Neuroprotective effect of 6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline mediated via regulation of antioxidant system and inhibition of inflammation and apoptosis in a rat model of cerebral ischemia/reperfusion

Article abstract of DOI:10.1016/j.biochi.2021.04.010

The aim of the study was the assessment of the neuroprotective potential of 6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline (DHQ) and its effect on inflammation, apoptosis, and transcriptional regulation of the antioxidant system in cerebral ischemia/reperfusion (CIR) in rats. The CIR rat model was constructed using the bilateral common carotid artery occlusion followed by reoxygenation. DHQ was administered at a dose of 50 mg/kg for three days. Histological staining was performed using hematoxylin and eosin. The level of S100B protein, 8-hydroxy-2-deoxyguanosine, and 8-isoprostane was assessed using an enzyme immunoassay. The intensity of apoptosis was assessed based on the activity of caspases and DNA fragmentation. The activity of enzymes was measured spectrophotometrically, the level of gene transcripts was assessed by real-time PCR. DHQ reduced the histopathological changes and normalized levels of S100B, lactate, pyruvate, and HIF-1 mRNA in the CIR rat model. In addition, DHQ decreased the oxidative stress markers in animals with a pathology. The tested compound also inhibited inflammation by decreasing the activity of myeloperoxidase, expression of interleukins and Nfkb2. DHQ-treated rats with CIR showed decreased caspase activity, DNA fragmentation, and AIF expression. DHQ changed activity of antioxidant enzymes to the control values, decreased the expression of Cat, Gsr, and Nfe2l2, which was overexpressed in CIR, and activated the expression of Sod1, Gpx1, Gsta2, and Foxo1. DHQ showed a neuroprotective effect on CIR in rats. The neuroprotective effect involve mechanisms such as the inhibition of oxidative stress, leading to a reduction in the inflammatory response and apoptosis and the modulation of the antioxidant defense components.

Full text of DOI:10.1016/j.biochi.2021.04.010

Biochimie 186 (2021) 130e146
Contents lists available at ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Neuroprotective effect of 6-hydroxy-2,2,4-trimethyl-1,2-
dihydroquinoline mediated via regulation of antioxidant system and
inhibition of inflammation and apoptosis in a rat model of cerebral
ischemia/reperfusion
E.D. Kryl'skii a, , E.E. Chupandina , T.N. Popova , Kh.S. Shikhaliev , V.O. Mittova ,
*
b
a
c
d
e
a
b
S.S. Popov , A.N. Verevkin , A.A. Filin
Department of Medical Biochemistry and Microbiology, Voronezh State University, Russia
Department of Pathological Anatomy, Voronezh State Medical University named after N.N. Burdenko, Russia
Department of Organic Chemistry, Voronezh State University, Russia
Department of Biochemistry, Voronezh State Medical University named after N.N. Burdenko, Russia
Department of Organization of Pharmaceutical Business, Clinical Pharmacy and Pharmacognosy, Voronezh State Medical University named after N.N.
a
b
c
d
e
Burdenko, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 March 2021
Received in revised form
The aim of the study was the assessment of the neuroprotective potential of 6-hydroxy-2,2,4-trimethyl-
1,2-dihydroquinoline (DHQ) and its effect on inflammation, apoptosis, and transcriptional regulation of
the antioxidant system in cerebral ischemia/reperfusion (CIR) in rats.
21 April 2021
The CIR rat model was constructed using the bilateral common carotid artery occlusion followed by
reoxygenation. DHQ was administered at a dose of 50 mg/kg for three days. Histological staining was
performed using hematoxylin and eosin. The level of S100B protein, 8-hydroxy-2-deoxyguanosine, and
Accepted 29 April 2021
Available online 5 May 2021
8
-isoprostane was assessed using an enzyme immunoassay. The intensity of apoptosis was assessed
Keywords:
based on the activity of caspases and DNA fragmentation. The activity of enzymes was measured
spectrophotometrically, the level of gene transcripts was assessed by real-time PCR.
Cerebral ischemia/reperfusion
Oxidative stress
Dihydroquinoline derivatives
Inflammation
Apoptosis
Antioxidants
DHQ reduced the histopathological changes and normalized levels of S100B, lactate, pyruvate, and HIF-
1 mRNA in the CIR rat model. In addition, DHQ decreased the oxidative stress markers in animals with a
pathology. The tested compound also inhibited inflammation by decreasing the activity of myeloper-
oxidase, expression of interleukins and Nfkb2. DHQ-treated rats with CIR showed decreased caspase
activity, DNA fragmentation, and AIF expression. DHQ changed activity of antioxidant enzymes to the
control values, decreased the expression of Cat, Gsr, and Nfe2l2, which was overexpressed in CIR, and
activated the expression of Sod1, Gpx1, Gsta2, and Foxo1.
