PROTECTIVE EFFECTS OF SULFORAPHANE AND/OR SELENIUM NANOPARTICLES AS WELL AS BROCCOLI JUICE ON ALCL3 INDUCED ALZHEIMER'S DISEASE IN RATS

PROTECTIVE EFFECTS OF SULFORAPHANE AND/OR SELENIUM

NANOPARTICLES AS WELL AS BROCCOLI JUICE ON ALCL

INDUCED ALZHEIMER'S DISEASE IN RATS

Ola Mohamed Samy Ahmad Sadek1, Amira Abd El Rhman1, Nehad Naem Hamed

Shosha1, Gehan Salah Eldin Moram Ali1*, Azza Abd El Fattah Ali2 and Nahla Hussein

Ali Ali1

1

Department of Biochemistry and Nutrition, Faculty of Women for Arts, Science and

Education, Ain Shams University, Egypt.

2

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al Azhar University,

Egypt.

ABSTRACT

Objectives: We examined the effects of Sulforaphane (SFN) and/or

Selenium (Se) nanoparticles (NPs) as well as fresh broccoli juice

(FBJ), on metabolic, epigenetic, antioxidant, inflammatory and

apoptotic markers in Alzheimer’s disease (AD) induced by AlCl3 in

rats. Methods: After characterization of the NPs and the determination

of FBJ’s bioactive components, sixty male Sprague Dawley rats were

divided into 6 groups (G1: healthy control, G2: untreated AD rats, G3:

SFN NPs, G4: Se NPs, G5: SFN +Se NPs and G6: FBJ). The

nutraceuticals were used as pre-and post-AD treatments; rats were first

given oral doses of the tested neutraceticals for 2 weeks then received

daily i.p. injections of AlCl3.6H2O (70 mg/kg/day) to induce AD, and

continued to receive the tested nutraceuticals along with the AlCl3 for

the next 5 weeks. Results: Our pre-and post-AD treatments significantly improved rats’

performances in Morris water maze task and the conditioned avoidance test. SFN NPs were

the most effective, by virtue of their small size and characteristic, followed by FBJ.

Epigenetic and antioxidant effects followed the same direction and were apparent through

significantly halting the effects of AlCl3 on histone deacetylase activity as well as the gene

expression of DNA methyltransferase-1 and heme oxygenase-1, further protecting the

Volume 8, Issue 8, 117-156. Research Article ISSN 2277– 7105

Article Received on 15 May 2019,

Revised on 05 June 2019, Accepted on 25 June 2019,

DOI: 10.20959/wjpr20198-15359

*Corresponding Author Prof. Dr. Gehan Salah Eldin Moram Ali

downstream endogenous antioxidant system. Levels of metabolic dysfunction, inflammation

and apoptosis markers also reflected the neuroprotective effects of our pre-and post-AD

treatments. These results were confirmed by histological and immunohistochemical

examinations. Conclusion: All tested pre-and post-AD treatments significantly exhibited

neuroprotective actions however, SFN NPs were the most effective.

KEYWORDS: Alzheimer’s, sulforaphane, selenium, broccoli, epigenetics, antioxidants.

1. INTRODUCTION

Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative disorder. AD

represents more than 70% of age-related dementia cases worldwide. With the increase in life

expectancy, Dementia is projected to grow to 82 million by 2030 and 152 million by 2050.[1]

According to the WHO report, Egypt ranks at number 33 in AD/dementia deaths. The age

adjusted death rate is 27.88 per 100,000 of population.[2]

AD was first identified as a pathological condition more than 100 years ago by the German

physician Alois Alzheimer,[3] however, AD should be differentiated from normal age-related

decline in cognitive functions. AD is characterized by loss of short term memory, followed

by subtle personality shifts and finally the patient may become incapable of self-care. Death

ultimately follows. From onset to mortality, the average course of AD is usually between 5 to

8 years.[4]

While the familiar form of AD represents less than 5% of all cases,[5] the major form of AD;

sporadic AD usually occurs due to age, environmental and lifestyle related epigenetic

alterations.[6-9]

The complexity of AD pathogenesis relies on the combination of several factors. One of the

fundamental hypotheses describing AD pathophysiology is the beta Amyloid (A) peptide

accumulation and subsequent toxicity. Aβ peptides are produced by cleavage of the

trans-membrane glycoprotein amyloid precursor protein (APP) by the β-secretase and

presenilin-dependent γ-secretase enzymes. Aβ peptides can exist in multiple aggregation forms. The

insoluble fibrils are responsible for various pathological effects. Aβ cascade hypothesis

suggests that not only is Aβ the major cause of neurotoxic insults in AD; it also activates a

number of biochemical pathogenic mediators, including oxidative stress and synaptic

For instance, the first Aβ toxicity targets are the astrocytes. The Aβ-induced astrogliosis

occurs via redox mechanisms and is characterized by fundamental changes in the astrocytic

functioning, affecting Ca2+ signaling, gliotransmitters production, neurovascular unit

functioning, mitochondrial performance; resulting in ROS overproduction,[12-15] and

neuroinflamation.[16-17] A plaques were also shown to surround by hypertrophic, reactive

astrocytes expressing high levels of glial fibrillary acidic protein (GFAP), mostly represented

in the hippocampus.[18]

Other than A, the other main pathological condition in AD is represented by intracellular

paired helical filaments of neurofibrillary tangles (NFTs). The major component of the NFTs

is abnormally phosphorylated and aggregated tau protein. Normally, tau protein is a

microtubule-associated family phosphoprotein and functions to maintain the stability of

microtubules. Hyperphosphorylation of the tau protein can result in the self-assembly of

tangles of paired helical filaments and straight filaments which causes failure of the binding

ability of microtubules and compromises axonal transport, ultimately leading to cytoskeletal

degeneration and neuronal death.[19-20]

Further, AD pathophysiology could be attributed to impaired cholinergic transmission,[21] or

cerebral microvascular abnormalities, leading to a disturbed cerebral blood flow, glucose

metabolism and oxygen consumption.[22] It is commonly known that the brain relies on

adequate blood supply (and thus oxygen) to maintain glucose perfusion and proper function.

