Buriachenko Semen
RESERCH OF THE NEUROPROTECTOR EFFECT OF NANOCRYSTALS
HALLOYSITE ON THE RESTORATION FUNCTION OF NEURONS
MICE BRAIN
NSC Institute of Experimental and Clinical Veterinary Medicine
of the NAAS of Ukraine
The neuroprotective interaction of halloysite nanocrystals on the restoration of
neurons, stimulating effect on central dopamine receptors, inhibition of aggregation
of amyloid proteins. Halloysite molecules are characterized by high lipophilicity,
strong electron affinity, large spheroid surface, and the ability to self-associate with
the formation of clusters, which determines its many biological effects. Pronounced
neuroprotective properties of halloysite nanocrystals are shown.
Key words: neuroprotective, nanocrystals, halloysite, antioxidant, nanotubes,
Amyloidogenesis
1. Introduction
Alzheimer's disease is a progressive neurodegenerative disease, the most
common form of dementia. The course of the disease is characterized by a steady
deterioration lasting for years. The patient's condition, rapid disability with the loss
of the ability to live independently and, ultimately, the death of neurons [1]. In within
the framework of the most common amyloid hypothesis, it is believed that the
peptide plays an important role in triggering irreversible changes in the patient's
brain Aβ42. This form is capable of forming oligomers and insoluble accumulations
of a significant number of monopeptides in the structure of the beta-fold, which are
called amyloid plaques [2]. Amyloid plaques cause pathological changes in the brain
patient, which are manifested in the formation of neurofibrillary tangles, impaired
synaptic transmission, neuronal death, and resulting dementia [3]. According to
modern concepts, Aβ42 triggers a complex set of processes at the biochemical and
cellular levels, which ultimately lead to neurodegenerative changes in the brain [4].
To reduce the level of Aβ42 a search is underway for drugs that prevent it formation
in the brain or remove already formed plaques in the tissues. These studies are being
carried out in three main areas: how to prevent the formation of Aβ42, how to clear
already accumulated plaques of Aβ42 and how to prevent Aβ42 oligomerization. In
1995, researchers succeeded to develop a line of transgenic mice with a mutant
human APP gene, in the brain of which amyloid plaques accumulated. These mice
performed worse on tasks that required memorization information, and they have
become a model for studying the effects of promising anti-amyloid drugs [5]. So far,
however, no drugs tested in mice have been shown to be effective in humans. One
of the possible reasons for the failure. The transfer of results from studies in mice to
humans may be the difference in the neurochemistry and pathophysiology of mouse
and human neurons. In this regard, studies of the fundamental mechanisms of AD
pathogenesis are of great importance. Understanding the molecular basis of
pathology is necessary not only for the development of therapeutic methods, but also
early diagnosis, which is extremely difficult due to the long course of the
asymptomatic stage of the disease. The plaques are formed mainly by beta-amyloid
peptide (Aβ) having a molecular weight 4 kDa and about 40 amino acid residues
long [6]. Aβ is a fragment of the transmembrane beta-amyloid precursor protein APP
(amyloidprecursorprotein) found in many tissues, including synapses of neurons.
Strategies for creating drugs aimed at preventing the accumulation of amyloid
plaques in Alzheimer's disease, include a decrease in the concentration of
amyloidogenic proteins [7].
2. Literature review
One of the factors leading to death nerve cells and cognitive impairment is the
pathological accumulation in the brain tissue of aggregates of βA peptide, which is
the main component of senile plaques - characteristic morphological signs of AD.
Aβ, being a product of proteolytic cleavage of APP, has pronounced fibrillogenic
properties, and its oligomers are toxic to nerve cells, causing them degeneration and
death [8]. This is due to the formation of highly organized fibrillar aggregates by
improperly folded proteins or peptides, or amyloid fibrils [9]. Amyloid fibrils are
long β-pleated sheets in which β-strands are perpendicular to the fibril axis (crossβ-structure). In connection with pathological and functional role of amyloid fibrils,
studies aimed at improving and search for methods for detecting fibrillar aggregates
and their recovery with the possibility of correcting the neuroprotective effect.
Neurotoxicity amyloid peptides is manifested by a violation of Ca2+ - homeostasis,
induction of oxidative stress, excitotoxicity, inflammatory processes, intensification
of apoptosis. The latter effect can be realized, in particular, by inducing the opening
of mitochondrial pores. In the latter case, cell death can also occur by the mechanism
of necrosis [10]. This disease is characterized by mutations of the mentioned genes
- APP, PSN-1 and PSN-2. Risk of sporadic form beta-amyloid proteins are currently
associated with increased expression in brain tissues of the gene apolipoprotein E
(APOE). It was shown that APOE binds to Aβ and together with it forms senile
plaques [11].
