experimental design and treatment groups

Twenty-five (25) rats were randomly assigned into five groups of 5 rats each as follows:

Group 1 (normal control) included rats receiving distilled water (10 ml/kg b.w.);

Group 2 (positive control) treated with HFD/ NaAsO₂ rats);

Group 3 rats treated with HFD/NaAsO₂ + N. latifolia roots extract – NlREq (200 mg/kg b.w.);

Group 4 rats treated with HFD/NaAsO₂ + N. latifolia roots extract – NlREq (400 mg/kg b.w.);

Group 5 rats treated with HFD/NaAsO₂ + Silymarin (50 mg/kg b.w.);

The high-fat diet (consisting of 40 grams of animal fat and 100 grams of pelletised rat chow), and drinking water containing NaAsO₂ was supplied (prepared from sodium arenite solution in deionized water to a final concentration of 10 mg/L). The doses of both HFD and NaAsO₂ were selected based on previous studies, which demonstrated their ability to induce organ toxicity in Wistar rats [19, 20]. The selection of N. latifolia at doses of 200 mg/kg and 400 mg/kg was informed by previous studies demonstrating its therapeutic efficacy and favourable safety profile. These doses have been shown to exert significant biological activity without inducing adverse effects on hepatic or renal function, thus supporting their suitability for use in this study [21,22].

Behavioural tests

A blinded investigator assessed behavioural abnormalities. Neurobehavioral tests (using Elevated plus maze and T-maze test) were conducted in a well-lit testing room and recorded using a digital video recorder. The recordings were analyzed with ANY-maze video tracking software (Kim & Friends Inc., USA), and the data were exported to excel for further evaluation [23].

Elevated plus maze

The EPM test is to evaluate anxiety-like behaviors in rats based on their preference for using an open field test [24,25]. The EPM for rats had two perpendicular open arms of size 21.5 × 7.5 cm and two closed ones 21.5 × 7.5 × 20 cm which were arranged from a central square platform of 7.5 × 7.5 cm. The platform, as well as the floored area of the maze, was made from wooden material; the lateral walls of the closed arms were made of wooden panels painted black. The whole maze was above the ground surface by a mean height of 38 cm [26].

In the course of each trial, one rat at a time was then put at the centre of the maze and the exploratory activities of the rat were tracked for five minutes. Some of the variables quantified included the totals of entries made into the open and closed arms as well as the amount of time spent in the open or closed arms. Data collected from rat activities was omitted for the time spent on the central platform. To avoid other scent clues affecting the subsequent experiments, the maze was washed with 70% ethanol and left to air dry in between trials.

T-maze spontaneous alternation

This experiment employed a T-maze apparatus made from black plexiglass to minimise any prospective fresh stimuli during the experiment. This included a long start arm with a width of 16 cm and length of 50 cm and two choice arms each with width of 10 cm and length of 10 cm located perpendicularly on the upper end of the start arm. A partition projected 15 cm from the start arm and forced the rat into one of the left or right goal arm without its reach the junction. A rat was taken separately and placed in the start arm, facing the junction, and the rat was trained to choose the appropriate arm – left or right. A correct alternation was defined as a time when the rat switched between goal arms, an incorrect choice was considered an error. An arm choice was recorded only if all four legs of the rat was placed in it. Every rat was given seven trials with 30 second intertrial interval; the maximum switch between the left and right arms were six as recommended by Jagadeesan et al. [27].

Biochemical studies

Wistar rat brain tissues were removed and prepared according to procedures elaborated by Zatta et al. [28] and Habila et al. [29]. Following anaesthesia, the treated and control animals were sacrificed by decapitation immediately after sample collection. Then, the brain tissues were immediately dissected out and dropped on an inverted Petri dish which rested on ice. These tissues were stripped and washed in physiological saline and then homogenized in PBS solution 0.7, pH 7:4. In order to pellet the cellular debris from the homogenized samples, they were further centrifuged at 5,000 X g for 15 minutes, and the resultant supernatant was aliquoted and kept at -20 °C for subsequent antioxidant (SOD, CAT, GSH and MDA) and Inflammatory (TNF-α and IL1-β) analysis.

Activity of superoxide dismutase (SOD)

SOD activity was measured following the methodology outlined by Kakkar et al. [30]. The assay involved a reaction mixture containing 2 mL of 0.052 mM sodium pyrophosphate buffer (pH 8.0), 0.1 mL of 186 μM phenazine methosulfate, 0.3 mL of brain supernatant, and 0.1 mL of 300 μM nitro blue tetrazolium. The mixture was subsequently incubated at 37°C for 5 minutes, after which 0.2 mL of NADH (780 μM) was introduced into the solution. The reaction was then allowed to proceed for 2 minutes at 37°C and stopped by the addition of 1.0 mL of glacial acetic acid (17.4 mol/L). The enzymatic activity of the resulting-coloured product was determined at 560 nm against reagent blank using spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan),and reported as units (U) protein^-1.

Determination of catalase activity (CAT)

CAT activity was assessed using a modified version of the Aebi, [31] method. The reaction mixture consisted of 2.5 mL of 50 mM phosphate buffer (pH 7.0), 0.4 mL of 5.9 mM hydrogen peroxide (H₂O₂), and 0.1 mL of enzyme extract. The activity was evaluated by monitoring the reduction in absorbance at 240 nM using spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan), with results expressed as units per milligram of protein.

