Neurobehavioral deficits, oxidative dysregulation, and inflammatory responses in male Swiss mice following sub-acute exposure to ethanol extract of Leiotrametes lactinea Berk

Abstract

Background

Leiotrametes lactinea (Berk.) Welti & Courtec. a mushroom reported to possess antimicrobial, antioxidant, antitumor and analgesic activities. However, there is limited information on its safety profile in the central nervous system. This study thus evaluates the neurotoxic effect of ethanol extract of Leiotrametes lactinea (EELL) in male Swiss mice following 30 days oral exposure.

Method

Twenty-four (24) Swiss male mice were divided into 4 groups (n = 6) and orally administered EELL for 30 days at doses of 50, 100, and 200 mg/kg; the last group served as the healthy control. Neurobehavioral assessment started on the 21^st day, and on day 30, the animals were euthanised. Brain tissues were collected for biochemical assays (Superoxide dismutase, Catalase activity, reduced glutathione, malondialdehyde and acetylcholinesterase activity). Tumour necrosis factor-alpha and interleukin-6 levels were also measured.

Results

There was a reduction in body weight gain of EELL administered groups (3.38 ± 0.68 - 4.28 ± 0.85 g) compared to the control group (7.21 ± 0.87 g), while relative organ weights were unaffected. Also, a significant reduction in locomotor activity and rearing behaviour was recorded with an increased anxiety-like behaviour in the extract treated groups. Cognitive performance evaluated with the Morris water maze demonstrated impaired retention, evidenced by reduced time spent in the target platform zone and increased proximity to the platform at higher doses. Biochemical evaluation of brain tissue showed pronounced oxidative stress, indicated by significant reductions in reduced glutathione (71.06 ± 4.74 - 124.36 ± 5.35 µg/mol vs 149.88 ± 7.07 µg/mol), catalase, and superoxide dismutase activities, alongside elevated malondialdehyde levels, especially at 100 and 200 mg/kg doses in comparison to the control. In addition, acetylcholinesterase activity was significantly increased in 100 and 200 mg/kg EELL treated groups compared to the control (0.121 ± 0.01 & 0.160 ± 0.01 µmol/min/g tissue vs. 0.049 ± 0.01 µmol/min/g tissue). Likewise, increased levels of pro-inflammatory cytokines (interleukin-6 and tumour necrosis factor-α) was recorded in the treated groups.

Conclusion

These findings demonstrated that sub-acute exposure to EELL induces neurobehavioral deficits, oxidative stress, and neuroinflammatory responses, suggesting potential neurotoxic effects at higher doses.

Background

Neurotoxicity refers to adverse effects on the structure, function, and neurochemical components of the central or peripheral nervous system resulting from exposure to chemical or physical agents (neurotoxins), either during development or at maturity [1]. Critical neuronal processes, such as cognition, memory, and learning, are particularly vulnerable to neurotoxic insults, often resulting in functional impairments and behavioural deficits [2]. Mushrooms are fungi with visible basidiocarps and are widely recognised for their diverse therapeutic properties, including immunomodulatory, antimicrobial, anticancer, and anti-inflammatory activities [3]. Consequently, their pharmacological potential has been explored over the years [4].

Leiotrametes lactinea (Berk.) is a white-rot fungus belonging to the family Polyporaceae [5]. Previous studies have demonstrated that L. lactinea possesses antimicrobial, antitumor, analgesic, and anti-ulcerative activities, among others [6, 7]. Despite these promising bioactivities, concerns regarding its safety profile are emerging, as reproductive toxicity has recently been reported [8]. However, there remains limited information on its effects on the central nervous system (CNS), underscoring the need for a comprehensive evaluation of its potential neurotoxicity.

Neurotoxicity is mediated by interrelated mechanisms, including mitochondrial dysfunction, oxidative stress, and neuroinflammation [9]. An imbalance between the antioxidant system and free radical production induces lipid peroxidation, cellular dysfunction, and, consequently, neuronal damage [10]. Neuronal damage can be characterised by neuronal inflammation, which leads to elevated levels of proinflammatory cytokines such as tumour necrosis factor-α and interleukin-6, which further exacerbate neuronal damage and oxidative stress [11]. Furthermore, impaired cognitive and behavioural functions associated with neurotoxicity result from disruption of cholinergic neurotransmission, leading to alterations in acetylcholinesterase activity [12].

