A study to determine the effect of nano-selenium and thymoquinone on the Nrf2 gene expression in Alzheimer’s disease

Abstract

Introduction

Alzheimer’s disease is a developing public health concern in aging communities that affects a sizable section of the global population. The risk of Alzheimer’s disease increases with age; it affects one-third of males and two-thirds of women^. This research attempts to assess the effect of nano-selenium and thymoquinone on Nrf2 gene expression levels in Alzheimer’s disease (AD).

Methods

There were five identical groups of 50 albino male rats: a control group that was healthy; an AD positive control group; an AD group that received nano-selenium (5 mg/kg); an AD group that received thymoquinone (2 mg/kg); and an AD group that received both. The duration of treatment was 4 weeks. The levels of Nrf2 in brain tissues were evaluated using real-time PCR.

Results

Nrf2 mean expression levels in the nano-selenium-treated rats, the thymoquinone-treated rats, and the rats that were given both treatments all increased significantly compared to AD rats with no treatment.

Conclusions

This study showed that nano-selenium and thymoquinone elevated Nrf2 gene expression levels in AD.

LAY ABSTRACT

Alzheimer’s disease (AD) is a common dementia causing cognitive decline. Nrf2 signaling may be a potential therapeutic target, while thymoquinone and nano-selenium could be effective neuroprotective agents for AD management, potentially reducing neuronal damage. In this study, the effects of thymoquinone and nano-selenium treatments on AD rat models were evaluated through behavior study by performing the Morris Water Maze test, estimation of different markers (Nrf2, Aβ-42, GSH, MDA, and TNF-α), and histopathological examination of brain tissue.

ARTICLE HIGHLIGHTS

• Alzheimer disease (AD) is the most prevalent form of dementia causing a steady deterioration in cognitive function. Thymoquinone and nano-selenium showed improvement in histopathology and behavior studies in the AD rats’ model.

• Alzheimer disease has no known treatment; however, medicines and other therapies can help with symptom management, improve quality of life, and slow down clinical decline.

• TQ and SeNPs demonstrated improvement in the levels of different biomarkers (Nrf2, Aβ-42, TNF-α, GSH & MDA) reversing them toward the normal levels.

• The combination of TQ and SeNPs showed more improvement than each of them as a single treatment.

GRAPHICAL ABSTRACT

1. Introduction

Over 25 million individuals globally are afflicted with Alzheimer’s disease, constituting between 50 and 70 percent of all cases of dementia [1]. The fact that current Alzheimer’s disease treatments mainly address symptoms has led to the search for new drugs that have fewer negative effects and side effects, are more affordable, and are compatible with long-term use [2]. Early diagnosis is essential to lessen the disease’s course and enhance the lives of those who suffer from Alzheimer’s [3].

Memory loss is among the initial indications of Alzheimer’s disease that patients and their carers notice. Early in the course of the disease, both working memory and long-term declarative memory are compromised. Multiple indicators of structural or functional brain integrity can be used to correlate a person’s pattern of memory impairment. Memory formation is hindered by Alzheimer’s disease at multiple levels, including the molecular level and the structure of brain networks [4]. Although dementia and neurodegeneration co-occur in Alzheimer’s disease, it is still unclear exactly how neuronal dysfunction and death are caused [5].

Alzheimer’s disease individuals who experience early cognitive decline also have measurable changes in their brain’s structural and functional connections. Early hippocampal atrophy decreased hippocampal synaptic density, and loss of synapses are all consequences of Alzheimer’s disease dysregulation of Aβ and tau metabolism [6]. The neuropathological signs of AD include neurofibrillary tangles (NFTs) and amyloid β (Aβ) aggregates outside of cells [7]. The discovery that the amyloid β protein (Aβ) is the primary fibrillar component of senile plaques, and that Aβ aggregation is the initial event in AD, marked the beginning of the “molecular age” for AD [8].

Increasing evidence points to the importance of increased neuroinflammatory processes in the emergence of Alzheimer’s disease [7].

There is evidence that the pathophysiology and neuronal damage of AD patients are significantly influenced by high levels of MDA and poor levels of antioxidant enzyme activity [9]. Lipid peroxidation results in the highly toxic and reactive metabolite MDA. Since MDA accumulation is a sign of increased lipid peroxidation and ROS generation, it is thought to be a marker of oxidative stress [10].

Additionally, postmortem examinations demonstrated a correlation between the deterioration in cognitive function in patients with severe AD and mild cognitive impairment (MCI) and reduced GSH and inversely elevated GSSG levels. GSH in particular is thought to be a biomarker for the early phases of AD since the ratio of GR to GPX activity is reduced in MCI and further reduced in the late stages of the illness, indicating that the ability to recycle GSH is gradually reduced as the disease progresses [11].

Alzheimer’s disease (AD) pathophysiology is significantly influenced by pro-inflammatory cytokines including TNF α and IL-6. When assessing pathological processes, normal biological processes, and biological reactions to a therapeutic intervention, these cytokines serve as biomarkers [12].

As of right now, AD is incurable; current treatments merely address its symptoms or stop it from getting worse. The blood-brain barrier is mostly to blame for the scarcity of drugs available to treat neurodegenerative illnesses since blood-brain drug molecules must pass via the brain compartment because of the brain’s complex structure [13]. Also, treatment problems for AD include inadequate efficacy of currently available medications and therapy approaches, as well as a lack of thorough knowledge regarding the molecular basis of the disease [14].

Existing medicines, such as monotherapies and combination medications, are unable to effectively treat the disease’s complex pathological process [15]. Moreover, due to issues with blinding, study design, and methodology, most small-molecule strategies that target tau are either in early-stage clinical studies or have been abandoned [16]. Also, several previous Aβ-targeting medication trials were unable to provide solid evidence of clinical benefit [17].