Glutathione peroxide (GSH) technique

Using Paglia and Valentine's [32] method, the activity of GSH was measured. The reaction mixture was prepared using 0.1 mL of 0.15 M reduced glutathione (GSH), 0.3 mL of 0.1 M sodium phosphate buffer (pH 7.4), 0.05 mL of 2.25 mM sodium azide, 0.2 mL of 0.85 mM NADPH, 0.05 mL of brain homogenate, and 0.05 mL of water. The mixture was placed on a vortex for 10 minutes Per sample. The experiment was kept at room temperature. To start the enzymatic reaction, 0.05 mL of hydrogen peroxide (H₂O₂, 0.0011 M) were added and the absorbance read at 340 nm for 3 minutesusing spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan). The data obtained for a particular enzyme was reported relative to milligrams of the extracted protein.

Lipid peroxidation by measuring the malondialdehyde (MDA) level

Oxidative stress was determined by estimating malondialdehyde (MDA) concentration, according to the method described by Ohkawa et al. [33]. Shortly, 1.5 mL acetic acid 20%, 0.2 mL sodium dodecyl sulfate 8.1%, 1.5 mL thiobarbituric acid 0.8% was mixed with 0.1 mL brain tissue. They added the mixture to heat at 100°C for one hour and finally cooled in running tap water. On cooling, distilled water 1.0 mL and 5.0 mL of n-butanol containing pyridine were also added. The mixture was centrifuged for 10 minutes at 4000 rpm and 4°C then the pink colored compound in the supernatant was carefully extracted for absorption measurement at 532 nm using spectrophotometer, model UV-2100, (Shimadzu Corporation, Kyoto, Japan).

Determination of inflammatory markers

Concentrations of TNF-α and IL1-β (inflammatory cytokines) in the samples were estimated using ELISA kits from Ray Biotech, Inc. USA. In this study, we quantified the cytokines in the blood serum in pg/ml in this study.

Histological studies

The brain was dissected out, the prefrontal cortex, hippocampus cerebellum was carefully excised, tissue was post-fixed in Neutral buffer formalin for 24 hours then processed for histological staining with H&E to demonstrate cortical histoarchitectural changes [34].

Statistical analysis

The data obtained in these investigations was analyse with help of the SPSS software, version 20, produced by IBM, USA. The statistical analysis was done using a one-way ANOVA and then Tukey post hoc test to ascertain the difference in control group and experimental groups. The results obtained were presented as means ± standard error of the mean wherever applicable. The measure of significance was at p < 0.05.

Results

Effects of N. latifolia on the gross morphology of HFD/NaAsO₂ exposed rats

Table 1, At the onset of the study, there was no statistically significant different (p > 0.05) in the weight of the Wistar rats across and within the experimental groups. At the end the experiment, HFD/NaAsO₂ exposure resulted in a substantial body weight loss (p < 0.05) in all rats treated with HFD/NaAsO₂ alone compared to rats in the treatment groups receiving 400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂. Altogether, body weight gain in HFD/NaAsO₂ treated rats was considerably (p < 0.05) less compared with control group and 400 mg/kg NlREq + HFD/NaAsO₂ treated rats. The change in body weight which was measured in percentage (%) was significantly low in the HFD/NaAsO₂ treated group compared to groups of rats that were treated with 400 mg/kg NlREq + HFD/NaAsO₂.

Table 1.

Protective effect of N. latifolia on body weight of the adult Wistar rats.

N. latifolia ameliorates anxiety-like behaviour in HFD/NaAsO₂-exposed rats

The data presented in Table 2 demonstrate significant differences across all groups regarding the number of closed and open arm entries by the rats. Similar patterns were observed in the percentage of entries into the closed and open arms and in the index of open arm avoidance. When compared to the control group, rats treated with HFD/NaAsO₂ exhibited reduced time spent in the open arms and increased time in the closed arms, accompanied by a significantly higher number of entries into the closed arms and fewer entries into the open arms.

Table 2.

Protective effect of N. latifolia on behavioural anxiety level of the adult Wistar rats.

Conversely, rats treated with 200/400 mg/kg NlREq + HFD/NaAsO₂ exhibited a significant, dose-dependent increase in the time spent in the open arms and a corresponding decrease in the time spent in the closed arms compared to the HFD/NaAsO₂-treated group. Furthermore, the index of open arm avoidance (%) was significantly reduced in HFD/NaAsO₂-treated rats compared to both the control group and the treatment groups (200/400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂).

Estimation of spontaneous alternation (%)

The statistical analysis of the mean values of spontaneous alternation is presented in Fig. 1. Rats treated with HFD/NaAsO₂ exhibited a significant reduction (p < 0.05) in spontaneous alternation, with a mean value of 43.41 ± 1.39, compared to the control group, which had a mean value of 71.46 ± 2.61. Treatment with 200 mg/kg and 400 mg/kg NlREq + HFD/NaAsO₂ resulted in mean values of 61.35 ± 1.54 and 68.72 ± 1.56, respectively, showing a significant (p < 0.05) increase in the percentage of spontaneous alternation compared to the HFD/NaAsO₂-treated group. Similarly, the 50 mg/kg SLY + HFD/NaAsO₂ treatment group demonstrated a significant improvement in spontaneous alternation compared to the HFD/NaAsO₂-treated group.

Figure 1.