Given the interplay among these mechanisms, assessing antioxidant enzymes (superoxide dismutase, catalase, and reduced glutathione), lipid peroxidation (malondialdehyde), pro-inflammatory cytokines, and acetylcholinesterase activity provides a comprehensive framework for evaluating neurotoxic effects and their underlying pathways. Therefore, based on the reported pharmacological activities of Leiotrametes lactinea and the limited data on its CNS safety, this study was designed to investigate the neurobehavioral and neurochemical effects as well as the safety profile of repeated EELL administration in male Swiss mice. We hypothesised that subacute exposure to EELL may induce neurotoxic effects, which could manifest as alterations in locomotor activity, anxiety- and depression-like behaviours, and cognitive performance. Furthermore, the study aimed to elucidate possible underlying mechanisms of the toxic effects by assessing oxidative stress markers, acetylcholinesterase activity, and pro-inflammatory cytokines in brain tissue.

Materials and methods

Mushroom collection and identification

Fruiting bodies of mushroom samples were randomly collected from the University of Ibadan environment by a member of the research team. The collected fruiting bodies were kept in a sterile paper bag and stored at −20 °C. The mushroom was molecularly identified [8] as shown in Fig. 1.

Fig. 1.

Dendrogram tree for Leiotrametes lactinea [8]

Extract preparation

The collected mushroom sample was identified using molecular biology techniques and subsequently freeze-dried. The dried sample was then pulverised and percolated in 70% ethanol for 72 hours with constant agitation to ensure efficient extraction of bioactive constituents. Following extraction, the mixture was filtered, and the filtrate was concentrated under reduced pressure at low temperature using a rotary evaporator. The resulting filtrate was further evaporated to dryness on a water bath at 45 °C. The ethanol extract was stored in a glass bottle at −20 °C until needed.

Experimental animals

Twenty-four (24) healthy male Swiss mice weighing 20–24 g were procured from the University of Ibadan Animal House and used in this study. The experiments were conducted in the Department of Pharmacology and Therapeutics at the University of Ibadan, Animal House. Animals were housed in polypropylene cages at a density of 6 mice per cage. Cages were lined with wood shavings as bedding, which were changed three times per week to prevent ammonia buildup and reduce the risk of respiratory distress. Cages were enriched with nesting material to promote natural behaviours. A 12-hour light–dark cycle was maintained throughout the study. The animal facility was maintained at 22 ± 2 °C and 50–60% relative humidity. Animals were provided with standard pelletized feed (ACE™ Feeds Nigeria Limited) and clean drinking water ad libitum. They were allowed to acclimatise for 7 days prior to the start of the experiment. These controlled housing conditions were implemented to minimise stress and environmental variability, which are known to influence behavioural and cognitive outcomes in rodents. The study protocol was approved by the University of Ibadan Animal Care and Use Research Ethics Committee (UI-ACUREC), with ethical number UI-ACUREC/051/2022A.

Experimental design

Sub-acute toxicity study

The sub-acute toxicity study was conducted according to the OECD 407 protocol [13]. A total of twenty-four (24) healthy male Swiss mice were randomly assigned to four groups (n = 6) using a simple randomization procedure. They were orally administered selected doses of EELL (50, 100, or 200 mg/kg) or 2 mL/kg distilled water for a period of 30 days. The doses were selected based on prior acute toxicity study carried out on the extract as reported by [8]. The Feed and water intake were checked daily, while the animals were weighed weekly.

Neurobehavioral study

From the 22^nd day of treatment, experimental mice were subjected to daily neurobehavioral evaluations such as cognitive functions, anxiety, depression and locomotor activity (Fig. 2). All behavioural studies were carried out between 7 am to 12 pm daily for six days in a quiet environment with minimal human movement during testing. Behavioural performance was captured using a webcam, and parameters were analysed on ANY-Maze video-tracking software (Version 7.0; WoodDale, IL). Videos were analysed using ANY-maze software, which enabled automated, objective quantification of locomotor activity, exploratory behaviour, anxiety-like behaviour, and cognitive performance. Investigators performing these analyses, as well as the biochemical assays, were blinded to group allocation to minimise observer bias.