DHQ showed a neuroprotective effect on CIR in rats. The neuroprotective effect involve mechanisms
such as the inhibition of oxidative stress, leading to a reduction in the inflammatory response and
apoptosis and the modulation of the antioxidant defense components.
©
2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights
reserved.
1. Introduction
energy deficiency in the ischemic area. Reoxygenation from reper-
fusion following ischemia can lead to additional tissue damage [2].
Strokes are the second leading cause of death worldwide, killing
The excessive generation of reactive oxygen species (ROS) in the
mitochondrial electron transport chain is considered to be the main
mechanism of neuronal cell death during reperfusion [3,4].
Cell protection against reactive molecules is provided by antiox-
idant enzymes [5]. The main transcriptional regulator of antioxidant
protection in mammals is the NF-E2-related factor 2 (Nrf2)-Kelch-
like ECH-associated protein 1 (Keap1) system. Under oxidative stress
about 6.7 million people every year [1]. Strokes occur due to
impaired cerebral circulation, leading to a decrease in oxygen levels,
the inhibition of oxidative phosphorylation in mitochondria, and
*
Corresponding author. 394018, Universitetskaya sq. 1, Voronezh, Russia.
E-mail address: krylskiy@bio.vsu.ru (E.D. Kryl'skii).
https://doi.org/10.1016/j.biochi.2021.04.010
300-9084/© 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
0

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Abbreviations
DC
diene conjugates
myeloperoxidase
MPO
TMB
SOD
NBT
GP
0
0
ROS
reactive oxygen species
3,3 ,5,5 -tetramethylbenzidine
superoxide dismutase
nitro blue tetrazolium
glutathione peroxidase
glutathione reductase
Nrf2
NF-E2-related factor 2
Keap1
AREs
FOXO1
Kelch-like ECH-associated protein 1
antioxidant responsive elements
forkhead box protein O1
GR
NF-kB
nuclear factor kappa B
GT
glutathione-S-transferase
DHQ
CIR
PASS
LD50
6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline
cerebral ischemia/reperfusion
Prediction of Activity Spectra for Substances
lethal dose 50
8-hydroxy-2-deoxyguanosine
biochemiluminescence
G6PDH
glucose-6-phosphate dehydrogenase
NADP-IDH NADP-isocitrate dehydrogenase
GSH
AIF
HIF-1
DAMP
PPP
reduced glutathione
apoptosis inducing factor
hypoxia inducing factor
damage associated molecular patterns
pentose phosphate pathway
aconitate hydratase
8
OHDG
BChL
POM
protein oxidative modification
2
,4-DNPH 2,4-dinitrophenylhydrazine
AH
TCA
trichloroacetic acid
conditions, the Nrf2-Keap1 complex is dissociated, the released Nrf2
translocates to the nucleus, heterodimerizes with one of the small
Maf proteins and recognize AREs (antioxidant responsive elements),
that are enhancer sequences present in the regulatory regions of
Nrf2 target genes [6]. Another regulatory protein playing an impor-
tant role in cell survival under oxidative stress is the transcription
factor forkhead box protein O1 (FOXO1) [7,8].
derivatives. One of the selected compounds, 6-hydroxy-2,2,4-
trimethyl-1,2-dihydroquinoline (DHQ), was synthesized and
tested by us as a neuroprotective agent.
In this study, we assessed the neuroprotective and antioxidant
potential of DHQ, and examined its effect on the inflammation,
apoptosis, and transcriptional regulation of the antioxidant system
in rats with cerebral ischemia/reperfusion (CIR).
Inflammation plays a key role in the pathogenesis of ischemia/
reperfusion. After ischemic injury, brain endothelium and astrocytes
secrete large amounts of chemokines and cytokines. These agents
stimulate the expression of adhesion molecules on the endothelium,
leading to the leukocyte adhesion and the degradation of endothe-
lium tight junction proteins and the degradation of the extracellular
matrix. The damaged blood-brain barrier promotes both the pene-
tration of peripheral inflammatorycells into the brain and an increase
in the secretion of mediators, leading to permanent damage to the
2. Materials and methods
2.1. DHQ synthesis
The synthesis of DHQ was carried out according to a well-known
method
by
dealkylation
of
6-ethoxy-2,2,4-trimethyl-1,2-
dihydroquinoline (ethoxyquin) when exposed to hydrobromic acid
(Appendix 1) [13]. The structure of the synthesized compounds was
confirmed by NMR, IR spectroscopy, and gas chromatography-mass
spectrometry.
barrier [9]. The NF-kB (nuclear factor kappa B) transcriptional acti-
vation pathway is considered the main mediator of inflammatory
responses in nervous and vascular tissues. In a steady-state condition
heterodimeric NF-
tory factor I in the cytoplasm. The I
phorylated and activated aftercerebralischemia/reperfusion, causing
phosphorylation and degradation of I . Thus, the p65/p50 dimer is
released and transported into the nucleus, where it promoting the
expression of proinflammatory cytokines [10].