T-Maze assessment of spontaneous alternation (%). Comparing the values within the bar charts that are superscripted 'a', 'b', and 'c' are significantly different (p < 0.05).

Effect of N. latifolia root on oxidative stress markers of HFD/NaAsO₂ exposed rats

The mean levels of oxidative stress biomarkers—SOD (4.10 ± 0.24), CAT (4.20 ± 0.22), and GSH (12.74 ± 1.18)—were significantly lower in the HFD/NaAsO₂-treated group compared to the control group, which showed mean levels of 15.38 ± 0.48 for SOD, 10.60 ± 0.58 for CAT, and 34.72 ± 1.73 for GSH, respectively. Rats treated with 400 mg/kg NlREq + HFD/NaAsO₂ showed a significant increase in SOD (9.42 ± 0.98), CAT (8.20 ± 0.26), and GSH (21.92 ± 1.46) levels compared to those treated only with HFD/NaAsO₂. Similarly, the 200/400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂-treated groups demonstrated significantly elevated CAT and GSH levels relative to the HFD/NaAsO₂ group. However, no significant differences in SOD levels were observed between the 200/400 mg/kg NlREq + HFD/NaAsO₂ and 50 mg/kg SLY + HFD/NaAsO₂-treated groups when compared with the HFD/NaAsO₂ group.

Additionally, the MDA levels in the HFD/NaAsO₂-treated rats (196.20 ± 3.59) were significantly higher (p < 0.05) than those in the control group (148.60 ± 4.56). Treatment with 200 mg/kg and 400 mg/kg NlREq + HFD/NaAsO₂ resulted in a dose-dependent significant reduction in MDA levels, with values of 179.34 ± 2.85 and 156.80 ± 1.83, respectively, compared to the HFD/NaAsO₂-treated group. Similarly, rats treated with 50 mg/kg SLY + HFD/NaAsO₂ exhibited a significant decrease in MDA levels relative to the HFD/NaAsO₂-treated group (Fig. 2).

Figure 2.

Comparisons of the bar charts of the oxidative stress parameters after 6 weeks of treatment. Different alphabets indicate significant difference in means of any two-barchart (p<0.05).

Effects of N. latifolia root on inflammatory markers of HFD/NaAsO₂ exposed rats

The statistical analysis of the mean values for oxidative stress biomarkers is presented in Fig. 3. Rats exposed to HFD/NaAsO₂ showed significant increases (p < 0.05) in TNF-α and IL-1β levels, with mean values of 91.21 ± 1.67 and 74.81 ± 2.46, respectively, compared to the control group, which had corresponding mean levels of 41.60 ± 2.06 and 40.73 ± 1.28. Treatment with 200 or 400 mg/kg NlREq in combination with HFD/NaAsO₂ significantly reduced TNF-α levels to 53.06 ± 4.00 and 51.64 ± 4.57, and IL-1β levels to 61.59 ± 5.19 and 62.23 ± 5.11, respectively, compared to the untreated HFD/NaAsO₂ group. Similarly, rats treated with 50 mg/kg SLY + HFD/NaAsO₂ showed a significant reduction in TNF-α and IL-1β levels, with values of 152.00 ± 2.66 and 182.80 ± 8.55, respectively, when compared to the HFD/NaAsO₂ group.

Figure 3.

Graphs of bar chats of inflammatory markers (A TNF-alpha concentration) and (B IL1-ꞵ ) after 6 weeks of treatment. Different alphabets indicate significant difference in means of any two-barchart (p<0.05).

Histological evaluation

According to histological changes, there were noticeable tissue changes in all groups of the experiment. In the control group the areas of interest showed hippocampus had normal architecture with well developed granular and pyramidal neurons in CA1 and CA3 regions as depicted in the Figure 4A. In contrast, rats placed on HFD/NaAsO₂ developed significant neuronal pathology in terms of shrunken nuclei, vacuolated cytoplasm or pyknotic nuclei or karyolysis in the CA1 region of the hippocampus. Further, areas devoid of pyramidal neurons were noted; and apoptosis in of pyramidal cells of the CA3 region were detected (blue arrow), and clumped neuronal fibrils (arrow) were also noticeable (Fig. 4B). Treatments with 200 mg/kg NlREq + HFD/NaAsO₂ led to mild damage on the granular cells in the CA1 area and the pyramidal cells in the CA3 area with intracellular vacuoles with more intense staining as shown in Fig. 4C. However, the group given 400 mg/kg NlREq+ HFD/NaAsO₂ exhibited near-normal structure or architecture of the brain plus nearly normal histology or arrangement of cells; with only a few pyramidal as well as granule cells remained vacuolated in both the CA1 and CA3 areas of the hippocampus (Fig. 4D). Supplementation with 50 mg/kg SLY along with HFD/NaAsO₂ induced relatively mild neurotoxicity characterized by degeneration of the granular and pyramidal cells as well as vacuolation in the CA1 and CA3 regions of the hippocampus.

Figure 4.

Composite photomicrographs of the hippocampus: Control group (A) was observed with packed layers of CA1 pyramidal cell with vesicular nucleus and standardized CA3 neurons each with central nucleolus and vesicular nucleus (arrow). The HFD/NaAsO₂-treated (B) group also has shrunken nuclei, vacuolated cytoplasm, and neuronal loss in CA1 and apoptosis together with fibril clumping in CA3 (arrow). The CA1 and CA3 of extract treated groups (C&D) (200/400 mg/kg NlREq + HFD/NaAsO₂) showed preservation of the pyramidal cells although with vacuolated pyramidal and granular cells were observed (arrow). The 50 mg/kg SLY + HFD/NaAsO₂ group (E) shows slighted necrotic pyramidal cells and granular cells at CA1 &CA3 (arrow) (H&E X 200).