Fig. 2.

Experimental design

Test for spontaneous locomotor (open field test)

The open field test (OFT), which measures locomotor activity and anxiety-like behaviours, was carried out according to [14] with slight modification. The test was conducted on the 22^nd day under natural daytime laboratory lighting conditions, and all tests were carried out within the same time period each day to maintain consistency. The arena was a 400 × 400 mm square wooden box divided into 16 equal squares, with the centre zone defined as the four central squares (25% of the total area). Tests were conducted under natural daytime lighting at the same time each day, and behaviour was recorded on camera and analysed using ANY-maze software. Locomotor activity parameters included total distance travelled, line crossings, and rearing frequency. The arena was cleaned with 70% ethanol between trials to eliminate olfactory cues. The mice were tested one at a time for 5 minutes each. Other exploratory parameters, such as grooming, rearing, and sniffing, were carefully observed, and the duration of time it took to perform each action was recorded by a mounted overhead camera (webcam). All parameters were automatically analysed using ANY-maze (Stoelting, USA) video-tracking software, which automatically tracks locomotion and exploratory behaviours. Rearing was counted each time the mouse stood on its hind legs, while grooming was scored as the duration (in seconds) spent licking, scratching, or cleaning fur. Sniffing was scored as the duration (in seconds) the mouse spent sniffing the arena floor or walls. Automated scoring from ANY-maze^TM was cross-checked with manual observations for a subset of videos to ensure accuracy and consistency.

Test for anxiety-like behaviour (light and dark box test)

Male Swiss mice were tested for anxiety-like behaviour using the light and dark box on the 22^nd day of the experiment. The specification for Light and Dark Box (LDB) is 600 × 450 × 400 mm with an open doorway (70 × 70 mm) on the wall used for partitioning of the two compartments (i.e., light, which was painted white and dark, which was painted black) with no covering on top of it. The test was carried out under natural daytime lighting; the light compartment was illuminated by the room light, while the dark compartment remained dim due to the black interior. Each animal was carefully placed at the central partition between the two compartments, facing the wall of the light compartment, and left in the box for 5 minutes. After each test, the box was cleaned thoroughly with 70% ethanol and dried before the next animal was placed in it. This test was recorded using a webcam. The time spent in the light/dark compartment and the number of transitions were automatically analysed using ANY-maze^TM (Steolting, USA) [15].

Test for depression-like behaviour (forced swim test)

The test was carried out on the 23^rd day of the study using the protocol of [16]. Clean water maintained at 25 ± 1 °C was poured into a transparent glass jar (200 high and 100 mm in diameter) to a depth of 150 mm. Each mouse was gently placed individually into the water and allowed to swim for 5 minutes. No pretest session was conducted prior to the experimental trial. After the test, each mouse was immediately removed, dried using a clean towel, and returned to its home cage. Behaviour was recorded with a camera positioned above the apparatus and analysed with ANY-maze^TM video-tracking software, which captured and quantified immobility time. Immobility was defined as the period during which the mouse remained afloat in the water without active movements, except those necessary to keep the head above water. This was used as the index of behavioural despair. Fresh water was poured for each mouse.

Cognitive function test (Morris water maze)

Spatial learning and memory were evaluated using the Morris Water Maze as previously described by [17] with minor modifications. The apparatus consisted of a circular pool with a diameter of 800 mm and a height of 370 mm, filled with opaque water at a controlled room temperature (25 ± 1 ºC), with a hidden escape platform submerged approximately 1–2 cm below the water surface. During the acquisition phase (training days 1–4), each mouse underwent four trials per day, with a 10 - 15-minute inter-trial interval to allow for rest. The starting positions were varied systematically across four quadrants of the pool (north, south, east, and west) in a semi-random order to prevent spatial bias. The hidden platform remained at a fixed location throughout the acquisition phase. Each trial lasted up to 60 seconds. If the mouse failed to locate the platform within this time, it was gently guided to the platform and allowed to remain there for 15–20 seconds before being returned to its home cage. On day 5, a probe trial was conducted in which the platform was removed, and each mouse was allowed to swim freely for 60 seconds. Memory retention was assessed by measuring time spent in the target quadrant, escape latency during acquisition, and proximity to the previous platform location. All behavioural sessions were recorded using a video tracking system and analysed with ANY-maze^TM software.

