Pharmacological evaluation of Embelin in Benzo[α]pyrene induced experimental model of cognitive and memory impairment in mice

doi:10.21203/rs.3.rs-1728539/v1

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Pharmacological evaluation of Embelin in Benzo[α]pyrene induced experimental model of cognitive and memory impairment in mice

https://doi.org/10.21203/rs.3.rs-1728539/v1

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Alzheimer's disease (AD) is a neurodegenerative disease that affects the neurons in the hippocampus, resulting in cognitive and memory impairment. The production of amyloid-beta (Aβ) plaques, neurofibrillary tangles, and neuroinflammation in neurons are the most prominent clinical characteristics of AD. It has been proven that Embelin (Emb) possess antioxidant, anti-inflammatory, and neuroprotective properties. Therefore, we assessed the therapeutic potential of Emb in Benzo(α)pyrene (BaP)-induced cognitive impairment in experimental mice. BaP (5 mg/kg, i.p) was given to mice daily for 28 days, and Emb (2.5, 5, and 10 mg/kg, i.p) was given for 14 days after the BaP treatment. Locomotor activity was evaluated using open-field and spatial working, and non-spatial memory was evaluated using novel object recognition tasks (NORT), Morris water maze (MWM), and Y- maze. At the end of the study, the animal tissue homogenate was used to check biochemicals, neuroinflammation, and neurotransmitter changes. BaP treated mice showed a significant declined in locomotor activity, learning and memory deficits and augmented oxidative stress (lipid peroxidation, nitrite and GSH). Further, BaP promoted the release of inflammatory tissue markers, decreased acetylcholine, dopamine, GABA, serotonin and norepinephrine and increased glutamate concentration. However, treatment with embelin at dose-dependently prevented biochemical changes, improved antioxidant levels, reduced neuroinflammation, restored neurotransmitter concentration and inhibited the NF-κB pathway. The current study's finding suggested that Emb improved cognitive functions through antioxidant, anti-inflammatory, neuroprotective mechanisms and inhibition of acetylcholinesterase (AChE) enzyme activities and Aβ-42 accumulation.

Benzo[α]pyrene

Embelin

Cognitive impairments

Oxidative stress

Acetylcholinesterase

Neurotransmitters

Alzheimer's disease (AD) was first mentioned in 1906 by Alois Alzheimer and was renamed by Emil Kraepelin many years later (Li et al. 2019). AD is a progressive age-related neurodegenerative disorder characterized by persistent memory loss and cognitive deficit. The pathogenesis of AD is multifactorial and caused by accumulation of β-amyloid peptide (Aβ), abnormal phosphorylation of the tau protein, oxidative stress, mitochondrial dysfunction, and degeneration of cholinergic neurons (Li et al. 2019). Extracellular deposition of amyloid β in the brain leads to activation of the microglia, which promote of release various pro-inflammatory cytokines, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Excessive oxidative stress, neuroinflammation, leads to energy failure, synaptic dysfunction and causes degeneration of the cholinergic neurons (Spuch et al. 2012). In addition, the accumulation of amyloid β triggers mitochondrial dysfunction through a number of pathways such as impairment of oxidative phosphorylation, the elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins (Rojas‐Gutierrez et al., 2017).

Oxidative stress has also been reported to play an essential role in the pathogenesis of AD (Fan et al. 2018). It has been recommended that cellular responses to increased stress cause aberrant activation of stress response molecules such as the nuclear factor-kappa b (NF-κB). It is a major cellular stress response protein activated by a number of stimuli, including UV light, cigarette smoke, viral and bacterial products, free radicals, carcinogens, neurotrophin, oxidative stress, and overload of endoplasmic reticulum protein (Oeckinghaus et al. 2009) and environmental pollutants. The NF-κB transcription factor family in mammals consists of five proteins, p65 (RelA), RelB, c-Rel, p105/p50 (NF-κB1), and p100/52 (NF-κB2) that associate with each other to form distinct transcriptionally active homo- and heterodimeric complexes (Wideman et al. 2018). The misproportion of NF-κB dimer formation between c-Rel and RelA might result in the pathological process in specific neurons. In addition to this, various other mechanisms cause neurodegeneration using NF-κB, such as microglial activation, Aβ accumulation, cholinergic, and mitochondrial dysfunction.

It is noticeable that neurotransmitters like acetylcholine, dopamine, serotonin, norepinephrine, GABA, and glutamate play a significant role in the brain's memory storage (Abraham et al. 2019). Synaptic plasticity, including long-term depression (LTD) and long-term potentiation (LTP) are essential learning and memory phenomenon influenced by neurotransmitter alteration (Xu et al. 2012). Indeed, there has been a decreased cholinergic and monoaminergic transmission (Sun et al. 2009) and a disparity of glutaminergic and GABAergic signalling in AD (Liu et al. 2020).