It has been proposed that hypoxia and ischemia resulting from cerebral hypoperfusion is a

direct contributor to the pathogenesis and development of AD, as decreased brain glucose

consumption is generally considered to be an early sign of AD and its accompanying brain

metabolic dysfunction.[23-26]

Another key aspect of AD pathophysiology relies on epidemiological evidence which

strongly suggests that potential external factors, including chemical exposures to increased

levels of metals such as Aluminium (Al) in the brain may link to epigenetic modifications of

gene expression leading to AD development and progression.[27-32]

In all cases, the ever increasing extent of AD burden declares the shortcomings of current

treatments.[33] The available pharmacologic treatments are characterized by limited

effectiveness, deleterious side effects, lack of selectivity and low cerebral uptake. This has

phytochemicals-rich medicinal plants as they exhibit remarkable characteristics such as

extraordinary chemical diversity. They are safe, cost effective, natural antioxidants with a

potential to target multiple pathways.[34-35]

Of the natural plant-derived neuraceuticals to be explored, broccoli is thought to be an

optimum candidate for AD. Broccoli (Brassica Oleracea Var.Italica) belongs to the family of

Brassicaceae; cruciferous vegetables. It is very rich in neutraceuticals such as micronutrients

and phytochemicals including Isothiocyanates (ITCs),[36] which have many protective

properties on top of their antioxidant activities.[37-40]

The most abundant glucosinolate in broccoli that generates the ITC Sulforaphane (SFN);

(1-isothiocyanato-4-methyl-sulfinyl-butane) is glucoraphanin.[41] It has been recognized to

warrant anti-inflammatory,[42] anti-oxidant,[43] and anticancer activities,[44] as well as

epigenetic modifications.[45]

Broccoli is also among the main crops that have the ability to accumulate Selenium (Se)

which allows for a greater synergism between the efficiency of Se and ITCs.[46] Se is

considered a trace element but it is crucial to our health.[17,47-48]

On the other hand, herbal medicines, or active compounds derived from natural sources

might suffer from the same limitations, especially the hurdle of delivery for drugs used in

AD. Other challenges include in vivo instability, poor bioavailability, and poor solubility,

poor absorption in the body, issues with target-specific delivery, and tonic effectiveness, and

probable adverse effects of drugs. Therefore, new research is focused on preparing

derivatives of these neutraceuticals that retain their activities and at the same time enhances

their physical properties to a more suitable form for pharmaceutical formulations in AD.[49-50]

Hence, nanotechnology plays a significant role in advanced medicine/drug formulations.[51-53]

Nanoparticles (NPs) have a size range from 1-100 nm. They can significantly impact AD as

novel therapeutics as they are sufficiently small enough to perfuse out of the bloodstream,

penetrate the vessels, and pass the blood brain barrier (BBB) to reach their site of action.[54]

Intriguingly, a large number of NPs can be isolated from broccoli. Edible SFN and Se NPs

2. MATERIALS

2.1. Animals

Healthy male 12 months old; aged albino rats of the Spargue-Dawley strain weighing (290 ±

10 g) were supplied from the Breading Unit of the Egyptian Organization for Biological

Products and Vaccines (Helwan, Egypt).

2.2. Chemicals

 Aluminum chloride: AlCl3.6H2O was purchased as yellowish white powder. Its purity

was 96%. It was freshly dissolved in distilled water.

 SFN and Se NPs: were purchased from Sigma Aldrich Company (USA).

 Broccoli plant: Broccoli florets were purchased from the Agriculture Research Center,

(Giza, Egypt).

 Trisol reagent: It was purchased from Invitrogen, CA (USA).

 Assay kits: DNA methyltransferase-1 (DNMT-1) and hemoxygenase-1 (HO-1) gene

expressions were measured by Real-time quantitative polymerase chain reaction

(qRT-PCR) using Accu Power Cycle Script RT PreMix reverse transcription Kit (Bioneer,

USA) and Light Cycler Fast Start DNA Master SYBR Green I (Roche Molecular

Biochemicals, Germany). Assay kits purchased from Biovision Company, USA were

used in determination of all biochemical measurements including: β-amyloid peptide,

Brain-derived neurotrophic factor, Glial fibrillary acidic protein, Acetylcholine esterase,

Lactate Dehydrogenase, Homocysteine, Asymmetric dimethylarginine, Nitric Oxide

synthase, Histone Deacetylases, Total Antioxidant capacity, reduced Glutathione,

Malondialdehyde, Glutathione S-transferase, Catalase, Glutathione Peroxidase,

Glutathione Reductase, Superoxide Dismutase, Tumor Necrosis Factor-, Interleukin

1-beta and Caspase-3.

3. METHODS

3.1. Characterization of SFN and Se NPs

NPs size distribution was determined by Dynamic Light Scattering (DLS) technique. Briefly,

SFN NPs were dispersed in Cyclo hexane, while the Se NPs were dispersed in distilled water

and their size distributions were assessed using Malvern Zetasizer at the Egyptian Atomic

3.2. Preparation of fresh broccoli juice (FBJ)

Broccoli florets juices were prepared by washing, cutting, squeezing; using a juicer and

filtration; using cheesecloth. The fresh filtrate was given orally by gavage tube at a dose of

(8.33g/kg/day).[60] This amount mimics the average broccoli intake of a normal adult [body

weight (bw) 60 kg].

3.3. Determination of the bioactive components of FBJ and its anti-oxidant activity

Samples of FBJ were analyzed for their total phenolic Content (TPC) using the Folin-

Ciocalteau method,[61] total flavonoid content (TFC) by aluminium trichloride colourimetric

method,[62] Total glucosinolate content (GLs) using sodium tetrachloropalladate,[63] as well as

the total antioxidant activity using the stable 1,1-diphenyl-2-picryl hydrazyl (DPPH•)

radical.[64] SFN content of FBJ was determined by HPLC,[65] while Se content was

determined using Perkin Elmer 2380, atomic absorption spectrophotometer.[66]

3.4. AD induction

AD was induced by daily intraperitoneal injections of AlCl3.6H2O at dose of 70 mg/kg/day

for 5 weeks.[67]

3.5. Animal trial

All rats were housed in stainless-steel cages and were maintained under controlled

environmental conditions; temperature 25ºC ± 5 ºC, air humidity 55% ± 10% and 12/12 hr.

light /dark cycle were held. Rats were given a balanced diet, with drinking water ad libitum

for the whole duration of the experiment (7 weeks). Rats were acclimatized to laboratory

conditions for 7 days before the commencement of the animal trial. The animals were divided

into six groups (𝑛= 10), as follows:

 Group I: (Healthy Control): Healthy rats received a daily oral dose of water at the same

manner for the whole duration of the experiment (7 weeks).

 Group II: (Untreated AD): Rats were given AlCl3.6H2O injections to induce AD as

already mentioned.

 Group III: rats were first solely pretreated with oral SFN NPs dose (0.5 mg/kg/day),[68]

for 2 weeks before AD induction and continued to receive the same dose of SFN NPstill

 Group IV: rats were first solely pretreated with oral Se NPs dose (0.5 mg/kg),[69] for 2

weeks before AD induction then continued to receive the same dose of Se NPstill the end

of experiment as treatment (Se NPs pre-and post-AD treated rats).

 Group V: rats were first solely pretreated with a combined oral dose of SFN and Se NPs

(0.25 mg/Kg + 0.25 mg/ Kg) for 2 weeks before AD induction then continued to receive

the same doses of SFN +Se NPs till the end of experiment as treatment (SFN +Se NPs

pre-and post-AD treated rats).

 Group VI: rats were first solely pretreated with FBJ (8.33g/kg),[60] for 2 weeks before

AD induction then continued to receive the same oral dose of FBJ till the end of

experiment as treatment (FBJ pre-and post-AD treated rats).

3.6. Behavioral assessments

Two experiments of behavioral assessments with different degree of stressfulness were

selected to formulate an integrative testing battery. The chosen battery of tests allows

measuring the most behavioral changes accompanying AD. Rats were taken to testing area

one hour before each experiment for adaptation after removing food and water from the home

cages. Experiments were usually carried out at a fixed time around 9A.M. - 2P.M.