Samples preparation and biochemical test

Animals were euthanised on the 30^th day using ketamine/diazepam (100/5 mg/kg). The whole-brain tissues were removed from the animals, weighed, and placed on ice. Harvested brain tissues were homogenised in cold homogenising buffer (0.1 M phosphate buffer, pH 7.4) at a ratio of 10 mL per gram of tissue. Homogenised tissue was centrifuged using a cold centrifuge. The supernatant obtained was stored in aliquots for biochemical and pro-inflammatory cytokine assays at −20 °C. A colourimetric assay following established protocols was used to measure the selected biochemical assays: Acetylcholinesterase [18], superoxide dismutase [19], catalase activity [20], reduced glutathione [21), and malondialdehyde [22].

Determination of brain pro-inflammatory cytokine (IL-6 and TNF-α) levels

The levels of IL-6 and TNF-α were measured in the brain tissue homogenates using the enzyme-linked immunosorbent assay kits (ELISA MAX^TM Deluxe Set for Mouse IL-6, Cat No: 431315 and ELISA MAX^TM Deluxe Set for Mouse TNF-α, Cat No: 430904) ordered from Biolegend, San Diego, USA. The manufacturer’s instructions were strictly followed when running the assay on the tissue samples. The result was expressed as ρg/mL.

Determination of chemical constituents of EELL using GC-MS analysis

Chemical constituents of EELL were quantified using GC-MS following a previously described method [23]. The instrument used was an Agilent Technologies 7890 GC system with a 5975 MC detector. The column was an HP5 MS 30 m in length, 0.320 mm in internal diameter, and 0.25 µm in thickness with, 99.9% pure helium as the mobile phase. A sample (1 µL) was injected into the column at 300 °C. To identify the compounds, the retention time and fragmentation pattern were compared to the NIST library database.

Statistical analysis

GraphPad Prism 8 software (San Diego, CA, USA version 8.04) was used for data analysis. Values are presented as Mean ± SEM. One-way analysis of variance (ANOVA) followed by a post hoc test (Dunnett’s) for multiple comparisons was performed for biochemical assays. For behavioural experiments, weight gain, as well as food and water intake that involves repeated measurement over time, a two-way ANOVA was applied, followed by Tukey’s multiple comparisons test. A p-value less than 0.05 was considered to be statistically significant.

Results

Survival of experimental animals

Two deaths were recorded during the 30-day study; one mouse died in each of the groups treated with 100 and 200 mg/kg EELL.

Effect of EELL on body weight and relative brain weight

A dose-dependent decrease in average weight gain was observed in mice in the treatment groups, ranging from 3.38 ± 0.68 to 4.28 ± 0.85 g compared to the control group (7.21 ± 0.87 g; F (3, 81) = 13.11, p ˂ 0.0001) during the 30 days of administration of EELL (Fig. 3A–B). The relative brain weight in the treated animals at all doses and the control group appear similar (Fig. 3C).

Fig. 3.

A-C: Effect of EELL on body weight and relative brain weight gain of male Swiss mice. All values are expressed as mean ± SEM (two-way followed by Dunnett’s post hoc test was carried out on weight gain data), n = 5; *p < 0.05, **p < 0.01 (significant difference) when compared with control group

Effects of EELL on feed and water intake

The reduction in the average feed intake of the animals treated with the extract during the 30 days of administration was significant (6.28 ± 0.22 - 8.41 ± 0.49 g) compared to the control group (10.01 ± 0.48 g; F (3,18) = 7.48, p = 0.001) (Fig. 4A). Likewise, the water intake decreased significantly in mice treated with 200 mg/kg EELL (4.75 ± 0.09 mL) relative to the control group (5.34 ± 0.36 mL; F (3,18) = 4.87, p = 0.009) as shown in Fig. 4B.

Fig. 4.