Cerebral ischemia and reperfusion can activate various cell
death pathways such as necrosis, apoptosis, or autophagy. Among
these, apoptosis is considered a key event in ischemic brain dam-
age. The activation of classical apoptosis occurs in two main ways.
One of these is the ligand-mediated pathway, which is initiated by
the activation of cell surface death receptors such as Fas, and
leading to the activation of caspase-8 or caspase-10. The other is the
mitochondrial apoptosis pathway, which originates from the
mitochondrial release of cytochrome C and subsequent activation
of caspase-9. Both pathways lead to signaling cascades that
converge in the activation of effector caspase-3, which ultimately
leads to apoptosis [11].
k
B p65/p50 complexes are bound with the inhibi-
2.2. Prediction of biological activity spectra and toxicity of DHQ
kBa
kB kinase is rapidly phos-
The biological activity of dihydroquinoline derivatives was
assessedusingthePredictionofActivitySpectraforSubstances(PASS)
biological activity analysis program [14e17]. The prediction of the
toxicityof the compound wascarried out by in silico analysis using the
PROTOX program [18,19]. This program predicts the lethal dose
(LD50) of the tested compound. According to the analysis, all sub-
stances are classified into six Globally Harmonized System of Classi-
fication and Labeling of Chemicals categories, depending on the
toxicity of the compound: Category I: LD50 ꢀ 5 mg/kg; Category II:
5 < LD50 ꢀ 50 mg/kg; Category III: 50 < LD50 ꢀ 300 mg/kg; Category
IV: 300 < LD50 ꢀ 2000 mg/kg; Category V: 2000 < LD50 ꢀ 5000 mg/
kg; Category VI: LD50 > 5000 mg/kg.
kBa
2.3. Animals and CIR model
Male Wistar rats 4e6 months old and weighing 200e250 g were
The analysis of compounds possessing simultaneously high
antioxidant and anti-inflammatory potential is expedient, since
oxidative stress plays a central role in the pathogenesis of ischemia/
reperfusion. These compounds include antioxidant ethoxyquin (6-
ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) [12]. Our in silico
analysis revealed more promising compounds for drug precursors
with the higher antioxidant activity among the dihydroquinoline
used in this study. Rats were kept under conditions of a 12 h light/
dark cycle. The animals were provided with food and water ad
libitum. The study's protocols were approved by the Institutional
Animal Care and Use Committee of Voronezh State University
(Voronezh, Russia) and correspond to EU Directive 2010/63/EU for
animal experiments. For the induction of CIR, anesthetized rats
were placed in a supine position on an operating table. The body
131

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
temperature of each rat was maintained with an incandescent
lamp. After shaving, a middle incision was made to expose both
common carotid arteries. The dissection was performed between
the sternocleidomastoid and sternohyoid muscles parallel to the
trachea. The incision area was regularly treated with novocaine.
Each common carotid artery was dissected from the adventitia
membrane and carefully detached from the vagus nerve. For the
induction of ischemia, both common carotid arteries were clamped
with a silk thread for 30 min, followed by reperfusion [20]. The
restoration of blood flow was monitored visually.
membrane and shrunken cytoplasm, surrounded by perineuronal
vacuoles, neuron size alteration, and triangular shape of the cells.
2.8. Assessment of the oxidative stress intensity
The oxidative stress intensity and the total antioxidant activity
in the sample were measured by biochemiluminescence (BChL)
induced by hydrogen peroxide with iron sulfate [21]. The BChL ki-
netic curve was recorded for 30 s using a BChL-07 bio-
chemiluminometer (Medozons, Russia), and the following
parameters were determined: the light sum of chemiluminescence
(S), maximum intensity (Imax), tangent of the BChL kinetic curve
2.4. Experimental design
slope (tg
a
2
). The reaction medium contained 0.4 ml of 0.02 mM
and
Groups (n ¼ 10 per group) were formed by the random
potassium phosphate buffer (pH 7.5), 0.4 ml of 0.01 mM FeSO
4
assignment of rats as follows: group 1 (control) of sham-operated
animals received 0.5 ml of physiological solution intraperitone-
ally; group 2 (CIR) of rats with CIR; group 3 (CIR þ DHQ) of rats
intraperitoneally administrated with DHQ at a dose of 50 mg/kg
dissolved in 0.5 ml of saline, 3 h after induction of CIR with an in-
terval of 24 h; group 4 (control þ DHQ) of sham operated rats
received DHQ at a dose of 50 mg/kg according to the above scheme.
The dosage of the tested compound was chosen based on the
therapeutic doses of the nootropic piracetam and the experimental
animals were dosed according to their body weight. On the fourth
day from the beginning of the experiment, rats were sacrificed,
brain and blood were extracted and immediately used for
biochemical analysis. In each experiment, the brain and blood of
two experimental animals were examined. Each investigated in-
dicator in the samples was analyzed in triplicate. The scheme of the
experiment is shown in Fig. 1.