The control group exhibited intact neurons and a normal cytoarchitecture in the prefrontal cortex and cerebellum (Fig. 5A), including well-preserved molecular, granular, and Purkinje cell layers (Fig. 6A).

Figure 5.

Representative photomicrographs of the prefrontal cortex. The control section (A) shows darkly pigmented neurons and an intact neuropil, indicating normal cortical architecture (green arrow). In the HFD/NaAsO₂-treated group (B), there is evidence of severe vacuolations, necrosis of neurons, and the presence of pericellular spaces surrounding necrotic and pyknotic neurons(red arrow). Treatment with 200/400 mg/kg of NlREq + HFD/NaAsO₂ (C & D) resulted in only mild vacuolations and preserved pyramidal and granular neuronal cells(green arrow). Similarly, the group treated with 50 mg/kg SLY + HFD + NaAsO₂ (E) showed a few vacuolated neurons with moderate degenerative changes(green arrow) (H&E stain, X200).

Figure 6.

Representative photomicrographs of the cerebellum. In the control section (A), the cerebellum displays a well-organized structure with intact molecular, granular, and Purkinje cell layers, along with healthy Purkinje cells (green arrow). In contrast, the HFD/NaAsO₂-treated group (B) shows degeneration of the Purkinje cells and disorganization of their layer (red arrow). Groups treated with 200/400 mg/kg of NlREq + HFD/NaAsO₂ (C & D) showed near-normal cerebellar layering and only mild alterations (green arrow). The SLY-treated group (E) exhibited some degenerative changes in the Purkinje cells and their layer (green arrow). (H&E stain, X200)

In contrast, the HFD/NaAsO₂ group had pronounced neuronal inflammation and loss of the neurons’cell, and most of the neurons were surrounded by widening of the perineuronal spaces (Fig. 5B), while for the cerebellum there is degeneration of the Purkinje cells (Fig. 6B). The rats, receiving 200/400 mg/kg NlREq, demonstrated neuronal preservation with multiple regenerated neurons and the decrease level of neuropil vacuolation(Fig. 5C&D), while for histology of the cerebellum revealed near normalorientation of the molecular layer, granular layer, and purkinje layer pyramidal and granule cells (Fig. 6C&D). The rats in the group receiving SLY of the 50 mg/kg backed up with HFD/ NaAsO₂ showed close to normal and crowded granular neuronal (Fig. 5E) cells and cerebellum densely populated granular layer, normal molecular layer and slight degeneration of the purkinje layer and cells (Fig 6E).

Discussion

The interplay between NaAsO₂ exposure and high-fat diets significantly influences body weight regulation and metabolic health. This study found that rats given NaAsO₂/HFD compared to controls, body weight gain dropped significantly (p < 0.05). Previous research indicates that HFD can disrupt energy homeostasis, leading to decreased body weight [35]. On the other hand, rats treated with 200 and 400 mg/kg with N. latifolia were able to prevent the HFD/NaAsO₂-induced adverse effects. N. latifolia mitigates weight loss in rats by eliciting recovery effects, as evidenced by a significant increase in body weight. The observed outcome was in agreement with the previous works done. N. latifolia has demonstrated potential in mitigating weight loss in rats through various mechanisms, particularly in the context of obesity and metabolic disorders. The studies indicate that extracts from this plant can influence body weight and lipid profiles, contributing to weight management without suppressing appetite [36,37].

In the study HFD/NaAsO₂ exposure have been shown to induce anxiety-like behaviours also impaired memory function in rats treated with HFD/ NaAsO₂ as observed in the result section (Fig.1 & Table 2). HFD are associated with changes in the gut microbiome, neuroinflammation, and alterations in brain signalling pathways, all of which contribute to increased anxiety [38, 39]. Likewise, NaAsO₂, is known to induce oxidative stress as also observed in this study, which can similarly affect brain function and behaviour [40, 41]. The interplay between HFD and NaAsO₂ exposure significantly impaired memory function, primarily through oxidative stress and neuroinflammatory pathways. Studies show that each of them reduces cognition individually and collectively, especially in the hippocampus, which is the part of the brain that deals with memory. Highrate of HFD consumption causes cognitive deterioration characterized by poor memory in rats after numerous days of a high fat diet accompanied by high IL-1β levels in the hippocampus[42]. Collectively, we reveal an HFD-induced alteration of synaptic plasticity and tau hyperphosphorylation, resulting in deficits in learning and memory [43]. NaAsO₂ exposure induces oxidative stress, leading to spatial learning and memory impairments in rats [12, 44]. Chronic arsenic exposure alters hippocampal neuron structure and function, correlating with memory deficits observed in behavioural tests. The simultaneous exposure to HFD and NaAsO₂ amplifies memory impairment, with studies showing that their combined effects are more detrimental than either factor alone [2].This experiment further demonstrated that N. latifolia improve memory function and mitigating anxiety level in rats’ model as shown in the result particularly the high dose (400 mg/kg), as revealed by the percentage of spontaneous alternation of rats in treated group in comparison to rats who received only NaAsO₂ alone. This might be as a result of the antioxidant potential of the N. latifolia, and this finding is consistent with the previous experiment.