Unquestionably, coordinating cellular function is the role of nuclear factor-erythroid-2-related factor 2 (Nrf2). Evidence suggests that this transcription factor controls many signaling pathways relevant to macromolecules, iron metabolism, xenobiotics, and redox homeostasis. The primary antioxidant system regulator, Nrf2, regulates cellular fate, affecting processes such as infectious disease success, cell proliferation, apoptosis, differentiation, resistance to treatment, and senescence. Dysregulation of Nrf2 signaling has been linked to cancerous occurrences, infectious disorders, and diseases associated with aging since Nrf2 is the main coordinator of the defensive mechanisms of cells [18].

Numerous scientific publications have demonstrated the effectiveness of targeting Nrf2 in the treatment of a wide spectrum of diseases. It is becoming more widely acknowledged that transcriptional upregulation of proinflammatory cytokine genes, which Nrf2 inhibits, is the primary cause of the elevated risk for the development of AD [19]. To shield tissues from the damaging effects of oxidation and stop the course of many diseases, pharmacological treatments that target Nrf2 are being developed [20]. Normally, the Nrf2 gene shields cells from stressful situations. Nrf2 is decreased in Alzheimer’s disease patients’ brains, despite we don’t know why, and this could account for why neurons are more vulnerable to damage in neurodegenerative illnesses [21].

Protein stability, translation, nuclear localization, and protein-protein interactions all play a role in controlling the transcription factor Nrf2’s activity during oxidative stress. Eight antioxidant and redox systems that remove reactive oxygen species are controlled by the Nrf2 transcription factor, which regulates the expression of critical components in these systems. The genes that are affected by Nrf2 status point to its role in mitochondrial turnover, tissue recovery, repair, or remodeling, metabolic reprogramming, and the restriction of proinflammatory cytokines [22].

Nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway controls the cellular central defense system against oxidative stress. Under homeostatic circumstances, Nrf2 is broken down by proteases. Nrf2 dissociates from Keap1 under oxidative stress circumstances, transports into the nucleus, attaches to antioxidant response elements, and ultimately generates SOD, HO-1, NQo1, and Catalase. Consequently, pro-inflammatory cytokine levels are reduced, and anti-inflammatory cytokine levels are raised. Consequently, elevated Nrf2 levels in AD may function as a neuroprotectant [23].

In trace amounts, selenium (Se), a naturally occurring metalloid element, is essential for the health of humans and animals. Se significantly contributes to how the human body works. It supports antioxidant defense mechanisms by being integrated into selenoproteins. Selenoproteins regulate reproductive processes, engage in the thyroid hormones’ metabolism, and possess neuroprotective capabilities [24].

The right amount of inorganic selenium supplementation is rather close to its toxic concentration, even though selenium is an essential element for living things. It has recently been established that biosynthesized SeNPs are less harmful and more advantageous to the host. As a result, they are thought to be a superior substitute for selenium supplements [25]. Also, clinical trials involving nanotechnology showed improvement in the administration of naturally occurring antioxidants by overcoming their low bioavailability, oxidation sensitivity, and difficulty crossing the blood-brain barrier [26].

Since selenium in nano form is less toxic and safer for biological systems, selenium toxicity issues have shifted the focus of research toward selenium nanoparticles (NPs). In comparison to selenium’s bulk form, all of its qualities are altered in nano form [27]. The distinct chemical, physical, and biological characteristics of selenium and its nanoparticles distinguish them significantly from one another. As an example, bulk elemental Se-NPs are somewhat water soluble, whereas Se is not soluble in water [28]. Current developments in nanotechnology could provide widespread access to affordable screening and treatment alternatives. Nanoparticles (NPs) may effectively pass the blood-brain barrier (BBB) for targeted drug delivery with few side effects [29].

SeNPs are frequently used in oxidative stress-induced disorders and have gained popularity in biomedical applications due to their excellent bioavailability and bioactivity. Studying SeNPs for the treatment of neurodegenerative illnesses primarily involves two areas of focus: inflammation and oxidative stress [30].

The vital role of elemental selenium nanoparticles in the treatment of neurodegenerative illnesses, especially Alzheimer’s disease, has drawn a lot of interest [31]. Due to their exceptional qualities, SeNPs are used in a variety of biological applications. Due to their connection with numerous moieties, including selenoproteins, selenocysteine, and selenomethionine, SeNPs are essential in a wide range of applications in biology. According to studies, SeNPs are quite effective at battling fatal conditions such as drug-induced toxicity, diabetes, cancer, Alzheimer’s disease, etc. Researchers are still quite concerned about the harm that nanoparticles can cause. In the near future, selenium nanoparticles may be used in nutritional supplements and therapeutic applications [32].

Thymoquinone (TQ), a small chemical with potential therapeutic effects against a variety of illnesses such as diabetes, cancer, and neurological disorders, is a key component of black cumin [33]. Thymoquinone comes in enol, keto, and mixed forms (2-isopropyl-5- methyl benzo-1, 4-quinone). The primary type of TQ that affects its pharmacological actions is keto [34]. Thymoquinone (TQ) has the versatility to lessen neurological deficits, making it a promising neuropharmacological agent. It has more advantageous effects on neurotoxicity and neuroinflammation brought on by toxins. In numerous models of neurological disorders, it demonstrates emergent functions and defends against several neurodegenerative disorders as well as other neurological conditions [35].

To prevent or slow the progression of many neurodegenerative disorders, including Alzheimer’s dementia, several nutraceuticals have been studied. Because of their potent anti-inflammatory and antioxidant properties, Nigella sativa (NS) and its isolated component thymoquinone (TQ) may be an efficient neuroprotective drug [36].

Numerous investigations confirmed TQ’s safety. According to one study, isolated mitochondria from the brain, heart, kidney, and liver did not exhibit any toxicity or abnormalities when exposed to different concentrations of TQ [37].

In cases of ischemia, epilepsy, encephalomyelitis, traumatic brain damage, and neurodegenerative disorders (Parkinson’s, Alzheimer’s), thymoquinone (TQ) is recognized as a medication with neuroprotective qualities. Antioxidant activity and enhanced production of neuroprotective genes and proteins, along with a decrease in pro-inflammatory cytokine activity—which is crucial for neuroinflammation—are the primary mechanisms of action [38].