Some of the well-recognized co-factors in neurodegenerative disorders are oxidative stress, mitochondrial dysfunction, and microglial and astrocyte activation. Further, exposure to harmful chemicals, environmental pollutions also like AD. Benzo[a]pyrene is a polycyclic aromatic hydrocarbon resulting from incomplete combustion of organic matter at temperatures between 300 °C and 600°C. The ubiquitous compounds can be found in coal tar, tobacco smoke, and many foods, especially grilled meats. Long-term exposure to benzo(α)pyrene promotes neuroinflammation, cognition, and memory, followed by neuronal death (Cummings et al. 2019). Recent evidence that the NF-κB signaling pathway played an essential role in the neuroinflammation and neurodegeneration induced by benzo(α)pyrene.

Current clinical trials for AD include disease-modifying medications, symptomatic agents, and cognitive enhancers to treat neuropsychiatric and behavioral changes. Anti-amyloid and anti-tau agents are disease-modifying therapies that contain molecules, biological agents, or monoclonal antibodies (Khazdair et al. 2019). There are 121 agents currently undergoing clinical trials for the treatment of AD. Twenty-nine agents are currently undergoing Phase 3 trials, 65 are currently undergoing Phase 2 trials, and 27 are currently undergoing Phase 1 trials (Khazdair et al. 2019). Twelve agents are being used for cognitive improvement, and twelve are used to treat neuropsychiatric and behavioral symptoms (Khazdair et al. 2019). Compared to previous, there is an improvement in the number of disease-modifying agents targeting pathways other than amyloid or tau in the 2020 pipeline compared to previous.

Several herbal compounds with neuroprotective effects have been widely studied in the last few decades to treat cognitive disorders, including neurodegenerative disorders such as AD and other memory-related disorders (Kundap et al. 2019). However, Embelin, a natural, safe hydroxy benzoquinone, is considered one of the main constituents of plant Embelia ribes (family: Myrsinaceae) and has been reported to possess beneficial effects on a range of CNS disorders (Shaikh et al. 2016). An available study has shown that embelin has neuroprotective activities by inhibiting the activation of astrocytes and microglia and decreasing the generation of TNF-α, iNOS, and IL-1β (Carola et al. 2002). Embelin also been reported to possess cholinesterase inhibitory, anti-amyloidogenic, and neuroprotective effects in vitro and silico studies, as shown in Fig. 1. Based on this evidence, the present study has been designed to investigate the neuroprotective potential of embelin as NF-κB inhibitor against Benzo(α) pyrene-induced cognitive and memory impairment in mice of behavioral, biochemical, neuroinflammation, neurotransmitters acetylcholinesterase, beta amyloids, and NF-κB pathways.

Experimental Animals

In the present study, experiments were carried out in male Swiss albino mice (30-35 gm) with 12 months were obtained from the central animal house of ISF College of Pharmacy, Moga, Punjab (INDIA). The animals were divided into five groups and housed in a polyacrylic cage (25 × 19 × 13 cm) and maintained under standard husbandry conditions (room temperature 22 ± 2º C and relative humidity of 55 - 60%). The animals were maintained on a commercial food diet in dry pellets and water ad libitium. All the behavioral assessments were taken between 9:00-11:00 AM and 2:00-5:00 PM. The experimental protocol was approved (ISFCP/CPCSEA/19/411) by the Institutional Animal Ethics Committee (IAEC), and experiments were carried out in accordance to Committee for the Purpose of Control and Supervision of Experimental on Animals (CPCSEA) guidelines for the use and care of experimental animals. All the experiments were performed for a given treatment using age-matched animals to avoid variability between experimental groups. The animal breeding and experimental facility are registered with the CPCSEA, Ministry of Environment and Forest and Climate Change, Government of India. Euthanasia was performed under sodium pentobarbital anesthesia by decapitation, and efforts were made to minimize the pain and suffering of the animals.

Drugs and Chemicals

Benzo(α)pyrene was purchased from Sigma–Aldrich (USA). Embelin was purchased from INDOFINE Chemical Company, USA. Benzo(α)pyrene was prepared in olive oil as a concentration of 1ml of olive oil per kg of mice, and embelin was always prepared afresh by suspending in 1 % Tween 80 (v/v) in a 0.9 % (w/v) saline at different doses (2.5, 5 and 10 mg/kg). The bodyweight of male Swiss albino mice was between 30-35 gm, and we calculated the dose of embelin based on the bodyweight of mice. Interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) kits were purchased from Krishgen Biosystem, India. All other chemicals used in the study are of analytical grade. The Solutions of the drugs and chemicals were freshly prepared before use.

Experimental procedure

Firstly, animals were divided into five groups, and seven mice were studied per group, whereas only six mice were in the control group. Mice were placed in individual polyacrylic cages (25 × 19× 13 cm), and in the control group, mice were injected with normal saline, and in the toxin group, mice are injected with BaP (5 mg/kg; i.p. for 28 days) whereas, in treatment groups, mice are injected with BaP as same as in toxin group along with the addition of embelin at different doses (2.5 mg/kg; i.p, 5 mg/kg; i.p, 10 mg/kg; i.p daily) from 14^th day 28^th days of a protocol which act as a treatment drug. Behavioral parameters are studied as per the given schedule as on specified days in the plan of work. On the 28^th day, the animals are sacrificed with the means of euthanasia and then proceed for biochemical, neuroinflammatory, neurotransmitters, and other protein estimations.