NaAsO₂ exposure leads to increased ROS production, which is linked to cognitive deficits and neurotoxicity. Studies show that arsenite treatment elevates lipid peroxidation and depletes antioxidant defenses, such as glutathione [44,45]. Chronic arsenic exposure disrupts mitochondrial function, decreasing the activity of key mitochondrial complexes and superoxide dismutase (MnSOD), resulting in further ROS accumulation [46]. High-fat diets can also exacerbate oxidative stress by increasing lipid peroxidation, which, when combined with arsenic exposure, may lead to synergistic neurotoxic effects [44]. In this study, N. latifolia stem-bark extract increases concentration of reduced glutathione and antioxidant enzymes notably. This finding affirms the earlier studies that N. latifolia extracts contain a high total antioxidant capacity with efficient DPPH scavenging and FRAP, revealing that plants could help combat free radicals [18]. Besides, the fact that N. latifolia contains phytochemicals such as flavonoids Saponins, alkaloids, vitamins C and E and phenolics also supports antioxidant properties of the brain [18].

In this study, NaAsO₂ combined with HFD in rats increased inflammation by upregulating pro-inflammatory cytokines such as TNF-α and IL-1β, thereby exacerbating oxidative stress and inducing neural cell necrosis, as further confirmed by brain histological analysis. NaAsO₂, an environmental toxicant, increase inflammatory responses [47, 48]. On the other hand, HFD incites chronic low-grade inflammation that activates pathways leading to neuroinflammation and the related cognitive decline by the C/EBPβ/AEP signalling [49]. In this study, N. latifolia was able to prevent the upsurge of the inflammation and it also prevent the cytoarchitecture of the histology of the brain (Cerebrum, cerebellum and Hippocampus), and this in line with other works where, N. latifolia have been shown to exhibits significant anti-inflammatory mechanisms primarily through its phytochemical constituents, which include flavonoids, phenolics, glycosides, and tannins. These phytochemicals also play a role in the anti-inflammatory effects by inhibiting pro-inflammatory pathways [17]. The plant is traditionally used to treat various ailments, including those associated with inflammation, suggesting its potential as a natural therapeutic agent [50].

Conclusions

The present investigation showed that aqueous root extract of N. latifolia reversed neurotoxicity and oxidative stress caused by NaAsO₂ and HFD in the Wistar rats. The extract added better behavioural performance, replenished antioxidant protection, lowered pro-inflammatory cytokines and provided structural protection to neurons which are the highest at increased doses. The results presented here affirm the viability of the N. latifolia for reducing neurotoxicity due to environmental toxins and unhealthy diet by modulating oxidative stress and neuro inflammation. However, more research must be done to understand how the compound operates at the molecular level and how it may be used in practice.

Notes

Acknowledgement

Mr. Bamidele, Senior Medical Laboratory Scientist, Department of Human Anatomy, ABU, Zaria, deserves a special mention for his efforts in performing the biochemical assay and processing the tissue samples.

Conflict of interest

There was no funding from public, commercial, or not-for-profit bodies for this project.

CRediT author statement

WM: Conceptualization, Methodology, Investigation, Funding acquisition, Writing – Original draft, Writing – Review & Editing; AA: Methodology, Funding acquisition, Writing – Review & Editing; OBO: Methodology, Investigation, Writing – Original draft; JTA: Methodology, Investigation; GKA: Project administration, Supervision; VKJ: Writing – Original draft, Writing – Review & Editing; AYS: Investigation, Writing – Original draft; BI: Funding acquisition, Writing – Review & Editing.

Funding

There was no funding from public, commercial, or not-for-profit bodies for this project

Ethics approval and consent to participate

The authors confirm that all animals were provided humane care during the experimental period, in compliance with the guidelines set by the Directorate of Academic Planning and Monitoring, ABU, Zaria, and the approved recommendations for laboratory animal care and use (ABUCAUC/2023/033).

Availability of data and material

The datasets utilized and/or examined in this study can be obtained from Wusa Makena or the corresponding author upon reasonable request.

Consent for publication

Not applicable

References

1. Ye Z, Xiong H, Huang L, Zhao Q, Xiong Z, Zhang H, et al. Mechanisms underlying the combination effect of arsenite and high-fat diet on aggravating liver injury in mice. Environ Toxicol 2024;39(3):1323–1334. https://doi.org/10.1002/tox.24037.

2. Alboghobeish S, Pashmforosh M, Leila Zeidooni L, Samimi A, Rezaei M. High fat diet deteriorates the memory impairment induced by arsenic in mice: a sub chronic in vivo study. Metab Brain Dis 2019;34(6):1595–1606. https://doi.org/10.1007/s11011-019-00467-4.

3. Ashraf S, Tahir A, Amjad A, Hameed S, Imtiaz R, Shabbir A, et al. Systemic health effects associated with sodium arsenite exposure: A reappraisal. Biological and Clinical Sciences Research Journal 2023;4(1):601. https://doi.org/10.54112/bcsrj.v2023i1.601.

4. Carmean CM, Kirkley AG, Landeche M, Ye H, Chellan B, Aldirawi H, et al. Arsenic exposure decreases adiposity during high-fat feeding. Obesity (Silver Spring) 2020;28(5):932–941. https://doi.org/10.1002/oby.22770.

