الفهرس 
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Abstract
Neurodegenerative diseases (NDs) of the central nervous system include, but are not limited to, Alzheimer’s disease (AD) and are given the umbrella term; Dementia. Occurrence of NDs is increasing all over the word due to the increasing proportion of old age population and their development is systematic and finally leads to death.
Genetic and environmental factors also play an important role in the progression of NDs. While common pathological features of these diseases are accumulation of certain aggregated proteins, oxidative stress and neuroinflammation, disruption of several metabolic pathways have been also thought to partly represent the cascades underlying their characteristic epigenetic alterations.
Due to the complexity of these multifactorial diseases, current treatment strategies are only useful as symptomatic relief agents rather than curbing the actual disease progression and they are not without side effects. Thus it is necessary to develop new, safe and more effective preventive strategies to combat these devastating diseases.
Consequently, the use of phytochemicals or herbal medicines in fighting chronic diseases, in general, and particularly NDs is being thoroughly investigated as they are shown to be protective against oxidative stress and neuroinflammation.
However, more futuristic approaches combine phyto and nanomedicines where the nanosized phytochemicals have increased efficiency and delivery to the target sites by several folds. This emerging feat of tailored nanomedicines represents the most promising areas of interest in the prevention of NDs, especially AD.
Hence, this study aimed to explore the neuroprotective potential of SFN and/or Se nanoparticles (NPs) as well as fresh broccoli juice (FBJ), against the metabolic, epigenetic, antioxidant, inflammatory and apoptotic changes accompanying the AlCl3-induced neurotoxicity, that may lead to the development of Alzheimer’s disease (AD) in rats.
This study had two broad sections:
First, SFN and Se nanoparticles (NPs) were characterized by Dynamic Light Scattering (DLS) and High Resolution-Transmission Electron Microscopy (HR-TEM). DLS revealed that the average hydrodynamic diameter of SFN NPs was approximately 55.8nm, while that of Se NPs was approximately 31 nm. HR-TEM analysis showed the quazi circular morphology of both NPs and confirmed that their sizes were within the DLS size distribution.
Analysis of bioactive components and the total antioxidant activity of (FBJ) revealed that it contained impressive amounts of flavonoids, glucosinolates, and other phenolic compounds.
The second part included the animal trial. Sixty 12 months old Spargue-Dawley rats weighing 290±10g were used after 7 days of acclimatization period. Animals were divided into 6 groups 10 rats each. G1: Healthy control, G2: untreated AD rats, G3: SFN NPs (0.5 mg/kg body weight/day), G4: Se NPs (0.5 mg/kg body weight/day), G5: SFN +Se NPs (0.25+ 0.25 mg/kg body weight/day) and G6: FBJ (8.33 mg/kg body weight/day). The nutraceuticals were used as pre/treatments; rats were first solely given oral doses of the tested neutraceticals for 2 weeks, then started receiving daily i.p. injections of AlCl3.6H2O (70 mg/kg body weight/day) while continuing to receive the tested nutraceuticals along with the AlCl3 for the next 5 weeks. Our results illustrated that the pre/treatments significantly exhibited neuroprotective actions;
1. At the end of the 7 weeks animal trial behavioral assessments were performed. Results of Morris water maze test and conditioned avoidance test showed that there was a significant (P <0.05) elevation in learning ability and memory function in pre/treated rat groups as compared to untreated AD rats. SFN NPs and FBJ were able to preserve the cognitive abilities of rats to remain comparable to that of the healthy control. Combined SFN +Se NPs trailed in their effectiveness followed by the singular Se NPs.
2. Shortly after the last behavior test, the animals were sacrificed and the effects of the tested pre/treatments were first examined by analysis of the total brain weight. All tested pre/treatments significantly (P <0.05) attenuated the loss of BBB integrity, and the resulting brain edema when compared to the untreated AD rats. These effects were most likely established through direct activation of the intrinsic antioxidant system as well as other mechanisms.