0.2 ml of a 2% hydrogen peroxide solution introduced immediately
before the measurement. The test material was added in a volume
of 0.1 ml before the introduction of hydrogen peroxide.
The method of Reznick et al. [22] with minor modifications was
used for the analysis of the protein oxidative modification (POM). The
method is based on the ability of the carbonyl amino acid residues
react with 2,4-dinitrophenylhydrazine (2,4-DNPH) with the forma-
tion of 2,4-dinitrophenylhydrazones. Briefly, the sample was diluted
with 100 mM phosphate buffer (pH 7.4), then 10 mM 2,4-DNPH dis-
solved in 2.5 M HCl was added, the mixture was incubated for 1 h and
then 20% trichloroacetic acid (TCA) was added. After cooling, the
samples were centrifuged at 3000 g, the protein precipitate was
washedwith10%TCA and amixtureofethanolandethylacetate(1:1),
and then dissolved in 2 ml of 8 M urea. The optical density of the
experimental sample was measured at 370 nm relative to the control
sample treated with 2.5 M hydrochloric acid. The molar extinction
ꢁ
1
ꢁ1
coefficient
x
¼ 22.000 cm ꢂ M was used for the calculation of the
2
.5. Assessment of the brain metabolic state of laboratory animals
content of carbonyl amino acid groups in proteins (nM).
For the analysis of the diene conjugates (DC) concentration,
heptane and isopropanol were added to the test sample, mixed and
precipitated by centrifugation at 3000g. The heptane phase of the
supernatant was diluted with ethanol and analyzed spectropho-
tometrically at 233 nm [23].
Determination of the lactate content in the brains of laboratory
animals was performed using the diagnostic kit “Lactat-01" (Olvex
Diagnosticum, Russia). Pyruvate concentration was detected using
the “Pyruvate UV-Abris þ" kit (NPF Abris þ, Russia).
2.6. Determination of S100B, 8-isoprostane and 8-hydroxy-2-
2.9. Enzyme activities assays
deoxyguanosine
Aconitate hydratase (AH) activity was estimated in a 50 mM Tris-
The S100B protein level was determined using an S100 Calcium
Binding Protein B ELISA Kit (Cloud-Clone Corp., USA). The 8-
isoprostane concentration was assessed using a Rat 8-isoprostane
ELISA Kit (Puda Scientific, China). The level of 8-hydroxy-2-
deoxyguanosine (8OHDG) was measured using an 8-hydroxy-2-
deoxyguanosine ELISA kit (Abcam, UK). The plates were washed
with a Stat Fax 2600 washer (Awareness Technology, USA); the
results were detected by a Stat Fax 4300 Chromate ELISA
photometer (Awareness Technology, USA).
HCl buffer (pH 7.8) containing 4 mM sodium citrate (“PanReac”,
0
0
Spain). Myeloperoxidase(MPO)activitywasmeasuredusing3,3 ,5,5 -
tetramethylbenzidine (TMB, Sigma). A 10 l sample was mixed with
80 l of 0.75 mM H (Sigma) and 110 l of a TMB solution (2.9 mM
m
m
O
2 2
m
TMB in14.5%DMSOand 150mM potassium-phosphate buffer pH5.4)
ꢃ
and the plate was incubated for 5 min at 37 C. The reaction was
stopped by adding 50
2 4
ml of 2 M H SO and the absorption was
measured at 450 nm [24]. Superoxide dismutase (SOD) activity was
measured by the indirect method developed by Nishikimi et al. based
on the reduction of nitro blue tetrazolium (NBT) [25]. In this assay,
SOD competes for the superoxide radicals generated in the presence
of PMS and NADH reducing the reduction of NBT. The reaction
mixture consisted of 0.33 mM EDTA, a 0.1 M phosphate buffer (pH
7.8), 0.8 mM NADH, 0.41 mM NBT, and 0.01 mM PMS. The absorbance
of the formed blue formazan was measured at 540 nm. Catalase ac-
tivity was assessed according to the spectrophotometric assay
developed by Goth [26]. The reaction mixture contained 0.08%
hydrogenperoxide in 0.1 M Tris-HCl buffer (pH 7.4) as a substrate. The
decomposition of hydrogen peroxide by catalase was terminated by
adding 4.5% ammonium molybdate. The intensity of the formed
yellow complex was measured at 410 nm. Glutathione peroxidase
(GP) activity was assayed according to Paglia and Valentina [27].