5. Cheng L, Zhang Y, Lv M, Huang W, Zhang K, Guan Z, et al. Impaired learning and memory in male mice induced by sodium arsenite was associated with MMP-2/MMP-9-mediated blood-brain barrier disruption and neuronal apoptosis. Ecotoxicology and Environmental Safety 2024;285:117016. https://doi.org/10.1016/j.ecoenv.2024.117016.

6. Lin L, Chen X, Zhou Q, Huang P, Jiang S, Wang H, et al. Synaptic structure and alterations in the hippocampus in neonatal rats exposed to lipopolysaccharide. Neurosci Lett 2019;709:134364. https://doi.org/10.1016/j.neulet.2019.134364.

7. Sun H, Yang Y, Shao H, Sun W, Gu M, Wang H, et al. Sodium arsenite-induced learning and memory impairment is associated with endoplasmic reticulum stress-mediated apoptosis in rat hippocampus. Front Mol Neurosci 2017;10:286. https://doi.org/10.3389/fnmol.2017.00286.

8. Kassab RB, El-Hennamy RE. The role of thymoquinone as a potent antioxidant in ameliorating the neurotoxic effect of sodium arsenate in female rat. Egyptian Journal of Basic and Applied Sciences 2017;4(3):160–167. https://doi.org/10.1016/j.ejbas.2017.07.002.

9. Silva-Adaya D, Ramos-Chávez LA, Petrosyan P, González-Alfonso WL, Gonsebatt ME. Early neurotoxic effects of inorganic arsenic modulate cortical GSH levels associated with the activation of the Nrf2 and NFκB pathways, expression of amino acid transporters and NMDA receptors and the production of hydrogen sulfide. Front Cell Neurosci 2020;14:17. https://doi.org/10.3389/fncel.2020.00017.

10. Spinelli M, Spallotta F, Cencioni C, Natale F, Re A, Dellaria A, et al. High fat diet affects the hippocampal expression of miRNAs targeting brain plasticity-related genes. Sci Rep 2024;14(1):19651. https://doi.org/10.1038/s41598-024-69707-7.

11. Zimmerman B, Kundu P, Rooney WD, Raber J, et al. The effect of high fat diet on cerebrovascular health and pathology: A species comparative review. Molecules 2021;26(11):3406. https://doi.org/10.3390/molecules26113406.

12. Gannon OJ, Robison LS, Salinero AE, Abi-Ghanem C, Mansour FM, Kelly RD, et al. High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner. J Neuroinflammation 2022;19(1):110. https://doi.org/10.1186/s12974-022-02466-2.

13. Xiong L, Huang J, Gao Y, Gao Y, Wu C, He S, et al. Sodium arsenite induces spatial learning and memory impairment associated with oxidative stress and activates the Nrf2/PPARγ pathway against oxidative injury in mice hippocampus. Toxicol Res (Camb) 2021;10(2):277–283. https://doi.org/10.1093/toxres/tfab007.

14. Wang C, Li H, Chen C, Yao X, Yang C, Yu Z, et al. High-fat diet consumption induces neurobehavioral abnormalities and neuronal morphological alterations accompanied by excessive microglial activation in the medial prefrontal cortex in adolescent mice. Int J Mol Sci 2023;24(11):9394. https://doi.org/10.3390/ijms24119394.

15. Abdel-Rahman NA. Nauclea latifolia (Karmadoda): Distribution, composition, and utilization. In: Mariod AA, editor. Wild fruits: Composition, nutritional value and products. Springer; 2019. 435-445.

16. Adepoju AJ, Esan AO, Olawoore IT, Ibikunle GJ, Adepoju VO. Nauclea latifolia stem bark extracts: Potentially effective source of antibacterial, antioxidant, antidiabetic and anti-inflammatory compounds. Journal of Applied Sciences and Environmental Management 2024;28(1):49–59. https://doi.org/10.4314/jasem.v28i1.6.

17. Balogun ME, Nwachukwu DC, Salami SA, Besong EE, Obu DC, Djobissie SFA. Assessment of anti-ulcer efficacy of stem bark extract of Nauclea latifolia (African Peach) in rats. American Journal of Biomedical Research 2016;4(1):13–17. https://doi.org/10.12691/ajbr-4-1-3.

18. Iheagwam FN, Israel EN, Kayode KO, DeCampos OC, Ogunlana OO, Chinedu SN. Nauclea latifolia Sm. leaf extracts extenuate free radicals, inflammation, and diabetes-linked enzymes. Oxid Med Cell Longev 2020;2020:5612486. https://doi.org/10.1155/2020/5612486.

19. Sun H, Yang Y, Gu M, Li Y, Jiao Z, Lu C, et al. The role of Fas-FasL-FADD signaling pathway in arsenic-mediated neuronal apoptosis in vivo and in vitro. Toxicol Lett 2022;356:143–150. https://doi.org/10.1016/j.toxlet.2021.11.012.

20. Chu F, Yang W, Li Y, Lu C, Jiao Z, Bu K, et al. Subchronic arsenic exposure induces behavioral impairments and hippocampal damage in rats. Toxics 2023;11(12):970. https://doi.org/10.3390/toxics11120970.

21. Gidado A, Ameh DA, Atawodi SE, Ibrahim S. Hypoglycaemic activity of Nauclea latifolia Sm. (Rubiaceae) in experimental animals. Afr J Tradit Complement Altern Med 2008;5(2):201–208. https://doi.org/10.4314/ajtcam.v5i2.31274.