One of the many key pathways controlling TQ’s therapeutic actions is the Nrf2 pathway. Indeed, when TQ interacts with other signaling pathways, Nrf2 pathways mediate the positive effects of TQ. The effectiveness of TQ in treating a number of illnesses suggests that it could be used in conjunction with other therapeutic approaches [39].

Nano-selenium particles and thymoquinone not only have antioxidant and anti-inflammatory effects that can help in the improvement of AD but also can modulate Nrf2 gene expression which has a neuroprotective role in AD.

The study aims to evaluate the effect of thymoquinone and nano-selenium treatments on AD rat models through estimation of brain tissue Nrf2 relative gene expression by Quantitative real-time PCR to investigate the possibility of using them as additive therapy in AD. Also, enhancing the results by performance of behavior study through Morris Water Maze (MWM) and estimation of amyloid beta-42 by ELISA technique in addition, Glutathione (GSH) and Malondialdehyde (MDA) by colorimetric method, and tumor necrosis factor-alpha (TNF-α) levels using the ELISA technique.

2. Materials and methods

2.1. Animals

This study was approved by the Committee of Scientific Ethics at Faculty of Pharmacy, Girls, Al-Azhar University, Cairo, Egypt (REC approval number: 257), and was carried out in accordance with its guidelines for animal use.

Experiments were carefully planned and prepared before the study project started, and needless animal exploitation was avoided.

50 albino male rats weighing between 150 and 200 g were purchased from Cairo University’s Faculty of Medicine’s experimental animal unit. Rats were kept in a pathogen-free, sterile, and temperature-controlled setting; given unrestricted access to water at 22 degrees Celsius, a 12-hour light/dark cycle, and a semi-purified diet containing (g/kg): 100 g Casein, 750 g Sucrose, 50 g Cellulose, 50 g Fat blends, 10 g Vitamin mix, and 40 g Mineral mix.

Randomly chosen rats were divided into 5 equal groups, each with 10 rats. The intended format for the experiment was as follows:

• A group of 10 rats was utilized as a healthy control (not injected by lipopolysaccharides) (group 1).

• To create an Alzheimer’s model, the remaining 40 rats received intraperitoneal injections of lipopolysaccharides at a concentration of 0.8 mg/kg for 1 week; After the first week, group 2, consisting of 10 rats, will serve as a positive Alzheimer’s disease control (only injected by lipopolysaccharide to induce AD model and not injected by any treatment) whereas the other three groups, each consisting of 10 rats, will get the following therapy:

• Group 3: will get a daily intraperitoneal injection of nano-selenium at a dose of 5 mg/kg throughout 4 weeks.

• Group 4: will get a daily intraperitoneal injection of thymoquinone at a dose of 2 mg/kg throughout 4 weeks.

• Group 5: will get both nano-selenium at a dose of 5 mg/kg and thymoquinone at a dose of 2 mg/kg throughout 4 weeks.

2.2. Selenium nanoparticle (SeNP) preparation and characterization

2.2.1. Preparation of fermented soy milk (FSM)

Organic soybeans and distilled water (1:10) were combined, cooked for 30 minutes at 100 °C, and then filtered to create soymilk. The Chung et al. method was used in the production of fermented soymilk (FSM) [40].

A variety of microorganisms were employed in fermentation of soymilk, including Saccharomyces cerevisiae (yeast), Bifidobacteria, Streptococcus lactis bacteria, and Lactobacillus acidophilus bacteria. The broth used for Saccharomyces cerevisiae (yeast) growth was Sabouraud dextrose medium (40 g glucose, 10 g peptone, 1-liter distilled water, pH 5.6) and the medium used for bacterium growth was TGY medium (pH 7.0, 1.0 g of glucose, 5.0 g of tryptone, 5.0 g of yeast extract, and 1.0 l of distilled water). Finally, heat sterilized and filtered the fermented soymilk.

2.2.2. Preparation of selenium nanoparticles (SeNPs)

The fermented soy’s aqueous portion served as a precursor for the creation of SeNPs. With vigorous stirring, the aqueous component of fermented soy (2 ml) was dropwise added to the 20-ml solution of SeO2 (10 mM). The mixture was incubated for 72 hours in the dark by being placed onto a rotator orbital shaker that was running at 5 × g and 30 °C. Using a UV-Vis spectrophotometer, the maximum wavelength of absorption was measured between 350 and 700 nm to track the reduction of selenium ions [41].

2.2.3. Se nanoparticles characterization

Using a spectrophotometer that is UV-Vis, the maximum wavelength of absorption was measured between 350 and 700 nm to track the reduction that occurred to selenium ions. The Zetasizer was used to analyze a sample of SeNPs using dynamic light scattering (DLS), which measures changes in the intensity of scattered light caused by Brownian motion particles [42]. VERTEX70 FTIR spectrometer (BRUKER) was used to investigate the functional groups that the sample of SeNPs presented on the nanoparticles’ surfaces. The findings are presented as a transmittance percentage, and the samples were scanned between the 400–4000 wave number cm⁻¹ range. The Shimadzu 1,700 UV–Vis spectrophotometer was used to scan the SeNPs sample for ultraviolet-visible (UV/Vis) absorption spectroscopy.

2.3. Sampling and preparation

First Morris water maze test was performed. After that and before the rats were beheaded, their venous blood was drawn from the retro-orbital vein, then after being allowed to clot for 30 minutes, they were centrifuged for 20 minutes at 10,000 × g. The serum was kept frozen until the ELISA method was used to assess the amounts of TNF-α. The animals were subsequently given anesthesia with sodium phenobarbital (60 mg/kg), and following their decapitation, brain tissue was removed.

The brains were extracted and split into three equal portions right away. For histological investigations, one part was fixed in 10% formalin, while the other two were lysed in different buffers to undergo various methods for the estimation of parameters:

• A part was set aside for the quantitative real-time PCR estimate of the relative gene expression of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2).

• The remaining part is used to estimate amyloid beta-42 using the ELISA technique in addition to the colorimetric approach for glutathione (GSH) and malondialdehyde (MDA).