3. Goals and objectives of the article
The purpose of the study is to study the neuroprotective and antioxidant properties
of nanotubular halloysite with the possibility of creating a new neuroprotective drug
for the treatment and prevention of neurodegenerative diseases of the brain based on
the composition of halloysite nanocrystals.
To achieve the goal, the following tasks were set:
1) to study the therapeutic effect on neurogenesis of neurons when using halloysite
nanocrystals;
2) to reveal the possibility of antioxidant protection of neurons from free radicals;
3) to study the molecular process of restoring neurons with halloysite nanocrystals;
4) to reveal the restoration of the synthesis of amyloid proteins;
5) to study the ability of halloysite nanocrystals to restore mutated APP, PSN-1
genes
and PSN-2;
6) to study the biochemical pathway of the influence of nanotubular halloysite on
neurogenesis.
4. Materials and methods
To achieve the results we in laboratory mice of the C57BL/6 line, a defect in the
aggregation of ß-amyloid proteins in the brain was reproduced, followed by the
death of neurons and a mutation in the APP, PSN-1 and PSN-2 genes. Got modified
nanotubular halloysite. Neuroprotective effect of nanocrystals halloysite on neurons
due to binding oxygen free radicals and antioxidant action. We investigated
halloysite nanocrystals for the possibility of antioxidant effects and the impact of
neuronal recovery in treatment neurodegenerative diseases in an experiment in
laboratory animals. Were first identified biological properties and molecular
mechanisms the influence of halloysite nanocrystals on the correction of cognitive
processes, the restoration of spatial memory in the brain and the restoration of
protein synthesis processes, amyloidogenesis. Halloysite nanocrystals are known to
have a unique spatial and electronic structure that causes pronounced donoracceptor, biological and photophysical properties. Halloysite is one of the common
clay minerals. The halloysite formula is Al2Si2O5(OH)4 x nH2O, where n=0 and 2,
however, the chemical composition of the mineral may vary slightly. The structure
and chemical composition of halloysite are similar to those of kaolinite, dikite, or
nakrite, but aluminosilicate layers in halloysite are separated water molecules, which
is the main difference. The water interlayer in halloysite is weakly bound and gives
a distance of about 10 Å between the layers, at dehydration of halloysite, we obtain
its 7 Å form, which is very close to kaolinite, as previously reported [1]. Any form
of halloysite (hydrated or dehydrated) has a greater tendency to intercalate organic
molecules. Halloysite particles can take various forms, the most common of which
is elongated tubule. Dominant the form of halloysite is tubular. Another form is
spherical halloysite with ball diameters ranging from 0.05 to ~0.50 mm. Pseudospherical or spherical particles are often found in volcanic ash and pumice [2]. The
studies used black laboratory mice C57BL/6 20 months of age in the number of 60
animals in which previously in at the age of 3 months, a defect in the aggregation of
ß-amyloid proteins in the brain was reproduced, followed by the death of neurons.
Animals were divided into experimental and control groups. In mice, attacks with
loss of consciousness, loss of sensitivity, death of neurons, increased excitability,
and neuropathy were observed. Line mice C57BL/6 were injected into the tail vein
twice a day halloysite nanocrystals pre-modified with montmorillonite. The
concentration of the nanoparticle solution was 0.1%. The length of the nanotubes
did not exceed 500 nm. In this work, there were studies have been carried out on
changes in amyloid after application of halloysite nanocrystals using fluorescent
detection and confocal microscopy to analyze changes in amyloid proteins in the
brain of mice. Conducted fluorescence detection amyloid aggregates. Fibrils were
obtained from the hippocampus, hemoglobin, and ribonuclease by incubating
proteins under denaturing conditions (glycine buffer, pH=2.0, 60°C) for two weeks.
Staining was carried out using the classical amyloid marker Thioflavin T (ThT).