Effect of EELL on daily food and water intake in male Swiss mice. A. Daily feed intake. B. Daily water intake. All values are expressed as mean ± SEM (two-way ANOVA followed by Dunnett’s post hoc test), n = 5; *p < 0.05, (significance difference) when compared with control group

Effects of EELL on spontaneous locomotor activity in male Swiss mice

The effects of EELL on spontaneous locomotor activity in terms of total distance travelled, line crossing and rearing frequency in male Swiss mice are presented in Fig. 5A, B & C, respectively. The total distance travelled was significantly reduced in the treatment groups in a dose-dependent manner (F(4, 14) = 4.60, p = 0.04), (F(4, 14) = 6.26, p = 0.01), and (F(4, 14) = 8.37, p = 0.00). Similarly, the number of line crossings decreased significantly in the groups treated with 100 and 200 mg/kg EELL (F(4, 14) = 18.25, p = 0.04; F(4, 14) = 21.08, p = 0.01) compared with the control group. There was also a notable decrease in rearing frequency in the groups treated with 100 and 200 mg/kg of EELL (F(4, 14) = 4.150, p = 0.13; F(4, 14) = 5.083, p = 0.10).

Fig. 5.

Effect of EELL on spontaneous locomotor activity of male Swiss mice in the open field. A. Total distance travelled. B. Number of line crossing. C. Rearing frequency. All values are expressed as mean ± SEM all values are expressed as mean ± SEM, followed by two-way followed by Dunnett’s post hoc test, n = 5; *p < 0.05, (significance difference) when compared with control group

Effects of EELL on anxiety-like behaviour in male Swiss mice

There was a significant increase in the time spent in the dark compartment of the Light and Dark box (LDB) (F (3,18) = 7.84, p = 0.001) (Fig. 6A). Also, the number of transitions made between the light and dark compartments of the LDB by the treated groups that received 100 and 200 mg/kg of EELL was significantly decreased compared to the control group (F (3,18) = 9.31, p < 0.001). (Fig. 6B).

Fig. 6.

Effect of EELL on anxiety-like behaviour in male Swiss mice. A: Effect of EELL on time spent in L/D compartment by male Swiss mice, B: Effect of EELL on number of transitions in L/D compartment by male Swiss mice. All values are expressed as mean ± SEM, followed by two-way followed by Dunnett’s post hoc test, n = 5;*p < 0.05, (significance difference) when compared with control group

Effects of EELL on depression-like behaviour in male Swiss mice

Repeated administration of EELL resulted in a significant increase in immobility time in the 100 and 200 mg/kg treatment groups compared with the control group (F (3,18) = 8.67, p < 0.001) (Fig. 7) as observed in the Forced Swim Test.

Fig. 7.

Effect of EELL on immobility time in male Swiss mice. All values are expressed as mean ± SEM (one way ANOVA followed by Dunnett’s post hoc test), n = 5; *p < 0.05, (significance difference) when compared with the control group

Effects of EELL on cognitive behaviour (learning and memory) in male Swiss mice

In the Morris Water Maze (MWM) test, the time taken for experimental animals to find the platform and escape from the maze (escape latency) is shown in Fig. 8A. The first three days of training showed a significant decrease in escape latency (F (3,18) = 5.42, p = 0.007). However, on the 4th day, there was a slight stabilisation of escape latencies, which may imply that there was a learning improvement by the animals to locate the hidden platform (Fig. 8B). Retention memory was assessed on the 5th day of the probe trial (without the hidden platform in the pool). A significant, dose-dependent decrease in time spent in the platform zone (F (3,18) = 6.89, p = 0.003) (Fig. 8C) was observed in the treatment groups compared with the control group. Also, a significant increase in average proximity to the platform zone, (F (3,18) = 4.76, p = 0.011) was recorded in mice that received 200 mg/kg of EELL when compared to the control group (Fig. 8D).

Fig. 8.