2.4. Behavior study

2.4.1. Morris water maze (MWM) general procedure

Rats were given the task of doing MWM after following the treatment regimen for a month to examine the impact of the treatments on the rats’ declining memory as a result of causing Alzheimer’s disease, which will be reflected in the rats’ performance in the maze. Rats were put in a blue, circular pool with a diameter of six feet and a water temperature of 26 ± 2 °C. In the center of the target quadrant, two centimeters below the water’s surface was positioned a circular escape platform where it might be exposed to water for a short while. during training to show the rats that there was a method to get help. Rats were trained to complete four trials a day, spaced 20 minutes apart, lasting 60 seconds each, for 4 days in a row. On the fifth day, an experiment was conducted in which the latency time (how long it took for every rat to get to the platform) was measured, considering behavioral changes and histological brain damage among the groups.

2.5. Biochemical tests

2.5.1. Quantitative real-time PCR to estimate nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)

Utilizing the SV Total RNA Isolation technique, total RNA was isolated from tissue homogenate. (Thermo Scientific, USA). The amount of total RNA that was produced was determined spectrophotometrically at 260 nm. Utilizing a reverse transcription system kit, reverse transcription into complementary DNA was done on extracted RNA (#K4374966, Thermo Fisher Scientific, USA), in which 2X reverse transcription (RT) master mix was prepared in compliance with the kit’s directions (for each sample). The master mix was composed of 2 μl 10X RT Buffer, 0.8 μl 25X dNTP Mix (100 mM), 2μl10X RT Random Primers, 1 μl multiScribe reverse transcriptase, 1 μl RNase Inhibitor, and 3.2 μl Nuclease-free H2O. Following that, 10 μL of 2 X RT RT master mixes were pipetted into each tube separately. Each tube was filled with 10 μL of RNA material, which was mixed by pipetting up and down twice. After sealing the tubes, they underwent quick centrifugation to force the contents to settle and remove any air bubbles. The last mixture was loaded into the programmed thermal cycler as follows (refer to Supplementary Table S1). Utilizing an Applied Biosystem with software (StepOne^™, USA) version 3.1, real-time qPCR amplification and analysis were carried out. The primer sets used in the qPCR experiment (Table 1) were optimized for the annealing temperature. Quantitative real PCR analysis was performed in 25 µl reaction volume comprising about 2× SYBR Green PCR Master Mix (12.5 μl), 0.3 μM of each primer, and water nuclease-free to 25 μl, after that, the master mix-containing PCR tubes were filled with the necessary volumes of template DNA (≤500 ng/reaction). To inactivate any potentially contaminated amplicons, uracil DNA glycosylase was incubated at 50 °C for 2 minutes before amplification, then the first denaturing cycle at 95 °C for 10 minutes, followed by 40 amplification cycles in which each cycle lasted for 15 seconds of 95 °C denaturation, 1 minute of 60 °C annealing, and 1 minute of 60 °C extension. Using the comparative Ct technique, the relative expression levels of Nrf2 and β-actin were calculated.

Table 1.

Sequences of the primers used for real-time PCR primer.

Gene	Primer sequence
Nrf2	Forward primer 5′-CCATGCCTTCTTCCACGAA-3′ Reverse primer 5′-AGGGCCCATGGATTTCAGTT-3′
β-actin	Forward primer 5′-CCCATCTATGAGGGTTACGC-3′ Reverse primer 5′-TTTAATGTCACGCACGATTTC-3′

2.5.2. Glutathione (GSH) and malondialdehyde (MDA) estimation using a colorimetric approach

Using a Biodiagnostic colorimetric test kit, GSH was measured. A yellow chemical product was produced by reducing 5, 5’-Dithiobis (2-nitrobenzoic acid) (DNTB) with GSH. The reduced chromogen’s absorbance was measured at 405 nm, and it was found to be directly correlated with the GSH content.

Using a Biodiagnostic colorimetric assay kit, MDA was measured. The technique measures the colored complex that is produced in an acidic media when lipid peroxidation products like MDA react with Thio barbituric acid (TBA). At 534 nm, the resultant color was measured.

2.5.3. Assessment of brain tissue amyloid beta (Aβ-42) and serum tumor necrosis factor-alpha (TNF-α) using the ELISA method

The quantitative sandwich enzyme immunoassay approach is used in this examination. An antibody specific to the measured marker (TNF-α, or Aβ-42) has been pre-coated on a microplate. Samples and standards were pipetted into the wells, and any detectable marker (TNF-α or Aβ-42) that was present was bound by the immobilized antibody. Once any unbound materials were eliminated, the wells were further supplemented with a biotin-conjugated antibody that was specific for the marker being evaluated (TNF-α, or Aβ-42). Following the wells’ washing, Horseradish Peroxidase (HRP) conjugated with Avidin was added. After washing to remove any remaining unbound enzyme-avidin reagent, the wells were then filled with a substrate solution. The intensity of color produced in the first phase is correlated with the amount of the measured marker (TNF-α, or Aβ-42) bound. At 450 nm, the color’s intensity was measured after the color development had stopped. From Cusabio, China, the kit was acquired.

2.6. Statistical analysis

To conduct the statistical analysis, SPSS (Statistical Package for Social Science; SPSS Inc., Chicago, IL, USA, version 21) was used. The following tests were carried out to assess the data’s distribution in relation to their mean values and to determine their central tendency: SD standard deviation, X mean. Test significance was assessed between the group variables using a one-way analysis of variance (ANOVA). To perform multiple comparisons, the post hoc Tucky test was used. To determine whether two numerical variables have a linear connection, the Pearson correlation coefficient test (r) was used. For a two-tailed test, statistical significance was determined by using P < 0.05.