Behavioral Parameters

Measurement of body weight

Bodyweight was performed on days 1^st, 7^th, 14^th, 21^st, and 28^th of the experiment schedule.

Open field task

To analyse the locomotor activity, open field task was performed. Each animal was tested for locomotor activity on days 1^st, 7^th, 14th, 21st and 28^th of the experimental schedule. Each locomotion activity of mice was analyzed using open field apparatus for 5 min and values expressed as box counts per 5 minutes (Vorhees et al. 200).

Morris water maze task

To evaluate the memory and cognitive impairment, Morris water maze test was performed (Morris, 1984). In this test, Escape latency was analyzed on the 22^nd, 23^rd, 24^th, and 25^th of the protocol schedule. Time taken by mice to reach the platform was considered as escape latency. For 120 seconds, mice were allowed to swim freely in the pool containing a hidden platform on the 26^th day, and accordingly, time spent in the target quadrant (TSTQ) was recorded. The TSTQ demonstrates a degree of memory consolidation which occurs after learning (Zhang et al. 2012).

Novel object recognition test

To analyze the memory and cognitive impairment novel object recognition testwas performed. Each animal was tested for memory impairment on day 1st and 28^th of the experimental schedule. Memory impairment was analyzed using a novel object recognition testfor 5 minutes and values were expressed as recognition index in percentage (Zhang et al. 2016).

The object recognition index was calculated with the following formula:

Recognition index = (time spent in new object) / (time spent in the new object + time spent in the

already known object).

Y-Maze test

Spontaneous alternation behavior in Y-maze was used to assess short-term spatial memory. On day 1^st, 7^th, 14^th, 21^st, and 28^th of the experimental schedule, mice were initially placed at the end of one arm and freely explored the three arms. The number of arm entries and the number of triads were recorded to calculate the percentage of an alternation. Over the course of multiple arm entries, the entry sequence (e.g., ABC, BCA, or CAB, where letters indicate code of arms) was recorded manually over 5 min. An actual alternation was defined as entries into all three arms consecutively, i.e., ABC, CAB, or BCA but not BAB. An entry was defined as placing all four paws within the boundaries of the arm (Zou et al. 2010). The percentage of alternation (% alternation) was calculated as spontaneous alternation/ (total number of arms entries-2) x100.

Cellular and molecular markers

Dissection and Homogenization

On day 29, the mice were sacrificed by cervical dislocation, and brain samples were removed and preserved at -80 °C in an ultra-low temperature freezer. On an experimental day, brains were removed from the deep freezer, and the hippocampus was isolated, weighed, and then homogenized using phosphate buffer solution (0.1 M, pH 7.4) containing 1 mmol Ethylene Diamine-Tetra-Acetic acid (EDTA), 0.25 M sucrose, 10 mM potassium chloride (KCL), and 1 mM Phenyl Methyl Sulfonyl Fluoride (PMSF). After brain homogenization, the sample was centrifuged for 15 min at 10,000 g, supernatants were separated and used to estimate biochemical parameters, acetylcholinesterase (AChE), NF-κB protein, amyloid Beta _1-42 (Aβ_1-42)level, oxidative stress, neuroinflammatory, and neurotransmitter estimations.

Determination of the expression levels of NF-κB protein

The expression level of NF-κBprotein was determined by using ELISA commercial kits (Krishgen diagnostics, India). This test was performed in brain homogenate as per the given standard procedure. The values are expressed as pg/ml protein (Singh et al. 2016).

Assessment of neurotransmitter levels

Assessment of brain acetylcholine levels

Acetylcholine was measured by using a diagnostic kit (Krishgen diagnostics, India). Mice brain homogenate, and all the reagents were prepared as per the standard procedure described in the kit. The optical density of the reaction mixture was determined at 540 nm in the microtiter plate.

Assessment of brain dopamine levels

The levels of dopamine in the brain sample were determined according to the method of Singh and co-workers. The dopamine activity in mice brain homogenate is expressed as ng/mg protein (Singh et al. 2016).

Assessment of brain serotonin levels

Serotonin level in brain homogenate was estimated using Singh and co-workers. First, it was estimated by HPLC using an electrochemical detector and C18 reverse-phase column. Then, the serotonin concentrations were calculated from the standard curve using a standard with a concentration of 10–100 mg/ml (Singh et al. 2016).

Assessment of brain norepinephrine levels

The levels of norepinephrine in the brain sample were determined according to the method of Singh and co-workers. The norepinephrine activity in mice brain homogenate is expressed as ng/mg protein (Singh et al. 201; Donzanti et al 1988).