Effects of ethanol extracts of Leiotrametes lactinea on cognitive behavior in male Swiss mice. A: Escape latency. B: ANY-maze track plot depicting the swimming trajectories of male Swiss mice (aspect ratio was maintained at approximately 3.33:1 (width:height), preserving the original 1:1 geometry of each circular tracking arena). C: Time spent in the platform zone. D: Average proximity to the platform zone border. All values are expressed as mean ± SEM all values are expressed as mean ± SEM, followed by two-way followed by Dunnett’s post hoc test, n = 5; *p < 0.05, (significance difference) when compared with control group

Assessment of the EELL on brain oxidative stress parameters in male Swiss mice

Reduced Glutathione (GSH) level was significantly lowered at in all the treatment group (71.06 ± 4.74 - 124.36 ± 5.35 µmol) when compared with the control group (149.88 ± 7.07 µmol) as depicted in (Fig. 9A). A significant increase was observed in MDA level in the treatment groups that received 100 and 200 mg/kg of EELL (0.054 ± 0.00 - 0.096 ± 0.01 µmol/mg protein) when compared to the control group (0.049 ± 0.00), Fig. 9B. The effect of repeated administration of EELL on Catalase (CAT) and Superoxide dismutase (SOD) activities as shown in Figs. 9 C and 9D respectively showed a significant decrease in treatment groups (100 and 200 mg/kg; p < 0.05) and (50–200 mg/kg; p < 0.05) respectively, when compared with their respective control groups.

Fig. 9.

Effect of EELL on brain oxidative stress parameters. A: GSH - reduced glutathione concentration. B: MDA – Malondialdehyde level. C: Catalase activity. D: SOD – Superoxide dismutase activity. All values are expressed as mean ± SEM (one way ANOVA followed by Dunnett’s post hoc test), n = 5; *p < 0.05, (significance difference) when compared with control group

Effect of EELL on acetylcholinesterase (AChE) activity in male Swiss mice

Acetylcholinesterase activity was significantly increased in the groups that received 100 and 200 mg/kg doses of EELL (0.121 ± 0.01 & 0.160 ± 0.01 µmol/min/g tissue) when compared to the control group (0.049 ± 0.01 µmol/min/g tissue), Fig. 10.

Fig. 10.

Effect of EELL on acetylcholinesterase (AChE) activity in male Swiss mice. All values are expressed as mean ± SEM (one way ANOVA followed by Dunnett’s post hoc test), n = 5; *p < 0.05, (significance difference) when compared with control group

Effects of ELL pro-inflammatory cytokine levels (IL-6 and TNF-α) in male Swiss mice

A significant elevation of IL-6 level was observed at 100 and 200 mg/kg (364.83 ± 14.32 ρg/mL tissue & 494.30 ± 62.70 ρg/mL tissue; Fig. 11A) compared to the control group (165.00 ± 17.55 ρg/mL tissue). Similarly, a significant increase in brain TNF-α level was observed (Fig. 11B) in the treatment group at 100 and 200 mg/kg (258.65 ±10.82 ρg/mL tissue & 325.09 ± 25.19 ρg/mL tissue) compared to the control group (148.61 ± 5.40 ρg/mL tissue).

Fig. 11.

Effect of EELL on brain pro-inflammatory cytokine level in male Swiss mice. A: IL-6 - Inteleukin-6 concentration. B: TNF-α - Tumour necrosis factor-alpha concentration. All values are expressed as mean ± SEM (one way ANOVA followed by Dunnett’s post hoc test), n = 5; *p < 0.05, (significance difference) when compared with control group

GC-MS analysis of the sample

GC-MS analysis of EELL revealed a lipid-dominated chemical profile, primarily comprising fatty acids and alcohols. The major constituents included palmitic acid (n-hexadecanoic acid), cis-vaccenic acid, linoleic acid (9,12-octadecadienoic acid, Z,Z-), stearic acid (octadecanoic acid), palmitoleic acid (9-hexadecenoic acid), along with minor fatty alcohols such as 1-hexadecanol and 8-dodecenol (Table 1).

Table 1.