Hydrogen peroxide (0.37 mM) and GSH (0.85 mM) were used as
substrates and coupled oxidation of NADPH (0.12 mM) by GR (1 U/ml)
2.7. Histological staining
Hematoxylin-eosin staining of brain was assessed in three rats
from each group. Rats were anesthetized, each brain was rapidly
removed and immersed in 10% formalin for 2 h, then washed three
times using the PBS. After being dehydrated and embedded with
paraffin, the brain tissues were cut into 6-mm-thick coronal sections
by rotary microtome HM-325 (Thermo Fisher Scientific, USA) for
the hematoxylin-eosin staining. The sections were counterstained
with hematoxylin in nucleuses and differentiated by eosin in
cytoplasm. High magnification images were captured using an
AxioLab A1 light microscope (Zeiss, Germany). A minimum of five
fields for each slide were evaluated. Identification of the dead cells
was done by morphological criteria by the blebbing in the plasma
132

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 1. The scheme of the experiment.
was determined at a wavelength of 340 nm. Glutathione reductase
and saturated potassium permanganate solution were added to the
supernatant. The resulting pentabromoacetate was extracted with
petroleum ether. The ether extract was then mixed with 2% thiourea
in a borate buffer. After phase separation, the lower layer was
analyzed at 430 nm using a Hitachi U-1900 spectrophotometer.
(
(
GR) activity was evaluated by measuring the oxidation of NADPH
0.16 mM) using oxidized glutathione as a substrate [28]. The reaction
mixture contained a 50 mM potassium phosphate buffer (pH 7.4),
.8 mM oxidized glutathione, 0.16 mM NADPH, and 1 mM EDTA.
0
Glutathione-S-transferase (GT) activity was assessed by the method
of Warholm et al. using GSH and 1-chloro-2,4-dinitrobenzene acid as
substrates [29]. The media for the determination of glucose-6-
phosphate dehydrogenase (G6PDH) activity consisted of a 50 mM
Tris-HCl buffer (pH 7.8) containing 3.2 mM glucose-6-phosphate,
The concentration of
a-tocopherol was determined using the
method based on photometry of the chromogenic complex com-
2
þ
pound Fe and o-phenanthroline [32]. Ethanol and hexane were
added to the test sample, centrifuged at 3000 g, after the hexane
layer was taken and the hexane was evaporated in a water bath.
Benzene and ferric chloride were added to the dry residue. After
5 min, 0.05% o-phenanthroline was added, then after 2 min the
optical density was measured at 510 nm.
0
2
.25 mM NADP, and 1.0 mM MgCl . The activity of NADP-isocitrate
dehydrogenase (NADP-IDH) was measured in a media consisting of
5
0
0 mM Tris-HCl buffer (pH 7.8), 1.5 mM isocitrate, 2 mM MnCl2, and
.4 mM NADP. The enzymatic activity was evaluated by the change in
optical density at 340 nm using a Hitachi U1900 spectrophotometer
Japan). The protein content was determined by Lowry's method.
2.11. Determination of the intensity of apoptotic processes
(
The enzyme activities of caspase-8 and caspase-3 were measured
using colorimetric assay kits purchased from BioVision (Milpitas,
California, USA). The assay kits detected caspase activation by the
spectrophotometric determination of p-nitroaniline produced via
hydrolysis of acetyl-Ile-Glu-Thr-Asp p-nitroaniline and acetyl-Asp-
Glu-Val-Asp p-nitroaniline by caspase-8 and caspase-3, respectively.
The para-nitroaniline produced in the reaction was determined at
2
.10. Determination of the content of non-enzymatic antioxidants
The concentration of reduced glutathione (GSH) was determined
by the reaction with 5.5-ditio-bis-(2-nitrobenzoic) acid (“Sigma
Aldrich”, USA), inwhich a thionitrophenyl anion havingayellowcolor
and a maximum absorption at 412 nm is formed in equimolar
amounts [30]. The samplewasmixedwitha 0.1 M phosphate bufferat
pH 7.4.20% trichloroacetic acid was added and then thoroughly
mixed. Then the mixture was left in the refrigerator for 15e20 min.
Then, the samples were centrifuged at 3000 g. The phosphate buffer
was added into the supernatant obtained by centrifugation. Ellman's
reagent and 96% ethanol were added to the experimental and control
samples, respectively. The content of the tubes was thoroughly mixed
and the optical density of the experimental and control samples was
405 nm. The caspase activity was expressed in picomoles of the
product formed per 1 min, per 1 mg protein.
Isolation of total DNA was performed using the K-Sorb kit
(
Syntol, Russia) in accordance with the manufacturer's instructions.
DNA samples were been normalized using the elution buffer to
0 ng/ l in the total volume of 100 l. DNA fragmentation was
5
m
m
detected by agarose gel electrophoresis of the samples using a Tris-
acetate buffer.
analyzed at l 412 nm against the phosphate buffer.