3. Results

3.1. Characterization of SeNPs

Upon UV–Vis spectroscopic examination of SeNPs, a surface plasmon absorption band was detected, with a maximum absorbance at 262.5 nm. This observation can be regarded as proof of SeNP synthesis (Figure 1a). The nanoparticles’ average size, as determined by DLS testing, with a high distribution was 90.68 nm, reaching 98.2%, as shown in Figure 1b. Fourier Transform Infrared Spectroscopy (FTIR) of SeNPs coated with fermented soy revealed strong peaks at 3318.10 cm − 1, 1636.93 cm − 1, and 602.12 cm − 1, respectively. Those peaks showed a single C-H or C-N bond, amide I bonds of proteins, and hydroxyl groups, illuminating the interactions between proteins and phytochemicals in fermented soymilk and nanoparticles. In summary, the proteins present in the fermented soy extract are absorbed as a layer on top of the synthetic SeNPs, which helps to stabilize the nanoparticles that are created by the proteins that are coupled to the surface (Figure 1c).

Figure 1.

Characterization of SeNPs (a) UV–Vis spectrum (b) Dynamic light scattering (c) Fourier transform infrared spectroscopy.

3.2. Behavior study through time consumed (second) in Morris water maze (MWM)

The mean of time consumed in MWM in group 3, group 4, and group 5 (52.90 ± 8.48, 53.89 ± 13.71, and 27.45 ± 11.96 seconds) respectively were significantly less than group 2 (104.01 ± 19.42 seconds) at p < 0.05. The mean time spent on MWM in groups 3 (52.90 ± 8.48 seconds) and 4 (53.89 ± 13.71 seconds) did not differ significantly at p > 0.05. In contrast, group 5’s mean MWM time of 27.45 ± 11.96 seconds was significantly lower than that of groups 4 (53.89 ± 13.71 seconds) and 3 (52.90 ± 8.48 seconds) at p < 0.05.

3.3. Biochemical traits of the subjects under study

According to Table 2 (refer to Supplementary Figures S1-S6).

Table 2.

Comparison of laboratory data statistics between the healthy control group, AD positive control group, AD + SeNPs group, AD + TQ group, and AD + SeNPs + TQ group.

	Healthy control group N = 10	AD positive control group N = 10	AD + SeNPs group N = 10	AD + TQ group N = 10	AD + SeNPs + TQ group N = 10
Variables	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
Nrf2	1.02 ± 0.03	0.24 ± 0.13a	0.62 ± 0.10b	0.64 ± 0.11b	0.81 ± 0.09b,c,d
amyloid beta-42 (pg/mg protein)	2.27 ± 0.87	11.21 ± 2.13a	4.86 ± 2.37b	5.80 ± 2.03b	3.91 ± 1.85b
GSH (mmol/mg protein)	59.67 ± 6.71	21.22 ± 4.62a	41.16 ± 5.87b	32.26 ± 9.14b	46.37 ± 11.64b,d
MDA (nmol/mg protein)	10.68 ± 3.19	73.23 ± 18.94a	27.37 ± 9.42b	29.23 ± 12.18b	23.28 ± 4.89b
TNF-α (pg/ml)	15.26 ± 2.29	105.65 ± 10.90a	63.03 ± 11.07b	66.05 ± 9.96b	36.41 ± 10.53b,c,d
Time consumed in MWM (sec.)	25.64 ± 7.77	104.01 ± 19.42a	52.90 ± 8.48b	53.89 ± 13.71b	27.45 ± 11.96b,c,d

^a Significant from the healthy control group at p < 0.05.

^b Significant from AD positive control group at p < 0.05.

^c Significant from AD + SeNPs group at p < 0.05.

^d Significant from AD + TQ at p < 0.05.

3.3.1. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) relative gene expression in brain tissue

The mean gene expression levels of nrf2 in group 3, group 4, and group 5 (0.62 ± 0.10, 0.64 ± 0.11, and 0.81 ± 0.09) respectively were considerably greater than that of group 2 (0.24 ± 0.13) at p < 0.05. The mean gene expression level of nrf2 did not significantly differ between groups 3 (0.62 ± 0.10) and 4 (0.64 ± 0.11), with p > 0.05. On the other hand, at p < 0.05, group 5’s mean gene expression serum level of nrf2 (0.81 ± 0.09) was considerably greater than that of groups 3 (0.62 ± 0.10) and 4 (0.64 ± 0.11).

3.3.2. Amyloid beta-42 (Aβ-42) (pg/mg protein) in brain tissue

Groups 3, 4, and 5 had mean brain tissue levels of amyloid beta (4.86 ± 2.37, 5.80 ± 2.03, and 3.91 ± 1.85 pg/mg protein), respectively, significantly lower than group 2 (11.21 ± 2.13 pg/mg protein) at p < 0.05. The mean amyloid beta level in brain tissue did not significantly differ between groups 3 (4.86 ± 2.37 pg/mg protein) and 4 (5.80 ± 2.03 pg/mg protein) at p > 0.05. Furthermore, group 3 (4.86 ± 2.37 pg/mg protein) and group 4 (5.80 ± 2.03 pg/mg protein) did not significantly differ from group 5 (3.91 ± 1.85 pg/mg protein) at p > 0.05 regarding the mean brain tissue levels of amyloid beta.

3.3.3. Glutathione level (GSH) (mmol/mg protein) in brain tissue

The mean brain tissue level of GSH in group 3, group 4, and group 5 (41.16 ± 5.87, 32.26 ± 9.14, and 46.37 ± 11.64 mmol/mg protein) respectively were significantly higher than that in group 2 (21.22 ± 4.62 mmol/mg protein) at p < 0.05. The mean GSH level in brain tissue did not significantly differ between groups 3 (41.16 ± 5.87 mmol/mg protein) and 4 (32.26 ± 9.14 mmol/mg protein), with p > 0.05. Furthermore, group 3’s mean brain tissue GSH level (41.16 ± 5.87 mmol/mg protein) and group 5’s (46.37 ± 11.64 mmol/mg protein) did not differ significantly at p > 0.05. It was shown that group 5’s mean brain tissue GSH level (46.37 ± 11.64 mmol/mg protein) was significantly greater than group 4’s (32.26 ± 9.14 mmol/mg protein) at p < 0.05.