Chemical composition of Leiotrametes lactinea using gas chromatography-mass spectrometry (GC-MS)

S/N	RT	Compounds	Abundance (%)
1	7.798	1-Hexadecanol	1.41
2	13.257	8-Dodecenol	4.70
3	16.686	n-Hexadecanoic acid	53.52
4	16.883	9-Hexadecenoic acid	2.11
5	18.113	Octadecanoic acid	6.65
6	18.305	cis-Vaccenic acid	14.17
7	18.461	Cyclododecane	3.34
8	18.678	9,12-Octadecadienoic acid (Z,Z)-	11.40

RT- Retention time

Discussion

This study evaluated the impacts of the EELL on the central nervous system. Food and water intake, as well as body weight changes, are vital indicators for assessing general health and evaluating the toxic effects or safety profiles of extracts in experimental animals [24–26]. Extract-treated animals exhibited a significant dose-dependent reduction in body weight compared with the healthy control group, which corroborates reports that some mushroom extracts may alter energy metabolism through appetite suppression or metabolic disruption [27]. Likewise, a corresponding dose-dependent reduction in food and water intake was recorded in the treated animals, suggesting that the extract might have induced appetite suppression, which also contributed to the observed reduction in weight gain. Also, mushrooms have a high polysaccharide content [28], which has been shown to be active in weight reduction [29]; this may also account for the observed weight loss in the treated groups in this study.

Relative organ weight is another important parameter in toxicity studies, providing insight into the potential adverse effects of extracts or compounds [30]. In this study, the relative brain weight of the treated animals did not differ significantly from that of the control group. This indicated that the extract did not induce brain atrophy or hypertrophy, although the death of one animal in each group was recorded in the groups administered 100 and 200 mg/kg EELL. This recorded death may have resulted from high-dose toxicity or systemic stress, as a study by [31] has also confirmed that high doses of herbal extracts may cause sporadic mortality.

The open field test revealed a reduction in total distance travelled, rearing frequency, and number of line crossings in the EELL-treated animals. This suggests that the extract may have a CNS-depressant effect, possibly due to sensory-motor integration or changes in excitatory neurotransmission (dopaminergic and glutamatergic pathways) [32; 33]. In the light-dark box test, anxiety-like behaviour was observed in animals that received 100 and 200 mg/kg of the extract, as they spent longer in the dark compartment and made only a few transitions between the light and dark compartments. This may be linked to oxidative stress and neuronal dysfunction [34; 35]. The forced swim test is used in measuring depressant-like activity, and in this study, a depressive-like response was observed in the treated mice that showed increased immobility time in a dose-dependent manner. This further supports the report from the open field test. In the Morris water maze (MWM) test, the treated animals showed increased escape latency and greater proximity to the platform zone. This indicates a problem with spatial learning and memory [36; 37]. Thus, the overall result from the neurobehavioral assessment suggests alterations in neurobehavioral functions following sub-acute exposure to EELL.

The observed neurobehavioral alterations appear to result from an imbalance between oxidative stress and the antioxidant system. The antioxidant systems, such as superoxide dismutase, catalase, and reduced glutathione, were reduced in the EELL-treated groups, leading to a concomitant elevation in malondialdehyde (MDA) levels, a product of lipid peroxidation. And because the brain has high lipid levels, uses more oxygen, and has a complex structure, it is highly predisposed to oxidative damage that can harm synapses and neurons [38–41]. Problems with cholinergic neurotransmission may also contribute to the observed cognitive problems. Since acetylcholinesterase breaks down acetylcholine, higher acetylcholinesterase activity recorded in the EELL-treated mice must have lowered acetylcholine levels, which can alter learning and memory [42–44] as observed in MWM.

Exposure to EELL also induces neuroinflammation, as evidenced by elevated levels of IL-6 and TNF-α in the brain. Oxidative stress can activate microglia, astrocytes, and neurons, leading to the release of pro-inflammatory cytokines [45; 46]. Also, high IL-6 levels have been linked to anxiety and depression [47]. This suggests that the anxiety-like and depressive-like behaviour observed in the animals treated with EELL may be due to overactive microglia and inflammation. Previous studies showed that polysaccharides and other active compounds from Trametes lactinea affect the immune system, depending on the dose. It was reported that the extract of T. lactinea enhanced nerve protection at lower doses, while higher doses or prolonged exposure induced inflammation [8; 26, 48].