Morris water maze (MWM) test: The measured parameters were learning ability [escape

latency (sec)] and memory trial [(Time spent in target quadrant (sec)]. It is a hippocampus

dependent spatial learning task.[70] The tests were carried out as previously described.[66]

Conditioned – Avoidance (CA) test: This test studies the conditioned reflexes involving

auditory as well as visual stimuli as an indication of cognitive performance as well as

evaluating learning ability and memory retention (short term memory) in high stressful

conditions.[71] The apparatus and procedure were set as previously described.[66]

3.7. Dissection and Tissue Preparation

Immediately after the final behavioral test, the animals were sacrificed by decapitation. On

the dorsal side of the skull, an incision was made to expose and rapidly remove the brain. The

whole brains were weighed immediately after separation and washed in a saline solution. The

hippocampi were separated and used for biochemical, histological and immunohistochemical

3.8. Real-time quantitative polymerase chain reaction (qRT-PCR)

qRT-PCR was performed as previously described.[72] Extraction of total RNA was done using

Trisol reagent (Invitrogen, CA). DNMT-1 and HO-1 genes’ expressions were measured in

four replicates. Beta Actin (-ACT) expression level was used as an internal control. Data

were normalized using the 2−ΔΔCt method.[73] The PCR conditions for DNMT-1 were 12

cycles at 22 C for 30s (primer annealing), 12 cycles at 45 C for 4 min (cDNA synthesis), 12

cycles at 55 C for 30s (melting secondary structure & cDNA synthesis) and finally 1 cycle at

95 C for 5 min (heat inactivation).

The forward primer was: 5 -CGTGCAGAGAATTCTGAGTTC-3. The reverse primer used

was 5-AGACGCTTTACGTAGTGCTG-3. The temperature transition rate was 20°C/second

except at the melting to cooling transition where it was 0.1°C/second. The primer Tm was

calculated according to the following formula, based on the nucleotide content of the

primer(s): Tm = 2°C (A + T) + 4°C (G + C). The PCR Primers used in HO-1 gene were:

forward: 5-CGTGCAGAGAATTCTGAGTTC-3 and Reverse 5

-AGACGCTTTACGTAGTGCTG-3.

3.9. Biochemical analyses

All biochemical assays were performed according to kit instructions in hippocampal tissue

homogenates.

3.10.Statistical analyses

Data were analyzed using the Statistical Package for Social Science (SPSS) program, version

17.0. The data were expressed as means  standard deviation (S.D) of the mean. Statistical

differences between groups were performed using one way analysis of variance (ANOVA).

The mean difference was significant at p < 0.05 level.[74]

3.11.DNA fragmentation assay

Briefly, about 50 mg of tissue were homogenized in lysis buffer pH 8.0 (10 mM Tris base, 1

mM EDTA and 0.2% triton X-100) and incubated on ice for 20 min. The cell lysate were

centrifuged at 12,000 r.p.m. for 30 min at 4◦C. The supernatant containing small DNA

fragments was separated. DNA fragmentation in samples was calculated as follow:

100]. The data were expressed as percentage of total DNA appearing in the supernatant

fraction.[75]

3.12.Histological examination

The tissues were fixed in neutral formal saline 10%, embedded in paraffin wax, fixed and

stained by Haematoxylin and Eosin then examined microscopically at 100X and 400X.[76]

3.13.Immunohistochemical study of phosphorylated Tau protein

Hippocampal tissues were sectioned then deparaffinized using xylene: ethanol (100%),

(95%), (70%) and (50%) for 3 min at each step. Antigen retrieval was carried out by placing

the slides in Tris-EDTA buffer solution (TBS) (pH 9.0) in a water bath at 100 C. The slides

were washed with TBS containing 0.025% triton 100X with gentle agitation. The sections

were then blocked in 10% normal serum with 1% Bovine Serum Albumin (BSA) in TBS and

incubated for 2 h for room temperature. Sections were then incubated in primary antibody;

phosphorylated tau monoclonal antibody (1:500) in TBS with 1% BSA overnight at 4 C and

were examined using light microscope.[29]

4. RESULTS AND DISCUSSION

4.1. Characterization of SFN and Se NPs

The size distribution of SFN and Se NPs are shown in figures (1-2). DLS analysis showed

that most of the SFN NPs had a diameter between 25.5 and 39.6 nm with a maximum size

distribution (mean ± one standard deviation) at 34.2 ±1 nm. On the other hand, Se NPs had a

diameter between 12.2 and 19 nm with a maximum size distribution (mean ± one standard

deviation) at 14.1±1 nm. Based on these results, we proposed that these NPs are capable of

crossing the BBB so they can exert their protective properties and decrease cell damage

directly in the brain.

4.2. Analysis of bioactive components and the anti-oxidant activity of FBJ

Results of the TPC, TFC, GLs, SFN and Se content of FBJ as well as the total antioxidant

activity are presented in table (1). Broccoli is known to contain different beneficial bioactive

compounds such as polyphenols and glucosinolates, which as mentioned are majorly broken

down by the endogenous enzymes; myrosinases into SFN. These attest to the high antioxidant

activity of broccoli. Our results of the phytochemicals’ analysis of FBJ confirmed those of

recent studies that investigated SFN, polyphenols and antioxidant activities changes of

4.3. Behavioral assessment

The results indicating the rats’ performance in MWM test are shown in figure (3). Measuring

the escape latency (sec.) was taken as an indication of learning ability in rats. Overall,

untreated AD rats (G2) had increased escape latency by 63% in day one, 62% in day two,

71% in day three and 76% in day four as compared to healthy control rats in G1. Untreated

AD rats spent a significantly (p<0.05) less time in the target quadrant than healthy rats; by

143%. On the other hand, SFN NPs pre-and post-AD treatment significantly decreased the

escape latency by 35%, 55%, 65% and 72% in the training sessions through four days,

respectively when compared to the untreated AD rats. As for the other groups, FBJ pre-and

post-AD treatment (G6) decreased escape latency progressively till it reached 64% by the

forth training day. It is worth mentioning that SFN NPs and FBJ were able to bring back the

escape latency time to near normal levels in all 4 days of training. Combined SFN +Se NPs

(G5) followed in their abilities to decrease the escape latency. However, singular Se NPs

(G4) showed a statistically significant ability to decrease the escape latency starting the

second day and continued to do so till it reached 55% in day four. The effects of the tested

pre-and post-AD treatments on the time spent in target quadrant (TTQ) are shown in figure

(3b).

In conditioned – avoidance (CA) test, untreated AD rats in G2 showed a significantly

(p<0.05) higher average in their number of trials performed before being able to avoid the

electric shock. In the first day, the % of change was 55%, while in the second day it reached

73% from that of the healthy control. However, the pre-and post-AD treatments, most

effectively SFN NPs, were able to significantly improve the cognitive performance in rats,

more clearly in the second day of testing, which was followed by FBJ. The effectiveness of

the pre-and post-AD treatments were equal to 59% in G3, 57% in G6, 49% in G5and 35% in

G4, when all were compared to untreated AD rats, as shown in figure (4).