Assessment of brain GABA and glutamate levels

The estimations of GABA and glutamate were done by the method described by Donzanti and Yamamotol (Donzanti et al 1988) with slight modifications. The Waters standard system consists of a high-pressure isocratic pump, a 20µl manual sample injector valve, and a C18 reverse phase column using an electrochemical detector. The mobile phase consisted of 100 mM disodium hydrogen phosphate anhydrous, 25 mm EDTA, and 22% methanol (pH- 6.5). The experimental electrochemical condition was +0.65 V, having sensitivity ranges from 5 to 50 nA. Separation was carried out at a flow rate of 1.2 ml/min, and the column temperature was maintained at 40ºC. Samples (20 μl) were injected manually. Brain samples were homogenized in 0.2 mol/l perchloric acid on the day of the experiment. Then the samples were centrifuged at 12,000 g for 15 min. The supernatant was derivatized using OPA/β-ME and then filtered through 0.22 mm nylon filters before injecting the HPLC sample injector. Data were recorded and analyzed with breeze software. The concentrations of amino acids were calculated from the standard curve using a standard with a concentration of 10–100 mg/ml.

Acetylcholine Estimation

The ACh levels in the brain were measured using a colorimetric technique. According to Lowry 1951, the various brain parameters were calculated by determining the total protein concentration (Lowry et al. 1951).

Measurement of neuroinflammatory biomarkers in mice brain homogenate

Measurement of TNF- α, IL-6, and IL-1β levels

The level of TNF- α, IL-6 and IL-1β were quantified by using mice TNF- α, IL-6, and IL-1β immunoassay kit (KRISHGEN BioSystem, USA). The activity of TNF- α, IL-6, and IL-1β in mice brain homogenate are expressed as pg/mg protein.

Estimation of Biochemical parameters

Preparation of mice brain homogenate

On the 28^thday of the protocol schedule, animals were sacrificed by decapitation, brains were removed and homogenized with ten times (w/v) ice-cold 0.1 M phosphate buffer (pH 7.4). The homogenate was centrifuged at 10,000×g for 15 minutes, supernatant separated, and aliquots were used for biochemical estimation.

Protein estimation

The protein content was measured using a Coral protein estimation kit (Biuret method).

Assessment of acetylcholinesterase (AChE) levels

The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01M sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide, and 0.10 ml of DTNB (Ellman reagent). The change in absorbance was measured immediately at 412 nm spectrophotometrically. The enzymatic activity in the supernatant was expressed as μM/mg protein (Ayyappan et al. 2016).

Assessment of catalase levels

Catalase activity was measured according to the method described by Takahara, Hamilton, Nell, Ogubra, and Nishimura (1960). The 0.2 ml of tissue homogenate was mixed with 1.2 ml of phosphate buffer (0.05 M, pH 7.0), and the enzyme reaction was started by adding 1.0 ml of hydrogen peroxide (0.03 M). The decrease in absorbance was recorded at 240 nm for 3 minutes, and the enzyme blank was run simultaneously with 1.0 ml distilled water instead of hydrogen peroxide. The catalase activity was expressed as micromoles of hydrogen peroxide decomposed per minute per milligram protein (Green et al. 1982).

Estimation of reduced glutathione levels

Reduced glutathione in the brain was estimated according to the method described by Ellman et al. 1959. First, 1 ml supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4°C for 1 hr. Then, the samples were centrifuged at 1200×g for 15 minutes. To 1 ml of the supernatant, 2.7 ml of phosphate buffer (0.1M, pH 8) and 0.2 ml of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were added. The yellow color that developed was measured immediately at 412 nm using a spectrophotometer. The concentration of glutathione in the supernatant is expressed as μM/mg protein (Ellman et al. 1959).

Assessment of nitrite levels

The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO) is determined by a colorimetric assay using Greiss reagent (0.1% N-(1- naphthyl) ethylene diamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid) as described by Green et al. 1982. Equal volumes of supernatant and Greiss reagent are mixed and incubated for 10 minutes at room temperature in the dark, and the absorbance was determined at 540 nm spectrophotometrically. The nitrite concentration in the supernatant is determined from the sodium nitrite standard curve and expressed as μM/mg protein (Wills 1966).

Estimation of malondialdehyde (MDA) levels

The quantitative measurement of MDA end product of lipid peroxidation in brain homogenate was performed according to the method of Wills. The amount of MDA was measured after its reaction with thiobarbituric acid at 532 nm using a spectrophotometer. The concentration of MDA was expressed as nM/mg protein (Galasko et al. 2010).

Estimation of amyloid Beta _1-42 (Aβ_1-42)level

The level of Aβ_1-42was estimated by using Aβ_1-42ELISA kit (Aβ_1-42ELISA kit protocol). It is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA), which uses a microtitre plate reader read at 450 nm. Concentrations of Aβ_1-42were calculated from a plotted standard curve (Jayasekara et al. 1992).