3. The levels of 3 major biochemical markers of AD development were determined. These were beta amyloid (A) peptide level, the neurotropine: brain derived-neurotrophic factor (BDNF) level and also level of the plaque cytoskeleton intermediate-filament protein; the glial fibrillary acidic protein (GFAP). Levels of A and GFAP were significantly (P< 0.05) decreased, while BDNF was elevated by all tested neutraceticals, most notably SFN NPs possibly due to their antioxidant profiles and their ability to quench cytotoxic free radicals; preventing multiple neuritic injuries including astrogliosis.
4. As for the effect of the tested pre/treatments on markers of metabolic dysfunction; the AlCl3 induced elevations in Acetylcholine esterase (AChE) activity, Lactate Dehydrogenase (LDH) activity, accumulated Homocysteine (HCYs) level and Asymmetric dimethylarginine (ADMA) level in untreated rats were effectively (P< 0.05) diminished in comparison by the neutraceuticals administration while Nitric oxide synthase (NOS) activity was positively elevated. SFN NPs surpassed the other tested pre/treatments in their protective effects on cholinergic transmission and metabolic functions as both are thought to be the main etiologic factors of the established memory deficit and cognitive impairment.
5. Results of the epigenetic markers confirmed that AlCl3-induced neurotoxicity was accompanied by a state of global DNA hypomethylation and histone hypoacetylation. These were instigated by a significant (P<0.05) reduction in DNA methyltransferase-1 (DNMT-1) gene expression and the elevation in histone deacetylase (HDAC) activity in untreated AD rats. On the contrary, the tested pre/treatments were able to significantly (p<0.05) restore the relative expression of DNMT-1 while expressively inhibiting the HDAC activity, when compared to untreated AD rats. Therefore, the role of those pre/treatments in orchestrating several neurobiological pathways was suggested to be dependent, at least in part, on the epigenetic regulation of AD related genes’ expression.
6. Concerning the oxidative stress markers, all tested pre/treatments significantly (p<0.05) neutralized the A and the mitochondrial dysfunction induced pro-oxidative state by impressively mitigating the plop down of the Heme oxygenase-1 (HO-1) mRNA expression and consequently elevating the levels of its enzymatic and non-enzymatic downstream antioxidant targets. In the present results, SFN NPs were shown to be the most effective HO-1 activators, endorsing their previously established role as potent inducers of its upstreatm promotor; the nuclear factor erythroid 2-related factor 2 (Nrf2). FBJ was also remarkably effective oxidative stress modulator due to its content of appreciable array of antioxidants. Se NPs were shown to significantly impart their antioxidant effects on the endogenous antioxidant system as well, though admittedly less pronounced when administered as a singular treatment. Concisely, SFN and/or Se NPs as well as FBJ significantly maintained the total antioxidant activity in the pre/treated rat groups by abolishing the accumulation of malondialdehyde and increasing the level of reduced glutathione as well as the activity of glutathione-S-transferase, superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase when compared to the untreated AD rats.
7. The results also confirmed 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 unchecked level of the proinflammatory cytokines; tumor necrosis factor- and interleukin 1- as well as the apoptotic marker; caspase-3 were all diminished in the pre/treated rat groups as compared to the untreated AD rats. The overwhelming consensus of the superiority of SFN NPs to the other pre/treatments were soundly confirmed by their effects on these markers as well as the percentage of DNA fragmentation.
8. Collectively, all previous results were supported by the demonstration of the effects of the tested pre/treatments on immunohistochemical and histopathological changes in the hippocampus. SFN NPs were able to lower the burden of accumulated Tau protein and also diminish the necrotic state as well as the pyknosis in the hippocampus. It was followed in effectiveness by FBJ, which itself surpassed the protective effects of the combined NPs and in turn the singular Se NPs.
9. All in all, our results established the remarkable neuroprotective effect of SFN NPs (G3), most likely by virtue of their small size and characteristics; enhanced bioavailability, absorption, uptake and delivery across the BBB. Following, the effectiveness of FBJ (G6) was considered to be due to its established content of various phytochemicals and its impressive antioxidant activity. Moreover, SFN and Se NPs (G5) combination was proven to be able to successful at nullifying the effects AlCl3 induced neurotoxicity, owing to the added neuroprotective effects of Se NPs to those established for SFN NPs which was clearly the all-around better neuroprotective agent. Nonetheless, Se NPs (G4) was marginally less effective when used as a singular pre/treatment.