These previous results demonstrate the decline in cognitive performance, learning ability and

memory impairment; all are hallmarks of AD development, which together attest to the

ability of Aluminum (Al) to interfere with long-term memory potentiation. The progressive

nature of these deteriorations is aligned with the hypothesis that the efflux of Al from the

brain is very slow; and as it gradually destroys the BBB, its concentration in the brain

deleterious effects, it is suggested that the pro-oxidant nature of Al is the main cause of its

neurotoxic effects, as confirmed by our following results.

On the other hand, Al is proposed to induce learning and memory defects through epigenetic

alterations of gene expression.[27] To normally regulate the memory process, neuronal activity

per se has to modify DNA methylation and histone modifications patterns. Consistently, for a

transient stimulus, such as the successive training sessions, to induce a lasting change in

behavior, cells must undergo a complex set of stimulus-specific cellular and molecular

changes that will consolidate a memory into an everlasting trace, thus epigenetic process of

gene expression is critical to learning and memory.

Previous studies have also reported learning and memory disturbance following Al

administration to rats.[28,66] Thus, to have successfully halted the observed deleterious effects

of Al on working memory in MWM and CA tests, our pre-and post-AD treatments are

suggested to combat the pro-oxidant nature of Al and thereby attenuating the loss of BBB

integrity, most likely through direct activation of the intrinsic antioxidant system as well as

other mechanisms dependent, at least in part, on the modulation of epigenetic regulation of

genes’ expression.

Regardless, the pre-and post-AD treatments were not found to be equally effective. Most

notably, and despite of our preceding results of the FBJ analysis which revealed its high

content of various phytochemicals, SFN NPs pre-and post-AD treatment were the most

successful at improving learning abilities and memory retrieval. We suggest that the

superiority of the nano-sized SFN’s efficacy to the FBJ was due to the enhanced

bioavailability, absorption, uptake and delivery of SFN NPs across the BBB. We even found

that using oral doses of merely 0.5 mg/kg/day the nanosized SFN and/or Se in this study

resulted in the same or even higher protection of the cognitive and memory functions of

animals shown by previous studies examining native SFN and/or Se containing compounds,

wither broccoli-driven or not.[66,79-82]

4.4. Effect of tested pre-and post-AD treatments on brain weight

Untreated AD rats had a significantly (P<0.05) increased brain weights (35%) as compared to

healthy control rats in G1, as shown in figure (5). Pre-and post-AD treatment of rats with

SFN NPs resulted in significant reductions in rat brain weights, which amounted to 26%.

combined NPs and the singular Se NPs pre-and post-AD treatments trailed by 22% and 21%,

respectively as compared to the untreated AD rats.

Since the brain’s weight is considered a putative indicator of edema, these results confirm

that exposure to Al for 5 weeks must have resulted in an inevitable neurotoxicity which led to

BBB injury; raising its permeability and promoting further influx of other macromolecules or

neurotoxic materials, such as plasma proteins, that might have gained access to the

parenchymal areas of the brain, eventually leading to brain edema of the vasogenic type. The

significant increment in total brain weight of untreated AD rats observed in this study and

confirmed by others.[13,83] Interestingly, the efficacy of the pre/treatments in decreasing the

brain weight was exactly in the same order previously observed when examining the

cognitive abilities and memory function results. Therefore, we postulate that BBB breakdown

could be an upstream detrimental factor for cognitive impairment and that our pre-and

post-AD treatments must have exerted their neuroprotective effects partly by preserving the

integrity of the BBB. Additionally, these rationalizations were justified by previous

studies.[56,80,84]

4.5. Effect of different pre/treatments on epigenetic and antioxidant genes’ expression

qRT- PCR results showing the relative mRNA expression of DNMT-1 and HO-1are

illustrated in figure (6). First off, untreated AD rats, had significantly (p<0.05) lower (-80%)

mRNA expression of DNMT-1 as compared to the healthy control. On the contrary, the

pre-and post-AD treatments were able to significantly (p<0.05) restore it by 161% in G3, 47% in

G4, 117% in G5 and 117% in G6, when compared to untreated AD rats. This disruption of

epigenetic networks is implicated in the etiology of AD as epigenetic regulation of gene

expression plays a critical role in orchestrating several neurobiological pathways.[9]

Mechanistically, Al exposure caused a global downshift in DNA methylation by down

regulating DNMT1, as it is known to be the major methylase acting for maintenance of DNA

methylation.[27-28,32] Methylation of a promoter downregulates the gene’s expression as the

transcription factors become unable to bind to that region. For that reason, Al administration

was thought to be responsible for disrupting the expression of influential genes instigating

AD; causing the observed alterations in cognitive abilities and their representative

biochemical markers of this study. The global DNA hypo-methylation in AD was previously

However, our findings of confirmed the abilities of SFN NPs pre-and post-AD treatment to

counteract the epigenetic alterations of Al on memory and cognition. SFN NPs were shown

to be the most effective DNMT-1 upregulators. Their effect possibly exceeded that of FBJ

most likely due to the NPs greater penetration of the BBB which makes them very targeted

and point specific treatments. FBJ itself could have targeted these post-translational

modifications through its content of SFN as the major GLCs as well as any of the multiple

phytochemicals it contains. As for Se, its effects on the DNA methylation reactions could

have possibly been achieved through direct interaction with the enzyme’s accessory

transcription factors, or by increasing the availability of the endogenous methyl donors,

especially S-adenosylmethionine (SAM). SFN and SFN-rich broccoli extract as well as Se

have all been previously shown to modulate the activity of DNMT-1.[8,43,86-87]

As for the HO-1 mRNA relative expression in untreated AD rats was at -64% of that of the

healthy control. SFN NPs pre-and post-AD treatment elevated HO-1 expression by 155%

relative to the untreated AD rats. FBJ restored the expression by 133%, followed by NPs

combination which resulted in 114% elevation relative to untreated AD rats. As for Se NPs

pre-and post-AD treatment, the mRNA restoration was at 75% of untreated AD rats.

These results were suggested to be a direct result of the activation of the nuclear factor

erythroid 2-related factor 2 (Nrf2). Nrf2 is a key redox-regulated gene. Under basal

conditions, Nrf2 is sequestered in the cytoplasm by its repressor protein Keap1. Keap1

contains several reactive cysteine residues that serve as sensors of the intracellular redox

state.[88-89] Nrf2 induces antioxidant and phase II detoxification enzymes’ genes including

HO-1 by recognizing an antioxidant response element (ARE) binding site within their

promoter regions. In our study, SFN and/or Se NPs as well as FBJ were shown to effectively

increase the mRNA of HO-1, presumably as a direct result of Nrf2 activation, through

abrogation of the binding between Nrf2 and keap-1, allowing Nrf2 translocation to the

nucleus to function as a transcriptional activator and enhance the expression of some

ARE-driven targets, including HO-1. This was postulated and confirmed by recent literature.