The concentration of citrate was evaluated according to the
Natelson method [31]. The experimental sample was mixed with 17%
2.12. RNA isolation, reverse transcription, and quantitative PCR
2 4
TCA, centrifuged at 4000 g, after 50% H SO , 1 M potassium bromide,
Total RNA was isolated using the ExtractRNA reagent (Eurogen,
133

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 2. Hematoxylin and eosin staining of the cerebral cortex tissues of rats. Neuronal damage was absent in the Control group (A, ꢂ100 magnification; B, ꢂ200 magnification), as
well as in sham-operated animals receiving DHQ (G, ꢂ100 magnification; H, ꢂ200 magnification). CIR modeling caused the development of pathological changes in neurons
(
(
C, ꢂ100 magnification; D, ꢂ400 magnification), while the administration of DHQ at a dose of 50 mg/kg under ischemia/reperfusion conditions reduced the pathological changes
E, ꢂ100 magnification; F, ꢂ400 magnification).
ꢁ
DDCt
Russia). The quality of RNA isolation was monitored using agarose
gel electrophoresis. Reverse transcription was performed in two
replicates using the MMLV RT kit (Eurogen, Russia) in accordance
with the instructions. The amount of mRNA of each gene was
2
method. The specificity of the reaction was evaluated based
on the melting curves.
2
.13. Statistical analysis
normalized to the level of Gapdh and Actb mRNAs used as house-
keeping genes (Appendix 2). Real-time PCR was performed using
qPCRmix-HS SYBR (Eurogen, Russia) with an ANK-32 device (Syn-
thol, Russia). The analysis of the results was carried out using the
Multiple groups were analyzed using a one-way ANOVA with
Tukey's post hoc test, p < 0.05 was considered to be statistically
significant. Statistical analysis was performed using the IBM SPSS
134

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 3. The effect of DHQ on the markers of cerebral ischemia in rats. The development of CIR in rats was accompanied by an increase in the concentration of lactate in the brain and
a decrease in the level of pyruvate, as well as the accumulation of Hif1a gene transcripts. The level of S100B in the serum increased in animals with the pathology. The admin-
istration of DHQ to rats with CIR changed these parameters towards the control values. Data in bars are presented as mean ± SD.
Statistics 25 software. All quantitative data were presented as the
mean ± standard deviation (SD).
nucleolus ectoped to the nuclear membrane and perinuclear
hyperchromatosis were visualized. Some neurons had
a
honeycomb-like cytoplasm, some neurons were totally dark-
colored, had narrower, elongated neurosome. Some neurons had
central tinctorial acidophilia. In addition, dystonia of the vascular
walls, thickening and coarsening of argyrophilic fibers, peri-
vascular edema, and rarely small perivascular hemorrhages were
observed.
3
. Results
3.1. Bioactivity and toxicity prediction of DHQ
The database of compounds of the Department of Organic
Chemistry of Voronezh State University [33] was used for the vir-
tual screening. A wide range of nitrogen-, oxygen-, and sulfur-
containing heterocyclic compounds are presented in the data-
base. The compounds are small drug-like molecules. The biological
activity spectra of hydroquinoline derivatives were predicted using
the computer program PASS (prediction of activity spectra for
substances). Appendix 3 represents the results of the prediction of
main types of biological activities of DHQ and the most promising
candidates. The predicted biological activity of the compounds was
compared to that of piracetam. DHQ, which has the highest anti-
oxidant and protective potential, was selected for research.
In silico analysis of toxicity showed that DHQ is of class 4 toxicity
with a predicted LD50 of 1450 mg/kg. Amino oxidase A (Avg
Pharmacophore Fit 34.97%) and progesterone receptor (Avg Simi-
larity Known Ligands 76.7%) are predicted among the targets of
toxic action.
In turn, DHQ administration at a dose of 50 mg/kg significantly
attenuated the histopathological changes in the brain cortex.
Therefore, DHQ may reduce brain damage caused by cerebral
ischemia and reperfusion.
3.3. Aerobic metabolism and the level of markers of cerebral
ischemia
For the assessment of the state of the aerobic metabolism in the
brains of animals we evaluated lactate and pyruvate levels in the
brain tissues of rats with CIR and subsequently injected with DHQ.
The accumulation of lactate, the end product of anaerobic glycol-
ysis, as well as a decrease in the pyruvate concentration, was found
in brain tissues of rats with pathology. The administration of DHQ
to animals with CIR changed the levels of these metabolites to-
wards the control values (p < 0.05, Fig. 3). In addition, the level of
S100B in the serum increased in rats with CIR. The development of
CIR was also associated with the induction of HIF-1 (Hif1a gene)
expression. The treatments with DHQ led to a decrease (p < 0.05) in
these parameters (Fig. 3).
3.2. Effect of DHQ on histopathological changes in the CIR rat model
For morphological analysis, sections of the brain stained with
hematoxylin and eosin were used (Fig. 2). As a result of the studies
carried out on animals with CIR, a sharp decrease in the blood
supply of the vessels of the microcirculation was found. The
number of functioning capillaries was significantly reduced. There
was a pronounced pericellular and perivascular edema, a large
number of dystrophically altered neurons with a predominance of
karyolysis. In places, hypertrophy and hyperchromicity of the
3.4. DHQ reduces oxidative stress in animals with CIR
We have previously shown that the modeling of CIR in rats led to
an increase in BChL parameters, DC content, POM level, and AH
inhibition [34]. In addition, the development of the pathology was
associated with an increase in such markers of oxidative stress as 8-
135

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 4. Effects of DHQ on oxidative stress markers in CIR. Administration of DHQ to animals with CIR significantly reduced DC content, POM level, concentration of 8-isoprostane and
-OHDG in the brain and blood serum.