Statistical Analysis

The results were expressed as mean ± Standard deviation (SD). Morris water maze and object recognition task were analyzed by repeated measure two-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons and others behavior and molecular, biochemical, neuroinflammatory, and neurotransmitters results were analyzed using one-way analysis of variance followed by Tukey’s post hoc test. Values with P<0.05 were considered to be statistically significant.

Effect of Embelin on change in body weight in BaP administered mice

BaP administered mice showed significant changes in body weight as compared to vehicle control. Treatment with embelin at different doses 2.5, 5, and 10mg/kg, significantly and dose-dependently attenuated BaP induced decrease in body weight. Moreover, embelin (10 mg/kg) treated mice showed better body weight than embelin (2.5, and 5 mg/kg). (Figure 2)

Effect of embelin on spontaneous locomotor activity in BaP administered mice

On days 1^st, 7^th, 14^th, 21^st, and 28^th BaP administered mice showed significantly reduced spontaneous locomotor activity than vehicle control. Treatment with embelin at different doses of 2.5, 5 and 10mg/kg significantly and dose-dependently showed spontaneous locomotor activity in BaP administered mice. Moreover, in the treatment of embelin (10 mg/kg) treated mice showed more improvement in spontaneous locomotor activity than embelin (2.5, and 5 mg/kg). (Figure 3)

Effect of embelin on memory performance in Novel object recognition task in BaP administered mice

On days 1^st, 7^th, 14^th, 21^st, and 28^th, BaP administered mice showed significant decreases in memory performance as compared to vehicle control. Treatment with embelin at different doses 2., 5 and 10mg/kg, significantly and dose-dependently showed improvement in memory performance in BaP administered mice. Moreover, embelin (10 mg/kg) treated mice showed better memory performance than embelin (2.5, and 5 mg/kg) treated mice. (Figure 4)

Effect of embelin on spatial learning and memory using Morris water maze task in BaP administered mice

On day 22^nd, 23^rd, 24^th, and 25^th of the protocol schedule, escape latency was observed. BaP treated mice showed a gradual increase in escape latency during the administration course at day 22^nd, 23^rd, 24^th and 25^th. Chronic treatment with embelin showed a significant decrease in escape latency than BaP treated groups. Long-term administration of embelin at different doses of 2.5, 5, and 10 mg/kg was more effective dose-dependently in BaP administered mice. (Figure 5)

On day 26^th, time spent in the target quadrant (TSTQ) was estimated. BaP administered mice show gradual decreases in TSTQ but chronic treatment with embelin at different doses 2.5, 5, and 10 mg/kg significantly increases TSTQ as compared to BaP treated groups. Long-term administration of embelin at different doses of 2.5, 5, and 10 mg/kg was found to be more effective in restoring TSTQ in BaP administered mice. (Figure 6)

Effect of embelin on learning and memory using Y-maze test in BaP administered mice

On days 1^st, 7^th, 14^th, 21^st and 28^th, BaP administered mice showed significant decreases in memory performance compared to vehicle control. Treatment with embelin at different doses 2.5, 5 and 10 mg/kg significantly and dose-dependently showed improvement in memory performance in BaP administered mice. Moreover, in the treatment of embelin (10 mg/kg), treated mice showed more improvement in memory performance than embelin (2.5, 5 mg/kg). (Figure 7)

Effect of embelin on of the expression levels of NF-κB protein

The expression level of NF-κBprotein was significantly increased in BaP intoxicated mice as compared to vehicle control. Treatment with embelin at different doses, 2.5, 5 and 10 mg/kg, significantly decreased expression levels of NF-κB protein compared to BaP treated group. Among the selected doses, embelin 10 mg/kg was most effective in the amelioration of BaP induced increased expression levels of NF-κB protein in BaP administered mice. The values are expressed as pg/ml protein (Jamali-Raeufy et al., 2020). (Figure 8)

Neuroprotective effect of embelin on amelioration of inflammatory cytokines in BaP administered mice

Neuroinflammatory cytokines such as TNF-α, IL-6 and IL-1β levels were significantly increased in BaP intoxicated mice compared to vehicle control. Chronic treatment with embelin at different doses 2.5, 5, 10 mg/kg significantly decreased inflammatory cytokines than BaP treated group. Among the selected doses, embelin 10 mg/kg was most effective in administering BaP induced increased levels of inflammatory cytokines in BaP administered mice. (Table 1)

Neuroprotective effect of embelin on reduction of oxidative stress markers in BaP administered mice

Oxidative stress markers like LPO, GSH, AchE, Nitrite, and catalase were quantified. There were LPO, nitrite, and AchE levels that were found to be increased significantly and reduction in the level of reduced GSH and catalase in chronic BaP administered mice compared to vehicle control. Chronic administration with embelin remarkably and dose-dependently decreased LPO, Nitrite, and AchE and restored the antioxidant defence system by increasing reduced GSH and catalase levels compared with BaP, administered mice. Among the selected doses of embelin, a high dose of embelin 10 mg/kg was significantly effective in reducing BaP induced oxidative stress markers in mice. (Table 2)

Neuroprotective effect of embelin on amelioration of neurotransmitters level in BaP administered mice

Neurotransmitters such as Ach, dopamine, NE, serotonin, GABA and glutamate were assessed at the end of the protocol schedule. BaP resulted in a significant decrease of Ach, dopamine, NE, serotonin, and GABA levels, whereas increased glutamate concentration in mice brains compared with vehicle control. On the other hand, chronic treatment with embelin significantly increased Ach, dopamine, NE, serotonin, and GABA levels and decreased glutamate levels in mice brain homogenate compared with BaP administered mice. Furthermore, embelin 10 mg/kg was most effective in restoring dopamine, NE, serotonin, Ach, GABA and glutamate neurotransmitter changes in BaP administered mice. (Table 3 and 4).