[45,90-95]

It has been reported that the regulation of Keap-1/Nrf2/ARE is more complex than previously

thought, with other mechanisms including epigenetic regulation of Nrf2 now known to be

involved. Intriguingly, sulfur containing compounds were found to play an integral role in

cysteine residues and is under oxidation-reduction (and alkylation) control via its highly

reactive thiol groups.[13,45,89,96]

4.6. Effect of tested pre-and post-AD treatments on biochemical markers of AD

development, metabolic dysfunction and histone deacetylase activity

The results presented in table (2) show that the untreated AD rats in G2 had a significantly (P

<0.05) higher level of accumulated β-amyloid (A) protein (55%), Glial fibrillary acidic

protein (GFAP) level (33%), Acetylcholine esterase (AChE) activity (37%), Lactate

Dehydrogenase (LDH) activity (25%), accumulated Homocysteine (HCYs) level (40%) and

Asymmetric dimethylarginine (ADMA) level (50%). However, untreated AD rats had

significantly lower level of Brain-derived neurotrophic factor (BDNF) (-138%) and Nitric

Oxide synthase (NOS) activity (-144%) in the hippocampus as compared to the healthy

control rats (G1).

Instead, SFN NPs administration significantly (p< 0.05) decreased A protein level by 37%,

GFAP level by 26%, AChE activity by 28%, LDH activity by 21%, accumulated HCys by

33% and ADMA level by 25%, while increasing BDNF by 66%) and NOS activity by 40%

when compared to the untreated AD rats. FBJ also amended the effects of Al on these

markers by 33%, 23%, 22%, 19%, 27%, 32%, 57% and 32%, in exactly the same order.

The effects of Se were significant though less pronounced, yet, its ameliorative effect was

still exceeded by its combination with SFN NPs in G5. The combined NPs decreased the A

protein level, GFAP level, AChE activity, LDH activity, HCys level and ADMA level by

32%, 22%, 19%, 17%, 23% and also 23%, in that order. All while significantly increasing the

BDNF level and NOS activity by 54% and 25% respectively.

Finally, the activity of the total Histone Deacetylases (HDACs) enzymes in G2 rats was

found to be significantly (P<0.05) elevated (by 52%) following AD induction. HDAC

inhibition was significantly (P<0.05) achieved in all pre-and post-AD treated groups when

they were compared to the untreated AD rats. However, the most effective pre- and post- AD

treatment was SFN NPs (35% inhibition), closely followed by FBJ at 32%, then finally Se

NPs trailed with 27% after the 28% reduction in these enzymes’ levels by the combined NPs.

As previously established, Al exposure led to its accumulation in the brain, more specifically;

proteolysis of normal amyloid precursor protein (APP).[28] Al alters the dynamics of Aβ,

causing it to accumulate as intracellular plaques due to alteration in glutamate receptors,

mitochondrial dysfunction and disturbance in signaling pathways related to synaptic

plasticity.[35]

Being a cation, Al could have also promoted the formation of an amyloid-Al complex; which

is more toxic than Al or amyloid peptides on their own as the observed elevation of GFAP

level in untreated AD rats clearly demonstrated that the sever Aβ toxicity further induced the

activation of astrocytes; astrogliosis, which is considered another major pathological feature

in AD.[97-99]

Aβ plaques also were shown to trigger a cascade that included neuritic injury due to the

significant drop in BDNF level, which is known to regulate synaptogenesis, neuronal

plasticity, induction and maintenance of long-term potentiation and memory formation as one

of the major neurotrophins, and its expression is critical for the cholinergic neurons.[100]

Our pre-and post-AD treatments are thought to have prevented the astrocyte hypertrophy due

to their antioxidant profiles and their ability to quench cytotoxic free radicals within the

hippocampal cells, thereby protecting them. Furthermore, neurons surrounded by mutant

astrocytes develop protein aggregates of Aβ and become more susceptible to cell death. It is

thereby suggestive, that the inhibition of astrocytes hypertrophy and mal-modification

underlies mechanisms of our pre-and post-AD treatments in preventing neurofilaments

degeneration and the accompanying neurotrophic damage in this study, however, these

reactive neurons and astrocytes may have been also responsible for the brain metabolic

dysfunction that is found to accompany AD development.[101]

On the other hand, pre-and post-AD treatment of rats with SFN and/or Se NPs as well as FBJ

were shown to have positively altered the level of accumulated A, partly prevented the

astrocyte hypertrophy possibly due to their antioxidant profiles and their ability to quench

cytotoxic free radicals within the hippocampal cells, thereby protecting them and lastly,

prevented the decline of BDNF level, which ensured its proper function in the signal

transduction pathways of neuronal survival and differentiation.

These pre-and post-AD treatments have also nullified the impaired cholinergic transmission

play here resulting in the memory deficit. Normally, cholinergic transmission is necessary for

the acquisition and retrieval of learning, memory and cognition.[102] AChE is a catabolic

enzyme responsible for degradation of acetylcholine (ACh); the main neurotransmitter

involved in learning and memory processes in synapse. Al is thought to have the ability to

produce direct effect on AChE activity by interacting with peripheral sites of the enzyme and

modifying its secondary structure and ultimately its active site, or Al could have elevated

AChE enzyme due to a direct action of Aβ, which binds to nicotinic receptors and increases

the activity of AChE within and around Aβ plaques. Al might have also exerted its

cholinotoxic effects by impairing the provision of acetyl-CoA which is required for ACh

synthesis. The excess acetyl-CoA could be shunted towards an increased but ineffective lipid

metabolism forced by the oxidative stress induced shift in the Krebs cycle intermediates as

shown by the significantly elevated LDH activity in the untreated AD rats.[29,103]

Accordingly, we determined LDH activity to serve as a marker of energy deficiency in the

brain and as well as a marker of toxic insults and damage to tissues; since LDH enzyme is

synthesized massively and released aberrantly into the cellular environment, especially where

there is an impairment of energy production.[98,104-105]

Our results confirmed the variable capabilities of our pre-and post-AD treatments to halt this

dysfunctional energy metabolism, through normoregulation of hippocampal LDH activities,

and further highlighted their inhibitory role in astrogliosis and subsequent prevention of

metabolic dysfunctions additionally represented by levels of HCYs, ADMA and NOS.

HCYs is known to be a pro-oxidant and an intermediate metabolite of the methionine cycle,

produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of

methylation reactions involving the methyl donor S-adenosylmethionine (SAM). Increased

level of Hcy is thus associated with an elevation of intracellular SAH, which is known to be a

potent inhibitor of methyl-transfer reactions and ultimately leads to DNA hypomethylation.