3.3.4. Malondialdehyde level (MDA) (nmol/mg protein) in brain tissue

At p < 0.05, the mean brain tissue MDA levels in groups 3, 4, and 5 (27.37 ± 9.42, 29.23 ± 12.18, and 23.28 ± 4.89 nmol/mg protein, respectively) were significantly lower than those in group 2 (73.23 ± 18.94 nmol/mg protein). The mean MDA level in brain tissue did not significantly differ between groups 3 (27.37 ± 9.42 nmol/mg protein) and 4 (29.23 ± 12.18 nmol/mg protein) at p > 0.05. Furthermore, group 5 (23.28 ± 4.89 nmol/mg protein), group 3 (27.37 ± 9.42 nmol/mg protein), and group 4 (29.23 ± 12.18 nmol/mg protein) did not significantly differ from one another in concerning mean brain tissue MDA levels at p > 0.05.

3.3.5. Tumor necrosis factor-alpha (TNF-α) levels (pg/ml) in serum

At p < 0.05, the mean serum levels of TNF-α in groups 3, 4, and 5 (63.03 ± 11.07, 66.05 ± 9.96, and 36.41 ± 10.53 pg/ml) were found to be considerably lower than those in group 2 (105.65 ± 10.90 pg/ml). The mean serum level of TNF-α did not significantly differ between groups 3 (63.03 ± 11.07 pg/ml) and 4 (66.05 ± 9.96 pg/ml) at p > 0.05. On the other hand, group 5’s mean serum TNF-α level was significantly lower (36.41 ± 10.53 pg/ml) than groups 3 and 4 (63.03 ± 11.07 pg/ml and 66.05 ± 9.96 pg/ml, respectively) at p < 0.05.

3.4. Correlations between different parameters in a group of rats treated with nano-selenium

As shown in Table 3 (refer to Supplementary Figures S7-S8): There is a significant positive correlation between amyloid beta and MDA (r = 0.184) at p < 0.05. On the other hand, nrf2 and MDA have a significant negative correlation (r = -0.694) at p < 0.05.

Table 3.

Correlations in Alzheimer’s group treated with nano-selenium.

	TNF-α	Amyloid beta	nrf2	MDA	GSH	Time consumed in MWM
TNF -α (r) (P value)	1.000 0.000	0.032 0.931	0.419 0.229	−0.093 0.799	−0.574 0.083	−0.538 0.109
Amyloid (r) beta (P value)	0.032 0.931	1.000 0.000	−0.430 0.215	0.184** 0.004	0.166 0.648	0.301 0.398
nrf2 (r) (P value)	0.419 0.229	−0.430 0.215	1.000 0.000	−0.694* 0.026	0.123 0.736	−0.162 0.655
MDA (r) (P value)	−0.093 0.799	0.184* 0.004	−0.694* 0.026	1.000 0.000	−0.163 0.654	0.316 0.374
GSH (r) (P value)	−0.574 0.083	0.166 0.648	0.123 0.736	−0.163 0.654	1.000 0.000	0.459 0.182
Time (r) consumed in (P value) MWM	−0.538 0.109	0.301 0.398	−0.162 0.655	0.316 0.374	0.459 0.182	1.000 0.000

^*Statistically significant difference at p < 0.05.

**Statistically significant difference at p < 0.01.

3.5. Correlations between different parameters in the group of rats given thymoquinone treatment

As shown in Table 4 (refer to Supplementary Figure S9): There is a significant negative correlation between amyloid beta and nrf2 (r = -0.649) at P < 0.05.

Table 4.

Correlations in Alzheimer’s group treated with thymoquinone.

	TNF-α	Amyloid beta	Nrf2	MDA	GSH	Time consumed in MWM
TNF -α (r) (P value)	1.000 0.000	−0.164 0.650	−0.051 0.890	−0.113 0.755	−0.088 0.808	0.386 0.270
Amyloid (r) beta (P value)	−0.164 0.650	1.000 0.000	−0.649* 0.042	0.080 0.827	−0.130 0.720	0.015 0.968
Nrf2 (r) (P value)	−0.051 0.890	−0.649* 0.042	1.000 0.000	−0.247 0.491	0.054 0.882	0.076 0.835
MDA (r) (P value)	−0.113 0.755	0.080 0.827	−0.247 0.491	1.000 0.000	0.276 0.441	−0.117 0.748
GSH (r) (P value)	−0.088 0.808	−0.130 0.720	0.054 0.882	0.276 0.441	1.000 0.000	0.300 0.400
Time (r) consumed in (P value) MWM	0.386 0.270	0.015 0.968	0.076 0.835	−0.117 0.748	0.300 0.400	1.000 0.000

^*Statistically significant difference at p < 0.05.

3.6. Correlations between different parameters in the group of rats treated with nano-selenium and thymoquinone

As shown in Figures 2 and 3 refer to Supplementary Table S2): There are significant negative correlations between each of (TNF and GSH) and (nrf2 and time consumed in MWM) (r = -0.651 and r = -0.654, respectively) at P < 0.05.

Figure 2.

Correlation between GSH and TNF in the group of rats with Alzheimer’s disease treated with nano-selenium and thymoquinone.

Figure 3.

Correlation between nrf2 and time consumed in MWM in the group of rats with Alzheimer’s disease treated with nano-selenium and thymoquinone.

3.7. Receiver-operating characteristics (ROC) curve

As shown in Table 5: Roc curves were carried out to assess sensitivity, specificity, positive predictive value (PVV), and negative predictive value (NPV).

Table 5.

ROC Curves of Nrf2, TNF- α, Aβ-42, MDA, and GSH.

	Nrf2	TNF-α	Aβ-42	MDA	GSH
Cutoff	< 0.735	> 53.3	> 5.91	> 30.25	< 39.6
Sensitivity %	100%	100%	100%	100%	100%
Specificity %	100%	100%	100%	100%	100%
PPV %	100%	100%	100%	100%	100%
NPV%	100%	100%	100%	100%	100%
AUC	1	1	1	1	1

3.8. Histopathological examination of brain tissue

As shown in Figure 4, sections of the dorsal striatum (caudate/putamen) stained with hematoxylin and eosin (H&E x400, scale bar 50 μm) were taken from:

Figure 4.