Effect of embelin on Aβ_-42level in BaP treated AD in mice

Vehicle control animals did not produce any significant changes in the level of Aβ_-42as compared with the normal group. The BaP induced cognitive impairment mice showed an increase in the level of Aβ_-42within the brain compared to a vehicle control group. However, treatment with embelin at different doses 2.5, 5 and 10 mg/kg, significantly decreased the level of Aβ_-42compared to BaP treated mice. Moreover, the oral administration of embelin (10 mg/kg) instigated a significant reduction in the level of Aβ-42 compared to a low dose of embelin (2.5, 5 mg/kg). (Fig. 9)

The present research showed the embelin has neuroprotective ability against BaP-induced neurotoxicity in Swiss albino mice by directly targeting the NF-κB pathway. Intraperitoneal (i.p) infusion of BaP has affected behavioral, biochemicals, neurotransmitters and molecular changes seen in AD (Maciel et al. 2014). In this study, administration of BaP has induced cognitive dysfunction, cholinergic deficit, an increased pro-inflammatory cytokines, oxidative stress and neurotransmitters alterations in mice brain. The reported improvements are aligned with available studies demonstrating significant behavioral and biochemical changes following injection of BaP into mice (Kandimalla et al. 2017). Although, the exact mechanism of cognitive impairments induced by BaP is unclear. However, BaP-induced cholinergic dysfunction, cerebro energetic failure, mitochondrial dysfunction, consequent glial cell activation, and majorly oxidative stress further contribute to the upregulation of the NF-κB pathway and glutamate excitotoxicity, correlated with cognitive dysfunction as well as other signs of AD.

In the present study, BaP caused a decline in locomotor activity in mice as observed in OFT at different time intervals (1,6,13,20,27 days) and caused significant learning and memory impairment as observed in MWM, Y-Maze and NORT similarly at different time intervals (1,7,14,21,28 days). It is known that neurotransmitters produce an impact on neuronal functioning and are strongly enriched with cholinergic, glutaminergic, GABAergic, and monoaminergic axon terminals, and these neurotransmitters are believed to play a vital role in the encoding, storage, and expression of memory (Luján et al. 2005). In this study, we have reported significant changes in neurochemistry after infusion of BaP in the brain of mice. BaP has caused a significant elevation in acetylcholinesterase activity (AchE) and a deficit in monoamine levels. AchE is responsible for the acetate and choline synthesis from Ach, thus, the elevated AchE level suggests an enhanced Ach metabolism. Therefore, the decline in neurotransmitter's extracellular concentration can be due to their increased metabolism. On the other side, BaP causes significant reductions in GABA and elevation in glutamate levels in mice's brains. Such differences in GABA levels and glutamate have been documented to contribute to excitotoxicity induced neuronal damage and cognitive impairment (Gupta et al. 2013).

Excitotoxicity due to glutamate is also reported to occur in brains with AD. It is shown with the support of ELISA that BaP is also responsible for the increase in the expression of NF-κB and is often stated to occur in AD brains. In this study, we found that BaP showed significant changes in the neurotransmitter concentration in the mice’s brain. BaP causes reductions in GABA, and elevated glutamate levels, such differences in GABA levels and glutamate contribute to excitotoxicity induced neuronal damage and cognitive impairment. Embelin therapy balanced the glutamate and GABA levels in check in a dose-dependent manner. Nevertheless, the current research demonstrates the neuromodulatory function of embelin and its immense capacity to down-regulate the NF-κB pathway responsible for AD. Our results showed that BaP treated mice have impaired spatial learning and memory functions, enhanced oxidative stress, damaged the cholinergic system, and activated the NF-κB signalling pathway in the hippocampus. The neuroprotective effect of embelin against BaP-induced AD inhibits the AChE activity and reduces the oxidative damage, pro-inflammatory cytokines (TNF-α IL-6, IL-1β) markedly of brain tissue via suppressing the NF-κB signalling pathway. Thus, the observed increase in cognitive functioning in the present study may be related to embelin's ability to restore the neurotransmitters of the brain and target the NF-κB pathway. There has been evidence that oxidative stress and neuroinflammatory cytokines play a critical role in several neurodegenerative diseases (Liu et al. 2015). There have also been records of mitochondrial dysfunction, increased production of free radicals, a compromised antioxidant defence, and neuroinflammation in AD (Block et al. 2007). Microglial cells function as the brain immune defense against brain injury; however, overactivation of microglia cells is harmful to the survival of neuronal cells and may contribute to neurotoxic events triggering the release of inflammatory cytokines and increased free radical production (Zawia et al. 2009). ROS and peroxides, on the contrary, play a significant role in host defence and may influence the number of transcription factors such as the production of NF-κB-dependent cytokine and activated protein 1, resulting in the output of pro-inflammatory cytokines triggering neuroinflammation (Palmer et al. 2012).