SAM is also known to be involved in formation of ADMA by catalyzing reaction of arginine

methylation.[25] However, when oxidative stress mediated proteolysis occurs, ADMA is

released in the cytosol, where it is regarded to act as an endogenous inhibitor of NOS, and

interferes with arginine-dependent NO production. Also, ADMA determines ―NOS

uncoupling,‖ a shift in NOS enzymatic activity from reductase to oxidase. In the absence of

its substrate, NOS transfers electrons to molecular oxygen, instead of arginine, leading to the

combines with NO producing peroxynitrite, a highly reacting intermediate and powerful

source of oxidative stress that entails DNA and protein oxidation and at high concentration,

cytotoxicity.[107]

Fortunately, pre-and post-AD treatments used in our study were shown to improve the

deregulated metabolic consequences of Al administration and decreased the predisposition to

subsequent oxidative stress induced injury. These findings are in cohort with the recent

literature.[17,23,37,108-112]

As mentioned, the machinery of DNA methylation does not act independently; DNMT1

repression could have also affected the process of histone acetylation through

hypomethylation, and thereby activation, of HDAC promoter region, as seen in untreated AD

rats. HDAC are known to control local DNA compaction, leading to site-specific chromatin

alterations and thereby control the access of transcription machinery to significant genes in

AD development and progression.[113]

In this study, we speculate that the pan hypoacetylation of the genome in untreated AD rats

following the elevated activity of HDAC has been associated with chromatin compaction

leading to transcriptional repression of some key genes including those responsible for Tau

phosphorylation, neurotropine production, astroglial function or Nrf-2 master gene which

could have preceded the observed disrupted oxidative state.

SFN and broccoli have been previously shown to inhibit the enzymatic activity of HDAC.

[114-116]

Similarly, Se was suggested to inhibit the cognitive impairment in AD by mediating

epigenetic regulation or modulating transcription factors and thereby influencing oxidative

stress, genomic stability, metabolic activities and cell cycle regulation.[117-118]

Though all pre-and post-AD treatments had significantly (P<0.05) restored the levels of these

parameters, as compared to the untreated AD rats, these favorable effects were not one and

the same. It was clear that SFN NPs were the most effective pre-and post-AD treatment and

was followed in efficiency by FBJ. We attributed this to the fact that the junctions between

the endothelial cells in the BBB are of course highly tight. That forces them to analyze the

molecules on the basis of their lipophilicity, size, surface charge, and hydrogen-bonding

potential. Therefore, these gateways must have given priority to the small lipophilic SFN NPs

Moreover, SFN +Se NPs pre-and post-AD treated rats (G5) proved that the synergistic effects

between SFN and Se NPs partly made up for the fact that these rats only received half the

amount of SFN NPs given to rats in G3, however since both neutraceuticals were used as

NPs, they exerted not just similar, but superior neuroprotection than their native counterparts

commonly used in other studies.[119-121]

4.7. Effect of tested pre-and post-AD treatments on biochemical markers of the redox

state

The redox state markers are presented in table (3). All in all, when compared to the healthy

control rats in G1, we observed a dramatically reduced antioxidant status in untreated AD rats

in G2. Reactive oxygen species (ROS) accumulation was estimated in terms of

Malondialdehyde (MDA) level. It was significantly (p<0.05) increased by 53%. This result

was further established by the plop down of the main endogenous non enzymatic antioxidant;

reduced Glutathione (GSH) by 113% and correspondingly, the total antioxidant capacity

(TAC) was reduced by 142%. Furthermore, all downstream enzymatic targets of HO-1 gene

had reduced activities in comparison to G1. There was a significant (p<0.05) reduction of

Glutathione S-transferase (GST) activity by 97%, Superoxide Dismutase (SOD) activity by

79%, Catalase (CAT) activity by 86%, Glutathione Reductase (GR) activity by 75% and

Glutathione Peroxidase (GPx) activity by 161%.

We sought to determine the antioxidant effects of our tested NPs compared to FBJ,

investigating whether they can mitigate the oxidative stress observed in untreated AD rats.

GSH level was elevated following the pre-and post-AD treatment of rats in G3 by 66%, G6

by 54%, G5 by 50%, and G4 by 39%, when all were compared to the untreated AD rats. The

TAC was similarly elevated in those groups by 68%, 54%, 48% and 30%, in the same order.

Additionally, SFN NPs, FBJ, combined NPs and singular Se NPs significantly reversed the

elevated MDA levels by 32%, 24%, 22% and 18%, in the same order. Dependably, the order

of effectiveness of these pre-and post-AD treatments in restoring the enzymatic antioxidants

measured in this study followed exactly the same pattern.

These results confirmed that the Al-induced neurotoxicity and the subsequent accumulation

of A were associated with a massive disruption in levels of oxidative stress markers and

cytoprotective molecules, confirming the pro-oxidant action of Al. Consistent with the results

mitochondria; decreasing the efficiency of ATP production, and inducing an increment in

ROS production; as higher proportion of oxygen was converted into free radicals. The

mitochondria themselves are susceptible to high ROS levels, eventually resulting in further

ROS production due to a mitochondrial membrane potential loss as Al bind with polar head

groups of membrane phospholipids leading to membrane deformation. As a result,

membranes become vulnerable to free radical induced damage. These consequences of events

are supported by studies that involved mitochondria preparations from several AD animal

models.[28,35,122]

Also, in a vicious cycle, Aβ has been shown to actually produce ROS itself, particularly in the presence of other free radicals. In fact, Aβ is now thought to be a redox-metal chelator

and its deposition could even represent a compensatory mechanism, aimed to isolate the

highly reactive metals accumulated in the AD brain. Unfortunately, the by-product of its

antioxidant activity; H2O2 could mediate the Aβ-related toxicity, accumulating in the brain

once the amyloid deposition increases.[30]

Our results suggested a particular vulnerability of hippocampal cells to the increased ROS

production, represented by MDA. The reason for this is probably due to their very high

oxygen consumption, high content of the highly susceptible polyunsaturated fatty acids, as

well as their low mitotic rate and low endogenous antioxidants concentration. The

unwarranted ROS in untreated AD rats predominantly decrease GSH and inhibited the

activities of GST, SOD, CAT, GR and GPx due to a concomitant exhaustion of Nrf2

activation and saturation of GSH detoxifying potential. GST is the other phase 2 response

enzyme, other than HO-1, measured in this study. The suppressed activity of GST recorded in

untreated AD rats led to the continual of the damaging effects of ROS and the accelerated

pathogenesis of AD. On the other hand, GSH is the most abundant intracellular non

enzymatic antioxidant. It is involved in direct scavenging of free radicals or serving as a

substrate for the GPx enzyme that catalyzes the detoxification of H2O2 to H2O. The drastic

depletion of brain GSH may be explained by an increased cytotoxicity of H2O2 following the

inhibition of SOD activity in untreated AD rats due to the formation of Al-SOD complex that

altered the conformation of the SOD molecule. The inhibition of O2– dismutation reaction

increased the cellular toxicity and overwhelmed the H2O2 neutralization machinery; CAT,

GR and GPx in the brain of untreated AD rats. The catalytic reduction of peroxides, either

groups, which in GPx is known to be selenium- dependent. So, low levels of GPx enzyme

within the hippocampal tissues may reflect failure in the detoxification of H2O2 within

neuronal cytosol. Arguably, excess H2O2 may have been converted to OH or hydroxyl-like

intermediates, while O2•- reacts with the diffusible gas nitric oxide, to form the potent

nucleophile oxidant and nitrating agent peroxynitrite (ONOO-). In turn, ONOO- which is

genotoxic directly to neurons by causing single and double-strand breaks in DNA may have

activated proteins involved in cell death as seen in this study. Accordingly, the observed

significant reduction in brain TAC in untreated AD rats was attributed to the depletion and

exhaustion of these antioxidant enzymes following the decrease in axonal mitochondria

transformation, impairment of golgi and reduction of synaptic vesicles and the continuous

release of oxidative products. These justifications were detailed by previous studies.[13,123-124]

Previous literature has confirmed that SFN-enriched broccoli and prominently SFN itself

have the potential to reverse the state of oxidative stress.[69,125-129] Potentially, through

donation of hydrogen atom to free radicals by metabolites of both pre/treatments, they formed

stable intermediate compounds instead, later removed by the cells. Endorsing the previously

expressed dominance of SFN NPs; they were shown here to be more effective at scavenging

those ROS and promoting the preservation of endogenous antioxidant systems than the

conventional well known antioxidants in FBJ. This was even though analysis of FBJ revealed

an impressive antioxidant capacity and the presence of a lot of bioactive compounds

including GLCs, ITCs other phenolic compounds and flavonoids.