Method fluorescence microscopy have been found suprafibrillar aggregates single
fibrils these proteins. These aggregates are presumably accumulations of amyloid
fibrils, which are formed in the process of their “aging”. Next, a confocal
microscopy was performed. Analysis of aggregation and localization of proteins
fused with blue CFP fluorescent protein and yellow fluorescent protein YFP, was
performed using a laser scanning confocal microscope Leica TCS SP5 “Leica
Microsystems GmBH” (Germany) and laser scanning confocal microscope Leica
TCS SP5 MP “Leica Microsystems GmBH” (Germany). Conditions: for CFP, 458
nm excitation laser, 462–510 nm cut-off filter, for YFP - excitation laser 514 nm,
blocking filter 518–580 nm, for DAPI – excitation laser 405 nm, blocking filter 425475 nm. Microscopy was performed on the second day of obtaining the material. To
prepare micropreparations, hippocampal cells were suspended in sterile water and
evenly distributed over the surface of the subject glasses “Polylysineslides”
(“Gerhard Menzel GmBH”, Germany). After the water dried, the preparations were
placed in VECTASHIELD Mounting Media. (“Vector Laboratories”, USA) or
“VECTASHIELD Mounting Media with DAPI” (“Vector Laboratories”, USA) for
DAPI staining, and covered cover glasses. To calculate the frequency of aggregation,
the number cells in which aggregates were detected were dividingon the total
number of cells in which fluorescent protein signal. For determining the frequency
of localization of aggregates, cells were selected, in which the signals of both studied
proteins were present, and the number of cells was determined, in which localization
of these signals coincides. At 1st, 3rd, 7th and 15th days of the study were carried
out blood sampling from the tail vein for determination concentrations of 8,12 isoprostane (IPF2A) by enzyme immunoassay in blood plasma, which is an
important biomarker product of fatty acid peroxidation and proportionally associated
with the level of Aβ. In parallel, we used intelligence and search tests exiting the
maze, trying to figure out if I've improved memory halloysitis and whether it
affected the number of plaques in hippocampal neurons, on the restoration of
cognitive functions.
5. Results of the study and their discussion
With the help of fluorescent detection and confocal microscopy, it was found
that the percentage of viable cells was 98% after the application of nanocrystals.
When studying the pharmacokinetics of halloysite nanocrystals, it was found that
after subcutaneous administration to mice at a dose of 5 ng/g of body weight rapidly
increased its concentration in the blood and in parallel in the cerebrospinal fluid,
which says about its free passage through the blood-brain barrier. In adult animals,
it increased the number of neuronal mitoses in the subventricular zone and olfactory
tracts, regulating current neurogenesis in a specific humoral way. Thus, halloysite
nanocrystals carry out a complex regulation of the generation of de novo brain cells.
A decrease was found amyloid aggregates from 98.9% to 2%. Halloysite
nanocrystals penetrate the cell and its cytoplasm, where they penetrate directly
through the cell membrane without damaging it. At In this case, the substance does
not show specific tropism for the organelles of neural progenitors. Nanocrystals are
completely metabolized in cells to water and molecular oxygen. A large number of
cells with a lack of signal localization indicates the death of neurons, impaired cell
metabolism, cell apoptosis, and impaired neuronal conduction of hippocampal cells.
Violation of calcium transport and accumulation of oxygen-containing radicals
initiates apoptosis and necrosis of neurons in the hippocampus and brain. Exactly
therefore, we began to study the possibility of influencing first of all for biochemical
correction of the system calcium transport and reduced exposure of free radicals to
neurons. These requirements are met by nanocrystalline halloysite modified with
montmorillonite, which has the ability to absorb free oxygen species. The
appearance of nanohalloysite in the brain really improved the memory of rodents
and cleared them hippocampus from fragments of beta-amyloids. First We have seen
positive effects on the first day after administration of nanohalloysite to mice. On
average, after 3 days, the concentration of beta-amyloids in the blood of animals
decreased from 127% to 60%, after 7 days up to 37%, after 15 days there were traces
of beta-amyloid. We believe that halloysite stimulates the production of a peptide the enzyme neprilysin, which is involved in the lysis of fragments of incorrectly
assembled peptides, including plaques from APP protein residues. Nanohalloysite
reduces the amount and at the same time time doubles the number of newly formed
neurons in the hippocampus. It has been shown to reduce inflammation and
oxidative stress in the brain hippocampus accompanying the disease.
6. Conclusions
The results of the study were the following data supporting neuroprotective
effects halloysite nanocrystals on mouse hippocampal neurons to reduce amyloid
proteins.
1. Change analysis data were obtained amyloid aggregates after application of
halloysite nanocrystals.
2. Proven to reduce the synthesis of amyloid proteins and their destruction.
3. The structure of neurons is restored.
4. It has been shown that under in vivo conditions the amount neurons with the
introduction of water-soluble nanocrystals of halloysite at a concentration of 20 nm
increases by almost 2 times compared with the control. At the same time, their
proliferative activity is preserved in throughout the entire study period.
5. The formation of neurospheres decreases.
6. Confocal ultrastructural analysis showed that halloysite nanocrystals penetrate
into cell through the cell membrane without damaging it, and localized in the
cytoplasm of the cell. This allows use nanocrystals in biotechnology and gene
therapy for diseases such as Alzheimer's, Parkinson's and epilepsy.
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