Likewise, elevated cytokine levels are also related to mitochondrial dysfunction and enhanced ROS production in neurodegenerative pathologies (Bourgognon et al. 2020). Cytokines (TNF-α and IL-1β) were associated with cholinergic dysfunction and cognitive decline as seen in ageing and AD. Enhanced formation of ROS and RNS, known to react with biomolecules including proteins, lipids, carbohydrates, DNA, and RNA, contribute to their cellular dysfunction and oxidative damage (Li et al. 2015). There has also been proof of overactivation of microglia cells, enhanced cytokine production, and oxidative stress following BaP administration in mice. BaP has substantially increased MDA, nitrite, and pro-inflammatory cytokine levels (TNF-α, IL-1β, and IL-6) in the current study and decreased GSH levels in mice's brains. In the current investigation, embelin inhibited BaP-induced oxidative stress and reduced the burden of the pro-inflammatory cytokine, indicating its antioxidant and anti-inflammatory capabilities. Furthermore, our study found that down-regulation of the NF-κB pathway after dosing with embelin was dose-dependent. Indeed, embelin has been reported to possess the antioxidant activity and anti-inflammatory ability and showing its neuroprotective role by downregulating the NF-κB pathway. Thus, the reported activities in the present study also play a significant role in improving cognitive impairment and memory dysfunction. Further studies are needed to confirm the role of NF-κB pathway in the pathophysiology of AD. However, research is still needed to understand the exact molecular approach and mechanisms of embelin against AD. In conclusion, BaP administration in mice induced significant cognitive and memory impairment, oxidative damage, increased pro-inflammatory cytokines, altered levels of neurotransmitters, and triggered upregulation of the NF-κB pathway. Treatment with the embelin dose-dependently attenuated cognitive dysfunction and other neurotoxic effects induced by BaP. The cognitive improvement in BaP administered mice found after embelin therapy could be attributed to its antioxidant and anti-inflammatory function and its ability to downregulate the NF-κB pathway, which is responsible for memory and cognitive impairment. Nonetheless, our findings indicate that the embelin would be a beneficial candidate drug molecule for restoring cognitive function associated with AD by directly targeting the NF-κB pathway.

Funding: This research did not receive any specific grant from funding agencies.

Conflict of interest: The authors declare no confict of interest.

Data availability: Data sharing not applicable to this article as no datasets were generated or analyzed in the current study.

Code availability: Not applicable

Author contributions: Akansh Goal: Conducted the experiment and wrote the manuscript. Khadga Raj: collected data and analysed by taking the help of Dr. Shamsher Singh. Dr. Shamsher Singh and Rimpi Arora: Designed, reviewed, and edited manuscript.

Ethical Approval: The experimental protocol was reviewed and approved by the Institutional Animal Ethics Committee (ISFCP/IAEC/CPCSEA/meeting NO.25/2019/ Protocol 411) and performed according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) for the use and care of experimental animals.

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Table 1: Neuroprotective effect of embelin on amelioration of inflammatory cytokines in BaP administered mice

S. No.	Groups	Neuroinflammatory markers
S. No.	Groups	TNF-α (pg/ml protein)	IL-1β (pg/ml protein)	IL-6 (pg/ml protein)
1.	Vehicle Control	36.49±3.31	75.89±4.88	54.40±5.42
2.	BaP 5 mg/kg	382.3±7.42^***	427.9±6.86^***	213.1±4.85^***
3.	Emb 2.5 mg/kg	323.1±4.76^@	374.6±9.12^@	184.5±6.31^@
4	Emb 5 mg/kg	252.8±6.55^@@#	268.3±7.56^@@#	152.5±3.66^@@#
5.	Emb 10 mg/kg	123.3±8.82^@@@##$$	151.4±4.06^@@@##$$	126.2±7.69^@@@##$

Effect of Embelin (Emb) on pro-inflammatory cytokines in Benzo(α)pyrene (BaP) induced Alzheimer’s disease (AD) mice. Values expressed as mean ± Standard deviation (SD), *** p<0.001 versus vehicle control; ^@p<0.05, ^@@p<0.01 and ^@@@p<0.001 versus BaP 5 mg/kg; ^#p<0.05 and ^##p<0.01 versus Emb 2.5 mg/kg; ^$p<0.05 and ^$$p<0.01 versus Emb 5 mg/kg [One-way Analysis of variance (ANOVA) followed by Tukey’s multiple comparison test].