Histopathological examination of rat’s brain tissue in (a) healthy control group (b) AD positive control group (c) AD + SeNPs group (d) AD + TQ group (e) AD + SeNPs + TQ group.

1. the healthy control group which demonstrated normal neurons (N), normal capillaries (c), and normal glial cells (g).

2. Alzheimer disease positive control group in which several darkly deteriorated neurons are visible. The neurons had disfigured and irregular nuclei or shrunken, pyknotic, nuclei that were deeply pigmented and had peri-neuronal vacuolations (V). There was also a noticeable widening of the perivascular gap (Virchow-Robin space), which divided the vessel from the neuropil (spiral arrow).

3. AD rats received the nano-selenium treatment group which showed a healthy structure of striatal neurons (N) and capillaries (c) where most neurons (N) in a healthy neuropil (neu) were observed to have vesicular nuclei and conspicuous nucleoli.

4. AD rats received the thymoquinone treatment group which displayed conserved architecture of the striatal neuronal and glial cells where only a few shrunken neurons could be observed.

5. AD rats received both nano-selenium and thymoquinone treatments group which showed hypocellularity of the striatum where it displayed few regenerating neurons (N) and pencil fibers of Wilson (W) that were exclusive to the striatum in an astrogliosis (As) image. A single layer of endothelial cells lined a narrow, delicate tube that made up certain capillaries (c). Obvious vascular dilatation and congestion (arrowhead) were observed.

4. Discussion

The most common neurological disorder in older adults is Alzheimer’s disease, which is also the primary cause of dementia [43]. Clinically speaking, the hallmark of Alzheimer’s disease is progressively declining memory and cognitive impairment that substantially interferes with patients’ daily activities [44].

Since there is presently no recognized treatment for Alzheimer’s disease, both individual health and the healthcare system are at risk due to the disease’s high prevalence and continued occurrence [45]. The current therapies for Alzheimer’s disease are only able to momentarily delay cognitive decline; they cannot stop or reverse the progression of dementia[46].

Neuroinflammation and oxidative stress are key mechanisms in Alzheimer’s disease (AD) [47]. Neuroinflammation and neuronal cell dysfunction can result from induced oxidative stress [48]. The inflammatory process, which is fueled by immune cells in the central nervous system (CNS), damages synapses and neurons [49]. Pro-inflammatory cytokines, such as interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 18 (IL-18), and tumor necrosis factor (TNF), chemokines, small-molecule messengers, such as prostaglandins and NO, as well as reactive oxygen species (ROS), are produced in large quantities. Neuroinflammation and tau protein tangle deposition are closely related [43, 50].

Pathological neuroinflammation responses in AD are facilitated by the activation of microglia and astrocytes, which includes the release of cytokines and immunological signaling pathways [51]. This ultimately results in neurotoxicity. There is great potential for the clinical application of neuroinflammation targeting in the treatment of AD [52]. According to several studies Nrf2 controls redox homeostasis and reduces inflammation in a variety of neurodegenerative diseases [44, 45, 53].

Thymoquinone (TQ), a bioactive volatile oil component derived from the seeds of black cumin (Nigella sativa) possesses substantial anti-inflammatory and antioxidant characteristics, indicating its potential as a neuroprotective medication [54]. Numerous studies with strong evidence suggested that TQ’s neuroprotective benefits might be linked to its ability to modulate oxidative stress, apoptosis, and inflammation.

One essential micronutrient that has numerous complex effects on human health is selenium (Se) [55]. Se is essential for Additionally, selenium serves as an antioxidant; its deficiency increases oxidative stress, which accelerates the onset of Alzheimer’s disease [56]. SeNPs exhibit an impressive range of bioactive characteristics. Their anti-inflammatory and antioxidant capabilities are two of these qualities, which may be beneficial for a variety of neurological illnesses. However, their unique usefulness becomes increasingly obvious in the treatment of AD, regulating synaptic neuronal activity [57].

The principal aim of this work is to evaluate the impact of nano-selenium and thymoquinone on Nrf2 gene expression levels (which protect cells from stressful environments, reducing neurodegeneration) in Alzheimer’s rats.

A master regulator of the cellular antioxidant response is Nrf2 [58]. The build-up of tau and Aβ in AD lowers Nrf2 levels, which lowers the antioxidant response. Decreased Nrf2 levels hinder the autophagy-mediated turnover of tau and Aβ, which leads to their continued accumulation [46]. Nrf2 activation guards against oxidative damage, neuroinflammation, neurodegeneration, and reduction of the accumulation of tau and amyloid-β [47].

According to recent research by Eftekharzadeh et al., Nrf2 activation by a variety of medications could reduce oxidative stress and toxicity in Aβ1-42-induced AD cell models, indicating that Nrf2 activation might be a useful therapy approach for AD. In order to counteract the damage that oxidative stress causes to brain neurons, it is necessary to activate endogenous antioxidant defense systems like Nrf2. The results of the trial indicated an improvement in AD symptoms [59].

The regulation of neuroinflammatory responses and preservation of redox homeostasis in AD are critically dependent on Nrf2. According to a study by Shang et al., activating the Nrf2 pathway might be a promising neuroprotective therapy for AD [60].

In an interesting finding, Kang et al. found that restoring Nrf2 in the microglia greatly inhibited the tauopathy in AD mini brains and prevented the activation of proinflammatory microglia [61].

In the present research, the mean expression level of nrf2 in brain tissue of rats treated with nano-selenium was significantly higher than that in the group of Alzheimer rats with no treatment at p < 0.05.

Zhu et al. noted that the role of Se-NPs in neurodegenerative illnesses has been thoroughly investigated and validated, which is consistent with the current findings. Selenium-modified rutin was able to considerably increase its antioxidant activity in vitro, as demonstrated by the way they processed rutin to create selenium-rutin nanoparticles (Se-Rutin). Nrf2 activation was linked to the protective mechanism, promoting the cytoplasmic to nuclear translocation of Nrf2. The pharmacological foundation for therapeutic application in the prevention or treatment of Alzheimer’s disease may be provided by the protective effects of selenium [62].