The reason given for this is that the entry of these native molecules to the brain could have

been restricted. Even very potent antioxidants need to have specific criteria of structural and

physicochemical properties in order to exert their beneficial effects in the brain. So, we

propose that the underscoring of FBJ, in vivo, compared to the SFN NPs was due to the fact

that not all FBJ components fell under the inclusion criteria to enter across the BBB, and

even if they did, there was still the high chance of being thrown out again by a number of

efflux transporters.

In either case, FBJ’s remarkable ability to upregulate all mediators of the antioxidative

pathways exceeded those of the singular Se NPs. Nonetheless, Se NPs played a significant

role as Se is known to be a cofactor necessary for these antioxidant enzymes to perform their

function,[48,81,130-132] However, its synergistic effect with SFN, is shown here to be more

4.8. Effect of tested pre-and post-AD treatments on markers of inflammation and

apoptosis

Results of the inflammatory and apoptotic markers confirmed that injection of AlCl3 for 5

weeks in G2 resulted in a statistically significant (P<0.05) elevation in Tumor Necrosis

Factor- (TNF-) level by 65%, Interleukin 1-beta (IL1-) level by 50%, Caspase-3 (Csp-3)

level by 45% and DNA fragmentation by 3 folds, when compared to G1. On the other hand,

pre-and post-AD treated rats in G3, G4, G5 and G6 showed a statistically significant

reduction in levels of all inflammatory and apoptotic markers, (P<0.05) shown in table (4).

For instance, levels of both TNF- and IL1- were decreased by 42% and 39% following

SFN NPs pre-and post-AD treatment, in the same order. Similarly, in G6, FBJ decreased

those inflammatory markers by 37% and 33%, respectively. Combined NPs (G5) and Se NPs

(G4) decreased TNF- level by 34% and 32% as well as IL1- level 31% and 26%, in the

same order. Results of the apoptotic marker Csp-3 were similar as SFN NPs pre-and post-AD

treatment most effectively decreased its level by 41%, while G6, G5 and G4 followed with

percentages of change equal to 33%, 30% and 20% in that order. Our chosen pre-and

post-AD treatments were also able to outstandingly limit the genotoxicity of Al. As shown in

figure (7), SFN NPs decreased the level of DNA fragmentation by 49%, followed by FBJ

with 44%, then SFN +Se NPs with 31% and finally Se NPs by 31%.

The previous results clearly confirm that the oxidative stress that correlated with changes in

behavior and cognitive impairment, metabolic dysfunction, epigenetic alterations in gene

expression and protein aberrations was also accompanied by considerable neuroinflammation

and finally resulted in DNA fragmentation and apoptosis. The neuro-inflammatory cascade

might have resulted from the established Aβ aggregation. A is known to attract and activate

microglial cells to form clusters at its deposition sites; to engage in its phagocytosis and

degradation.[133] Such phagocytic function was however ineffective in untreated AD rats and

the activated microglia contributed to the release of neuroinflammatory markers.

On the other hand, neuroinflammation, as a consequence of oxidative stress, could have

preceded Aβ, and tau, pathology and the inflammation-activated microglia did not properly phagocytize Aβ, leading to plaque accumulation rather than plaque clearance.

Regardless, there is an overwhelming consensus on the adverse impacts of

increased production of IL-1β and TNF- α led to further Aβ deposition and contributed to the

already established metabolic and oxidative dysfunctions.[134-135] Consistent with this notion,

these cascades of have inevitably induced apoptotic cell death. Apoptosis is known to be the

ultimate mechanism of neuronal loss in AD. It is regulated by caspases, a family of protease

enzymes. There are two major pathways through which apoptosis is induced, but both

pathways converge on caspase-3 activation.[99]

Almost certainly, activated microglia had increased susceptibility to apoptosis under high

metabolic demand and post-mitotic nature.[26] Also, the previously mentioned accumulation

of abnormally folded tau and Aβ proteins most likely provoked apoptosis. Another possibility

involved the epigenetic modulation of DNA as the upregulation of HDAC could have

catalyzed the hypoacetylation of tumor suppressor genes, thus hampering their expression.

Our results also link the Al induced biochemical alterations with DNA damage. As

previously mentioned, Al is a trivalent cation which means it has a high affinity for

negatively charged groups such as phosphates and phosphorylated proteins in nucleic acids.

Therefore, Al may bind to DNA leading to strand breakage and DNA fragmentation.[136]

Nonetheless, our pre-and post-AD treatments were postulated to inhibit TNF-α and IL-1β,

most likely through regulating the mitogen-activated protein kinase (MAPK) signaling and

c-Jun N-terminal kinase (JNK) phosphorylation levels, which subsequently reduces NF- kB and

AP-1 signaling and translocation.[82,137] As a result, the expression of these proinflammatory

cytokines was decreased as these pathways are confirmed by recent studies.

[42-43,55,126-128,138-140]

In addition to this, the activation of Nrf2 and HO-1 is known to potentiate the production

of anti-inflammatory cytokines, thereby indicating the anti-inflammatory potential of these

proven powerful redox modulators.

4.9. Effect of the tested pre-and post-AD treatments on immunohistochemical and

histopathological changes in the hippocampus

Figures (8-9) show the immunohistochemical staining of Tau protein and other

histopathological alterations in the hippocampus at 100x and 400x magnifications. The

healthy control showed no expression of Tau protein and no histopathological changes, while

untreated AD rats showed strong positive expression of Tau protein (immunopositivity

indicated by brown color) and severe necrosis and atrophy of hippocampus neurons. SFN

state as well as the pyknosis in the hippocampus, followed in effectiveness by FBJ, which

itself surpassed the protective effects of the combined NPs.