Table 2: Neuroprotective effect of embelin on reduction of oxidative stress markers in BaP administered mice

S. No.	Groups	Catalase (µM H₂O₂decomposed / mg of tissue/ min)	AchE (U/mg protein)	MDA (µmol/mg protein)	NO (µmol/mg protein)	GSH (µmol/mg protein)
1.	Vehicle Control	16.25±0.27	14.93±0.41	1.57±0.01	1.56±0.01	0.086±0.0017
2.	BaP 5 mg/kg	44.89±0.67^**	53.59±0.57^***	3.40±0.04^**	0.48±0.01^**	0.015±0.0024^*
3.	Emb 2.5 mg/kg	35.87±0.32^@	45.88±0.57^@	2.97±0.02^@	0.67±0.009^@	0.027±0.0018^@@
4.	Emb 5 mg/kg	29.80±0.27^@@#	38.26±0.46^@@#	2.55±0.01^@@#	0.88±0.01^@@#	0.040±0.0019^@@#
5.	Emb 10 mg/kg	22.03±0.22^@@@##$	27.06±0.18^@@@##$	2.08±0.01^@@@##$	1.16±0.01^@@@##$	0.064±0.0033^@@@##$

Effect of Embelin (Emb) on oxidative stress in Benzo(α)pyrene (BaP) induced Alzheimer’s disease (AD) in mice. Values expressed as mean ± Standard deviation (SD), * p<0.05 and ** p<0.01 and *** p<0.001 versus vehicle control; ^@p<0.05, ^@@p<0.01 and ^@@@p<0.001 versus BaP 5 mg/kg; ^#p<0.05 and ^##p<0.01 versus Emb 2.5 mg/kg; ^$p<0.05 versus Emb 5 mg/kg [One-way Analysis of variance (ANOVA) followed by Tukey’s multiple comparison test].

Table 3: Neuroprotective effect of embelin on amelioration of neurotransmitters level (Dopamine, Serotonin, Norepinephrine) in BaP administered mice

S. No.	Groups	Neurotransmitters level
S. No.	Groups	Dopamine (ng/mg protein)	Serotonin (ng/mg protein)	Norepinephrine (ng/mg protein)
1.	Vehicle Control	102.0±1.58	27.83±0.80	34.91±0.90
2.	BaP 5 mg/kg	24.82±2.05^**	5.01±0.64^**	7.40±0.32^**
3.	Emb 2.5 mg/kg	32.98±1.05^@	10.93±0.63^@	12.85±0.69^@
4.	Emb 5 mg/kg	46.73±2.48^@@#	16.41±1.02^@@#	20.93±0.87^@#
5.	Emb 10 mg/kg	58.49±1.29^@@@##$	23.18±0.65^@@@##$	28.84±0.57^@@@##$

Effect of Embelin (Emb) on neurotransmitters level in Benzo(α)pyrene (BaP) induced Alzheimer’s disease (AD) in mice. Values expressed as mean ± Standard deviation (SD), ** p<0.01 versus vehicle control; ^@p<0.05, ^@@p<0.01 and ^@@@p<0.001 versus BaP 5 mg/kg; ^#p<0.05 and ^##p<0.01 versus Emb 2.5 mg/kg; ^$p<0.05 versus Emb 5 mg/kg [One-way Analysis of variance (ANOVA) followed by Tukey’s multiple comparison test].

Table 4: Neuroprotective effect of embelin on amelioration of neurotransmitters level (GABA, Glutamate, Ach) in BaP administered mice

S. No.	Groups	Neurotransmitters level
S. No.	Groups	GABA (ng/mg protein)	Glutamate (ng/mg protein)	Ach (ng/mg protein)
1.	Vehicle Control	76.76±1.00	75.83±0.80	8.84±0.51
2.	BaP 5 mg/kg	27.32±1.28^**	175.70±0.82^***	1.19±0.63^**
3.	Emb 2.5 mg/kg	35.80±1.54^@	155.30±0.80^@	2.87±0.27^@
4.	Emb 5 mg/kg	47.21±1.37^@@#	134.70±0.80^@@#	4.85±0.32^@@#
5.	Emb 10 mg/kg	57.16±1.45^@@@##$	104.70±0.88^@@@##$	6.94±0.42^@@@##$

Effect of Embelin (Emb) on neurotransmitters level in Benzo(α)pyrene (BaP) induced Alzheimer’s disease (AD) in mice. Values expressed as mean ± Standard deviation (SD), ** p<0.01 and *** p<0.001 versus vehicle control; ^@p<0.05, ^@@p<0.01 and ^@@@p<0.001 versus BaP 5 mg/kg; ^#p<0.05 and ^##p<0.01 versus Emb 2.5 mg/kg; ^$p<0.05 versus Emb 5 mg/kg [One-way Analysis of variance (ANOVA) followed by Tukey’s multiple comparison test].

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Pharmacological evaluation of Embelin in Benzo[α]pyrene induced experimental model of cognitive and memory impairment in mice

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