8
isoprostane and 8-OHDG. The administration of DHQ to rats with
CIR led to a significant (p < 0.05) decrease in the markers of
oxidative stress and restoration of AH activity (Fig. 4, Table 1).
NF-kB (Nfkb2 gene). The level of cyclooxygenase-2 transcripts (Ptgs
gene) in the CIR þ DHQ group changed insignificantly. Among other
things, DHQ reduced (p < 0.05) MPO activity in tissues of animals
with CIR (Fig. 5).
3.5. Anti-inflammatory effect of DHQ in CIR rats
3.6. DHQ administration to CIR animals reduces apoptosis in brain
CIR is associated with the activation of an inflammatory
response. As our studies shown, the administration of DHQ to an-
In previous studies, we have demonstrated that CIR in animals is
imals with CIR led to a significant (p < 0.05) decrease in the
associated with the increased activity of caspase-3, caspase-8, and
DNA fragmentation [34]. In addition, in the present work, it was
shown that the level of apoptosis inducing factor (AIF) transcripts
expression of proinflammatory cytokines, such as TNF-
a, IL-1b, IL-6
(
encoded by the Tnf, Il1b, and Il6 genes), and the transcription factor
136

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Table 1
Effects of DHQ on BChL parameters and AH activity in CIR animals. Administration of DHQ to animals with CIR led to a decrease in Imax and S, showing the intensity of free
radical-induced oxidation, as well as tg 2, characterizing the degree of antioxidant system mobilization. In addition, DHQ promoted the restoration of AH activity inhibited
under CIR conditions. Data are presented as mean ± SD. * - p < 0.05 compared to Control; ** - p < 0.05 compared to CIR.
a
Groups
Blood serum
Control
Brain
CIR
CIR þ DHQ
Control þ DHQ Control
CIR
CIR þ DHQ
Control þ DHQ
5.29 ± 1.19*
I
max, mV
S, mV*s
tg
AH, U/ml serum, U/g brain tissue 1.12 ± 0.35
AH, U/mg protein
1.72 ± 0.45
3.68 ± 0.61*
2.23 ± 0.51**
13.11 ± 2.66** 9.48 ± 1.84*
1.87 ± 0.41** 1.81 ± 0.33*
1.52 ± 0.23*
6.12 ± 1.46
13.61 ± 3.04* 5.83 ± 1.06**
11.66 ± 2.45 26.85 ± 6.8*
1.93 ± 0.45 4.75 ± 1.14*
0.521 ± 0.11* 1.047 ± 0.42** 1.15 ± 0.51
33.10 ± 8.99 58.99 ± 15.77* 38.36 ± 8.82** 29.77 ± 6.28*
1.09 ± 0.19
2.18 ± 0.72
a
2
2.63 ± 0.67*
0.831 ± 0.25* 1.683 ± 0.51** 2.22 ± 0.75
1.02 ± 0.34**
0.90 ± 0.19*
0.065 ± 0.021 0.013 ± 0.004* 0.052 ± 0.015** 0.068 ± 0.020 0.368 ± 0.115 0.156 ± 0.051* 0.205 ± 0.071** 0.375 ± 0.112
(
gene Aifm1) increased in the brains of animals with the pathology.
brain were shown in CIR rats. The level of these changes signifi-
cantly decreased after the administration of DHQ. The main regu-
lator of the metabolism, sensitive to the oxygen level, is the hypoxia
inducing factor (HIF-1), which, as our data confirmed, is induced
The present study showed, that the administration of DHQ to CIR
rats led to a significant (p < 0.05) decrease in the activity of caspase-
3
and caspase-8 and Aifm1 expression (Fig. 6).
under ischemic conditions. During hypoxia, the HIF-1
a protein
3.7. DHQ decreases the mobilization of antioxidant enzymes in CIR
undergoes rapid stabilization, leading to its nuclear translocation,
binding with the aryl hydrocarbon receptor nuclear translocator,
As we have shown in previous studies, the modeling of CIR in
also designated as hypoxia-inducible factor (HIF)-1b with the for-
rats led to the activation of SOD, catalase, GP, GR, and GT, which was
of a compensatory effect [35,36]. The administration of DHQ to
animals with the pathology led to a decrease in the severity of
oxidative stress and load on antioxidant enzymes, which contrib-
uted to a decrease (p < 0.05) in their activity (Appendix 4). Enzyme
activity, expressed as E/mg protein, changed in a similar way
mation of the HIF-1 transcription factor complex. Mature HIF-1
modulates the expression of more than 200 target genes and me-
diates the formation of hypoxia tolerance and a neuroprotective
effect [38]. At the same time, HIF-1 activates the expression of
vascular endothelial growth factor and matrix metalloproteinases,
which have been shown to be involved in the disruption of the
blood-brain barrier [39]. In addition, there is evidence that HIF-1, in
addition to its neuroprotective effect, interacts with the p53 protein
and causes delayed neuronal death caused by ischemia [40]. The
(
Appendix 5).