Consistent with the current investigation, Du et al. discovered that supplementing male Kunming mice exposed to Cd-induced hepatotoxicity with nano-selenium decreased the acute liver damage caused by Cd by increasing the Nrf2 pathway’s expression [52]. 

These outcomes were consistent with those of Xiao et al., who demonstrated that biogenic nano-selenium particles synthesized by phosphorylation of Nrf2 enhanced the accumulation and activation of Nrf2 [53]. 

Furthermore, these results were consistent with those of Xu et al., who found that biogenic selenium nanoparticles particles activate Nrf2 signaling and upregulate antioxidant enzymes to shield cells from oxidative stress. In cells treated with SeNPs, Nrf2 and its downstream target genes were shown to be upregulated [63].

Moreover, Ge et al. observed that Including sodium selenite, selenium nanoparticles, and selenium-rich yeast in the diet may decrease the adverse effects of cadmium-induced cardiotoxicity, with Se nanoparticles having the strongest antagonistic effect. By initiating multiple pathways, including the Nrf2 signaling pathway, nano-Se therapy demonstrated beneficial properties of mitigating oxidative damage and cadmium-induced histopathology and ultrastructure morphological abnormalities [55]. 

Additionally, Mohamed et al. observed that the neuroprotective effect of rutin-loaded selenium nanoparticles was also responsible for the enhanced antioxidant defense in the hippocampus, lowered pro-inflammatory cytokine levels, and improved neuronal survival in epileptic mice by significantly upregulating Nrf2 [56]. 

In the current study, the mean expression level of nrf2 in brain tissue of rats treated with thymoquinone was significantly higher than that in the group of Alzheimer rats with no treatment at p < 0.05.

These findings were supported by the research conducted by Hofni et al., which discovered that giving rats treated with thymoquinone to prevent streptozotocin (STZ)-induced diabetic nephropathy increased the expression of the nuclear factor-E2-related factor (Nrf2). This suggested that thymoquinone could prevent STZ-induced diabetic nephropathy by modifying the Nrf2 signaling pathway [57]. 

Furthermore, when monosodium glutamate (MSG) was used to generate Attention-deficit/hyperactivity disorder (ADHD)-like behavior in rats, Abu-Elfotuh et al. observed that thymoquinone administration increased Nrf2 mRNA expression [58]. 

These outcomes were in line with those of Farkhondeh et al. who observed an increase in nuclear accumulation of the protein nuclear factor (erythroid-derived 2)-like 2 (Nrf2), in which LPS-induced neuroinflammation was reduced by thymoquinone. Thymoquinone also boosted Nrf2’s transcriptional activity [64]. Thymoquinone demonstrated anti-inflammatory properties by reducing many cytokines, such as TNF-α, NF-κB, and IL-6, and modifying the oxidant-antioxidant system by elevating the amount of antioxidants, such as GSH [65].

Adinew et al. discovered that thymoquinone could demonstrate a noteworthy degree of antioxidant activity through the regulation of numerous oxidative stress-antioxidant defense enzymes. Thymoquinone’s cytoprotective effects were mediated by these enzymes, which Nrf2 could modify either directly or indirectly. Considering that inflammation and oxidative stress in the tumor microenvironment aggravate several cancers, thymoquinone might be a potential Nrf2 activator used in the treatment of breast cancer [60].

The results of this study were consistent with those of a study carried out in 2023 by Fath et al., who found that thymoquinone restored urea and creatinine levels, oxidative indicators, and reduced renal dysfunctions caused by diazinon by overexpressing Nrf2, which also increased the expression of heme oxygenase-1 and NFκB in renal tissue [61].

This study explored the potential of a combination therapy involving SeNPs and thymoquinone to mitigate neurotoxicity in an animal model of AD. The combination showed significant improvement in the behavior study (time consumed in MWM), the biochemical markers (Nrf2 and TNF-α), and the histopathological study compared to the single treatment.

5. Conclusion and future perspective

This study investigated and demonstrated that nano-selenium and thymoquinone enhanced the expression of Nrf2, which has a broad neuroprotective effect, so several studies have explored the potential application of Nrf2 as a medicinal target in Alzheimer’s disease. Thymoquinone and nano-selenium showed improvement in histopathology and behavior studies in the AD rats’ model. Also, demonstrated improvement in the levels of different biomarkers (Aβ-42, GSH, MDA, and TNF-α) reversing them toward the normal levels. The combination of TQ and SeNPs showed more improvement than each of them as a single treatment.

Further studies can be performed on aluminum chloride (AlCl3)-induced Alzheimer’s disease in rats to evaluate the effect of TQ and SeNPs treatments. Further investigations with a greater number of rats could be done to determine better the exact role of thymoquinone and nano-selenium as treatments in AD. Exploration of new therapeutic approach for AD by targeting Nrf2 which appears to have a role in the disease improvement.

Furthermore, there is a limitation in translating these results from a rat model to human clinical applications because diseases that are engineered in a controlled laboratory setting and artificially induced in animals differ considerably from human diseases in natural course, so the outcomes in a human clinical application may differ. This limitation could be addressed by conducting “co-clinical trials,” in which ongoing human phase I and II studies are expressly paralleled by preclinical trials, and a translatability scoring system is developed to find biomarkers that more precisely predict therapeutic outcomes. The delivery route and timetable of the therapeutic agents must also be considered to make them comparable to those that will be utilized with humans.

Ethics approval

The protocol for treating animals was done following the moral guidelines for animal facilities, Faculty of Pharmacy, Girls, Al-Azhar University, Cairo, Egypt (REC number: 257).

Authors’ contributions

This work was carried out in collaboration between all authors. Doha Ellakwa worked on the methodology, data collection analysis, and paper writing. Noha El-Sabbagh contributed to editing the work, writing-review, and supervision.

Disclosure statement

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.