The observed pattern of impaired memory performance, increased anxiety-like behaviour, elevated malondialdehyde and pro-inflammatory cytokines, alongside reduced glutathione and superoxide dismutase levels, suggests that the neurotoxicity induced by EELL was mediated primarily through oxidative stress–driven neuroinflammatory mechanisms. The elevation of malondialdehyde indicates enhanced lipid peroxidation, which in turn leads to oxidative damage to neuronal membranes, while the concomitant depletion of endogenous antioxidants such as glutathione and superoxide dismutase implies a compromised antioxidant defence system. This redox imbalance promotes excessive generation of reactive oxygen species, which has been shown to disrupt neuronal integrity and synaptic function, ultimately leading to cognitive deficits [49]. Furthermore, oxidative stress is closely linked to the activation of microglia and astrocytes, resulting in the release of pro-inflammatory cytokines that exacerbate neuronal injury and impair synaptic plasticity, which is essential for learning and memory [50].

In addition, the increased acetylcholinesterase activity observed in the study may contribute to cholinergic dysfunction by excessive hydrolysis of acetylcholine, thereby impairing neurotransmission in brain regions such as the hippocampus, which are critical for memory formation [51]. Evidence suggests that oxidative stress and neuroinflammation can further aggravate cholinergic deficits, creating a vicious cycle that accelerates cognitive decline [52]. The interplay between oxidative stress, inflammation, and cholinergic dysfunction may also induce mitochondrial impairment and excitotoxicity, thereby contributing to neuronal loss and behavioural abnormalities, including anxiety-like responses and memory deficits [53]. These findings thus indicate that the neurotoxic effects of EELL are driven by the interaction between oxidative stress, neuroinflammation and cholinergic dysregulation, ultimately leading to structural and functional neuronal damage.

The GC-MS analysis of the extract aligns with previous reports, indicating that mushrooms contain high proportions of unsaturated fatty acids, particularly linoleic acid, a dominant structural lipid in fungal membranes [54]. Unsaturated fatty acids, such as linoleic and oleic acids, are associated with structural, metabolic, and signalling functions rather than neurotoxicity [55]. Therefore, the observed neurotoxicity might not be directly mediated by the volatile lipid fraction. Although not primary neurotoxins, the identified fatty acids may contribute indirectly to oxidative stress under conditions of metabolic imbalance. Linoleic acid, as a polyunsaturated fatty acid, is highly susceptible to lipid peroxidation, generating reactive oxygen species and malondialdehyde (MDA), a widely recognised biomarker of lipid oxidative damage [56]. Similarly, palmitic acid has been shown to induce lipotoxicity at elevated concentrations, characterised by mitochondrial dysfunction, oxidative stress, and inflammatory signalling [57]. High doses of some of these lipids might contribute to redox imbalance and partially explain the increased oxidative stress observed in the present study. However, lipid-mediated oxidative stress alone is unlikely to fully account for the magnitude of the neurobehavioral and inflammatory changes observed at high doses of EELL. Thus, future studies will focus on non-volatile constituents, including polysaccharides, lectins, phenolic compounds, and other secondary metabolites, which possess strong immunomodulatory and neuroactive properties [58].

Conclusion

Sub-acute oral exposure to the ethanol extract of Leiotrametes lactinea in mice induces oxidative stress, which subsequently disrupts cholinergic neurotransmission and initiates neuroinflammation. This sequence of events results in motor impairments, anxiety-like behaviours, memory deficits, and depressive-like symptoms. male Swiss mice at higher doses or with extended use.

Abbreviations

EELL

Ethanol extract of Leiotrametes lactinea

GSH

Glutathione

SOD

Superoxide dismutase

MDA

Malondialdehyde

ACHE

Acetylcholinesterase

IL-6

Interleukine-6

TNF-α

Tumor necrosis factor-α

CNS

Central nervous system

OFT

Open field test

LDB

Light and dark box

MWM

Morris water maze

Author contributions

OOA: conception, study design and manuscript revision, TBO: data acquisition and analysis; AAA; data acquisition and manuscript writing and revision, CBO: manuscript revision, IVA: manuscript revision, PUE: data acquisition, analysis and manuscript revision. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

The data presented are available upon request from the corresponding author.

Declarations

Ethical approval

The study protocol was approved by the University of Ibadan Animal Care and Use Research Ethics Committee (UI-ACUREC) with the Ethical number UI-ACUREC/051/2022A. The procedures for the use and care of animals were in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH, 1985).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.