Table (1): Analysis of active component of tested fresh broccoli juice. Bioactive component

Total glucosinolates 115.82 ± 3.63

Total flavonoids (as quercetin equivalent) 159 ± 8

Total phenolic content (as gallic equivalent) 193

Sulphorafane 210.6

Selenium 0.052±11

Antioxidant activity by DPPH (%) 36.51±0.43/100 mL juice

T ab le ( 2 ): E ff ec ts of u sin g S FN a n d

/or Se N

Ps or FBJ as p re -an d p ost -AD t re at m en ts on b rain b iochem ical m ar k er s of AD d eve lop m en t, m eta b oli c d ysfun ction a n d H DA C ac tivity . HDAC (ng/ m g p rot ein ₎ 97.42 ± 3.59 a 164.66± 5.19 b

107.21± 6.3

c

120.89 _± 4.07

d

117.94

±

5.11

d

111.89 _± 3.33

c

Dat

a is re

p re se n te d as m ean  S D. T h er

e is n

o si gn ificant d iff er en ce b etw ee n me a n s h avin g the same let te r in t h e same c olu m n (p< 0.05) NOS _(pmol/ m in /n g) 4.58 ± 0.69 a 1.87 ± 0.12 b 2.61 ± 0.36 c 2.23 ± 0.8 b ,c 2.33 ± 0.42 b ,c 2.48 ± 0.36 b ,c AD M A (ng/ m g p rot ein ) 79.42 ± 10.69 a 157.66 ± 7.76 b 107.93 ± 9.15 c 127.36 ± 6.99 d 121.76 ± 8.73 d 118.03 ± 11.98 c,d

HCys _{(pmol/ m}in

/n g) 98.96 ± 8.36 a 166.11 ± 6.62 b 110.84 ± 6.46 c 132.85 ± 4.02 d 128.02 ± 3.28 d 120.84 ± 6.01 e L DH

(nmol/ _min

/ml)

84.6 ± 1.24

a

113.11 _± 1.41

b

88.82± 1.8

c

97.72 ± 1.23

d

93.72 ± 0.98

e

91.89± 1.35

f AC h E (m U/ m g p rot ein ) 0.18 ± 0.07 a

0.29 ± 0.05

b

0.21 ± 0.01

c

0.26 ± 0.08

d

0.24 ± 0.01

e

0.23 ± 0.01

e

GFAP _(ng/

m g p rot ein )

98 ± 9.7

a

145.41 ± 10.92

b

107.68 ±

9

a

,c

125.66 ± 6.75

d

114.13 ± 10.34

c

112.11 ± 7.11

c B DN F (pg/ mg p rot ein ₎

8.28 ± 0.26

a

3.48 ± 0.5

b

5.77 ± 0.35

c

4.92 ± 0.47

d

5.37 ± _0.35

c, d 5.48 ± 0.98 c,d A β (p g/ mg p rot ein )

74.08 ± 8.76

a

163.28 ± 19.5

b

102.92 ± 1.2

c

118.22 ± 8.78

d

110.72 ± 3.93

c,d

109.29 ± 3.8

c, d Par ame te rs Group s GI: ( Healt h y con tr ol) GII : ( u n tr eat ed AD r at s) GII I: ( S FN N Ps p re -an d p ost -AD tr eat m en t)

G IV: (

S e NPs p re -an d p ost -AD tr eat m en t) G V: ( S FN + S eNPs p re -an d p ost -AD tr eat m en ts )

G VI: (

T ab le ( 3 ): E ff ec t of u sin g S FN a n d

/or Se N

Ps or FBJ as p re -an d p ost -AD t re a tm en ts on b rain oxid at ive st at u s p ar ame te rs GR (m U/ g )

15.42 ± 1.37

a

8.8

1 ±

0.99

b

12.93 ± 1.31

c, d

11.42 ± 1.18

c

11.83 ± 1.23

c, d

12.5 ± 0.39

d

Dat

a is re

p re se n te d as m ean  S D. T h er

e is n

o si gn ificant d iff er en ce b etw ee n me a n s h avin g the same let te r in t h e same c olu m n (p< 0.05) CAT (U /g)

59.22 ± 3.94

a

31.78 ± 1.07

b

45.95 ± 1.88

c

39.77 ± 4.88

c

41.23 ± 4.06

c, d 44.57± 3.73 d GPX (m U/g )

16.58 ± 0.85

a

6.35 ±

1.49

b

10.38 ± 1.91

c

9.59 ± 3.96

c

9.83 ± 1.84

c

10.32 ± 1.39

c

GS

T

(U

/g)

28.41 ± 2.54

a

14.44 ± 1.57

b

23.5 ± 1.86

c

20.14 ± 2.01

d

22.02 ± 3.55

c, d

22.96 ± 0.94

c S OD (m U/ g )

5.37 ± 0.42

a

2.99 ± 1.06

b

4.78 ± 0.8

c

4.41 ± 0.35

c

4.6 ± _0.32

a

,

c

4.72 ± _0.33

a , c M DA (m m ol/ g) 15.87± 1.36 a 33.95± 1.34 b

22.98 ± 2.97

c

27.78 ± 2.12

d

26.48 ± 3.12

d

25.77 ± 1.46

d GS H (ng/ g )

6.65 ± 0.28

a

3.

11 ± _0.6

b

4.99 ± 0.55

c

4.34 ± 0.53

c

4.67 ± 0.59

c

4.81 ± 0.83

c T AC (m m ol/ g) 39.35± 2.27 a

16.26 ± 3.39

b

27.3 ± 2.87

c,d

21.15 ± 5.57

d

24.13 ± 2.77

c,d

25.04 ± 3.15

c,d Par ame te rs Group s GI: ( Healt h y c on tr ol ) GII : ( u n tr eat ed AD r at s) GII I: ( S FN NPs p re -an d p ost -AD t re at m en t)

G IV: (

S e NPs p re -an d p ost -AD t re at m en t) G V: ( S FN + S eNPs p re -an d p ost -AD t re at m en ts )

G VI: (

T ab le ( 4) : Eff ec t of u sin g S FN a n d

/or Se N

Ps or FBJ as p re -an d p ost -AD t re at m en

ts on b

rain in fla m m at or y an d ap o p tot ic m ar k er s CSP -3 (ng/ g) 2 ± 0.19 a

3.63 ± 0.59

b

2.13 ± 0.57

a

2.92 ± 0.34

c

2.53 ± _0.71

a

,

c

2.42 ± 0.62

a

,c

Dat

a is re

p re se n te d as m ean  S D. T h er

e is n

o si gn ificant d iff er en ce b etw ee n me a n s h avin g the same let te r in t h e same c olu m n ( p < 0.05) IL 1 -  (ng/ g) 19.82 ± 0.95 a 39.43 ± 7.66 b 24.15 ± 3.01 a ,c 29.06 ± 2.13 d 27.33 ± 1.72 c,d 26.58 ± 3.26 c,d T NF - (ng/ g)

6.78 ± 0.44

a

19.58± 4.5

b

11.42 ± 3.76

c

13.35 ± 1.61

c

12.94 ± 1.82

c

12.39 ± 2.31

c Par ame te rs Group s GI: ( Healt h y c on tr ol) GII : ( u n tr eat ed AD rat s) GII I: ( S FN N Ps p re -an d p ost -AD tr eat m en t)

G IV: (

S e NPs p re -an d p ost -AD t re at m en t) G V: ( S FN +SeN Ps p re -an d p ost -AD tr eat m en ts)

G VI: (