22. Alabi QK, Olukiran OS, Adefisayo MA, Fadeyi BA. Effects of treatment with Nauclea latifolia root decoction on sexual behavior and reproductive functions in male rabbits. J Diet Suppl 2018;15(5):649–664. https://doi.org/10.1080/19390211.2017.1380105.

23. Adeniyi PA, Ishola AO, Laoye BJ, Olatunji BP, Bankole OO, Shallie PD, et al. Neural and behavioural changes in male periadolescent mice after prolonged nicotine-MDMA treatment. Metab Brain Dis 2016;31(1):93–107. https://doi.org/10.1007/s11011-015-9691-z.

24. Pellow S, Chopin P, File SE, Briley M. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14(3):149–167. https://doi.org/10.1016/0165-0270(85)90031-7.

25. Chioca LR, Ferro MM, Baretta IP, Oliveira SM, Silva CR, Ferreira J, et al. Anxiolytic-like effect of lavender essential oil inhalation in mice: participation of serotonergic but not GABAA/benzodiazepine neurotransmission. J Ethnopharmacol 2013;147(2):412–418. https://doi.org/10.1016/j.jep.2013.03.028.

26. Deák F, Lasztóczi B, Pacher P, Petheö GL, Kecskeméti V, Spät A. Inhibition of voltage-gated calcium channels by fluoxetine in rat hippocampal pyramidal cells. Neuropharmacology 2000;39(6):1029–1036. https://doi.org/10.1016/s0028-3908(99)00206-3.

27. Jagadeesan S, Chiroma SM, Baharuldin MTH, Taib CNM, Amom Z, Adenan MI, et al. Centella asiatica prevents chronic unpredictable mild stress-induced behavioral changes in rats. Biomedical Research and Therapy 2019;6(6):3233–3243. https://doi.org/10.15419/bmrat.v6i6.550.

28. Zatta P, Ibn-Lkhayat-Idrissi M, Zambenedetti P, Kilyen M, Kiss T. In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase. Brain Res Bull 2002;59(1):41–45. https://doi.org/10.1016/s0361-9230(02)00836-5.

29. Habila N, Inuwa HM, Aimola IA, Lasisi OI, Muhammad A, Okafor AI, et al. Acetylcholinesterase activity in the brain and blood of mice infected with Naja nigricollis venom. Biological Segment 2012;3(1):BS/1565.

30. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984;21(2):130–132.

31. Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121–126. https://doi.org/10.1016/s0076-6879(84)05016-3.

32. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70(1):158–169.

33. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95(2):351–358. https://doi.org/10.1016/0003-2697(79)90738-3.

34. Romero-Sarmiento Y, Soto-Rodríguez I, Arzaba-Villalba A, García HS, Alexander-Aguilera A. Effects of conjugated linoleic acid on oxidative stress in rats with sucrose-induced non-alcoholic fatty liver disease. Journal of Functional Foods 2012;4(1):219–225. https://doi.org/10.1016/j.jff.2011.10.009.

35. Fang LZ, Vidal JAL, Hawlader O, Hirasawa M. High-fat diet-induced elevation of body weight set point in male mice. Obesity (Silver Spring) 2023;31(4):1000–1010. https://doi.org/10.1002/oby.23650.

36. Vincent ME, Ohine AY, Desmond BA. Hypocholesterolemic effect of Nauclea latifolia fruit on glucose changes and lipid profile of alloxan-induced diabetes in albino rats. International Journal of Advances in Scientific Research and Engineering (IJASRE) 2018;4(10):13–20. https://doi.org/10.31695/IJASRE.2018.32896.

37. Makena W, Ishaku B, Solomon AY, Etukudo EM, Aminu A, Onimisi OB, et al. Extracts of Nauclea latifolia (African Peach) roots attenuate oxidative stress, inflammation, and hepatic and renal damage in Wistar rats induced by arsenic and high-fat diet. Journal of Food Biochemistry 2025;2025:5783346. https://doi.org/10.1155/jfbc/5783346.

38. Noronha SSR, Lima PM, Campos GSV, Chírico MTT, Abreu AR, Figueiredo AB, et al. Association of high-fat diet with neuroinflammation, anxiety-like defensive behavioral responses, and altered thermoregulatory responses in male rats. Brain Behav Immun 2019;80:500–511. https://doi.org/10.1016/j.bbi.2019.04.030.

39. Noronha SSR, de Moraes LAG, Hassell Jr JE, Stamper CE, Arnold MR, Heinze JD, et al. High-fat diet, microbiome-gut-brain axis signaling, and anxiety-like behavior in male rats. Biol Res 2024;57:23. https://doi.org/10.1186/s40659-024-00505-1.

40. Sharma S, Zhuang Y, Gomez-Pinilla F. Diet transition to a high-fat diet for 3 weeks reduces brain omega-3-fatty acid levels, alters BDNF signaling and induces anxiety & depression-like behavior in adult rats. Nature Precedings 2012. https://doi.org/10.1038/npre.2012.6950.1.

41. de Pinho Tavares Leal PE, da Silva AA, Rocha-Gomes A, Riul TR, Cunha RA, Reichetzeder C, et al. High-salt diet in the pre- and post-weaning periods leads to amygdala oxidative stress and changes in locomotion and anxiety-like behaviors of male Wistar rats. Front Behav Neurosci 2022;15:779080. https://doi.org/10.3389/fnbeh.2021.779080.

42. Sobesky JL, Barrientos RM, de May HS, Thompson BM, Weber MD, Watkins LR, et al. High-fat diet consumption disrupts memory and primes elevations in hippocampal IL-1β, an effect that can be prevented with dietary reversal or IL-1 receptor antagonism. Brain Behav Immun 2014;42:22–32. https://doi.org/10.1016/j.bbi.2014.06.017.

43. Yi W, Chen F, Yuan M, Wang C, Wang S, Wen J, et al. High-fat diet induces cognitive impairment through repression of SIRT1/AMPK-mediated autophagy. Exp Neurol 2024;371:114591. https://doi.org/10.1016/j.expneurol.2023.114591.

44. Gong X, Ivanov VN, Hei TK. 2,3,5,6-Tetramethylpyrazine (TMP) down-regulated arsenic-induced heme oxygenase-1 and ARS2 expression by inhibiting Nrf2, NF-κB, AP-1 and MAPK pathways in human proximal tubular cells. Arch Toxicol 2016;90(9):2187–2200. https://doi.org/10.1007/s00204-015-1600-z.

45. Medda N, Patra R, Ghosh TK, Maiti S. Neurotoxic mechanism of arsenic: Synergistic effect of mitochondrial instability, oxidative stress, and hormonal-neurotransmitter impairment. Biol Trace Elem Res 2020;198(1):8–15. https://doi.org/10.1007/s12011-020-02044-8.

46. Prakash C, Soni M, Kumar V. Biochemical and molecular alterations following arsenic-induced oxidative stress and mitochondrial dysfunction in rat brain. Biol Trace Elem Res 2015;167(1):121–129. https://doi.org/10.1007/s12011-015-0284-9.

47. Singhirunnusorn P, Moolmuang B, Lirdprapamongkol K, Ruchirawat M. Arsenite exposure potentiates apoptosis-inducing effects of tumor necrosis factor-alpha- through reactive oxygen species. J Toxicol Sci 2018;43(2):159–169. https://doi.org/10.2131/jts.43.159.

48. Umare MD, Bajaj KK, Wankhede NL, Taksande BG, Upaganlawar AB, Umekar MJ, et al. High-fat diet-induced cellular neuroinflammation: Alteration of brain functions and associated aliments. In: Martin CR, Patel VB, Preedy VR, editors. Diet and nutrition in neurological disorders. Academic Press; 2023. 613-629.

49. Liu P, Wang ZH, Kang SS, Liu X, Xia Y, Chan CB, et al. High-fat diet-induced diabetes couples to Alzheimer’s disease through inflammation-activated C/EBPβ/AEP pathway. Molecular Psychiatry 2022;27:3396–3409. https://doi.org/10.1038/s41380-022-01600-z.

50. Taïwe GS, Bum EN, Talla E, Dimo T, Weiss N, Sidiki N, et al. Antipyretic and antinociceptive effects of Nauclea latifolia root decoction and possible mechanisms of action. Pharm Biol 2011;49(1):15–25. https://doi.org/10.3109/13880209.2010.492479.

Article information Continued

This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Groups	Initial Weight	Final Weight	Weight Diff. (%)
Groups	Mean ± S. E	Mean ± S. E	Mean ± S. E
Control	148.60 ± 4.56	208.80 ± 2.94^b	40.81 ± 2.56^c
HFD/NaAsO₂	159.00 ± 3.59	178.00 ± 5.03^a	11.97 ± 2.34^a
200 mg/kg NlREq + HFD/NaAsO₂	152.20 ± 2.85	185.40 ± 4.46^a	15.22 ± 1.5^a
400 mg/kg NlREq + HFD/NaAsO₂	156.80 ± 1.83	201.60 ± 5.07^b	28.51 ± 2.05^b
50 mg/kg SLY + HFD/NaAsO₂	152.00 ± 2.66	182.80 ± 8.55^b	20.13 ± 4.20^a
F	1.647	7.329	18.390
P-Value	0.202	< 0.001	< 0.001

Groups	No. of entries into close arm	No. of entries into open arm	Time spent in close arm	Time spent in open arm	Index of open arm avoidance (%)
Groups	Mean ± S. E	Mean ± S. E	Mean ± S. E	Mean ± S. E	Mean ± S. E
Control	6.00 ± 0.63^a	10.20 ± 0.73^c	84.60 ± 6.44^a	136.20 ± 13.33^c	36.87 ± 2.40^a
HFD/NaAsO₂	10.40 ± 0.51^c	2.00 ± 0.32^a	173.60 ± 5.12^c	38.40 ± 14.46^a	83.99 ± 2.30^c
200 mg/kg NlREq + HFD/NaAsO₂	7.80 ± 0.58^c	6.60 ± 0.24d	120.40 ± 5.38^b	91.80 ± 2.26^b	53.92 ± 1.92^b
400 mg/kg NlREq + HFD/NaAsO₂	6.80 ± 0.37^a^b	8.40 ± 1.21^cd	134.60 ± 17.62^b	104.60±16.24^b^c	50.33 ± 2.48^b
50 mg/kg SLY + HFD/NaAsO₂	8.20 ± 0.73^c	5.60 ± 0.24^b	154.20±14.31^b^c	102.80 ± 2.35^c	54.78 ± 0.61^b
F	8.298	21.595	9.395	9.570	69.790
P-Value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001