3.8. DHQ provides a normalization of non-enzymatic antioxidant
administration of DHQ led to a decrease in the HIF-1a expression,
concentrations in the tissues of CIR rats
which could be a consequence of the DHQ neuroprotective activity
and the normalization of energy metabolism. The positive effect of
DHQ was also confirmed by a change in the concentration of S100B
in the serum, which is the biomarker of brain damage. With
physiological concentrations, S100B acts as a neurotrophic factor,
but an increase in its concentration may lead to the opposite effect.
In addition, there is evidence of a positive correlation between the
S100B level and neuron-specific enolase, a marker of brain
neuronal loss [41]. Histopathological findings confirmed the neu-
roprotective effect of DHQ. The administration of DHQ significantly
reduced the damage of neurons caused by CIR, which was associ-
ated with the leveling of morphological changes in neurons and an
increase in cell survival.
CIR in rats was associated with an increase in the serum and
brain levels of GSH and citrate, as well as the depletion of the a-
tocopherol pool. The administration of DHQ to animals at the same
time led to a significant (p < 0.05) decrease in the concentration of
GSH and citrate, as well as the restoration of the
in tissues (Fig. 7).
a-tocopherol level
3
.9. Effects of DHQ on the transcriptional regulation of antioxidant
defense in CIR
As we have shown earlier, one of the most important mecha-
nisms in the activation of antioxidant defense enzymes in CIR was
the induction of their expression at the transcriptional level [34]. In
particular, there was an increase in the transcript levels of SOD,
catalase, GP, GR, and GT, and factors Nrf2 and Foxo1 (genes Sod1,
Cat, Gpx1, Gsr, Gsta2, Nfe2l2, Foxo1, respectively). DHQ had a
multidirectional effect on the expression of these genes. Thus, the
analyzed compound led to a decrease (p < 0.05) in the expression of
Cat, Gsr, and Nfe2l2, while the expression of Sod1, Gpx1, Gsta2, and
Foxo1 was increased in CIR þ DHQ rats (p < 0.05; Fig. 8).
Oxidative stress is the leading cause of tissue damage in CIR.
Oxidative stress is characterized by the overproduction of ROS,
causing mutations in mitochondrial DNA, damaging the mito-
chondrial respiratory chain, altering membrane permeability,
2þ
affecting Ca
homeostasis and mitochondrial defense systems.
Commonly, ROS are generated endogenously from molecular oxy-
gen by cellular oxidases, mono- and dioxygenases of the mito-
chondrial electron chain transport system or peroxidases, and are
involved in damage to nerve cells after ischemia/reperfusion
[
42,43]. Oxidative stress can activate the NF-
to synthesize of pro-inflammatory mediators such as nitric oxide,
TNF- , IL-6, promoting inflammation in neurons, and matrix
kB signaling pathway
4
. Discussion
a
In the course of this study, we analyzed the neuroprotective
metalloproteinase-9, causing the destruction of the extracellular
matrix and tight junction proteins with subsequent damage to the
blood-brain barrier [44]. As our results demonstrated, in addition to
an increase in oxidative stress markers, such as DC, POM, 8-OHDG,
mechanisms of the DHQ effect and evaluated its regulatory effect
on the antioxidant defense system in CIR rats. Our results demon-
strated that the tested dihydroquinoline derivative reduced
oxidative stress and modulated the functioning of the antioxidant
defense system, contributing to the inhibition of inflammation and
apoptosis, improvement of aerobic metabolism, and reducing his-
topathological changes in the cerebral cortex.
As is known, ischemia leads to the inhibition of aerobic meta-
bolism and the formation of lactic acidosis [37]. In our studies,
lactate accumulation and depletion of the pyruvate pool in the
8
-isoprostane, and BChL parameters, inhibition of the AH activity,
which is a sensitive target of ROS in CIR rats was also observed [45].
DHQ significantly reduced oxidative stress in rats with the pa-
thology, probably due its antioxidant activity. Thus, a high reducing
ability for this compound was predicted by in silico analysis
(
p ¼ 0.888). Protective and antioxidant properties are also known
137

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 5. The effect of DHQ on the inflammatory response in animals with CIR. The level of gene transcripts, represented in the form of a heat map, demonstrating that the
administration of DHQ to animals with CIR led to a decrease in the expression of the proinflammatory cytokines TNF-
reduced MPO activity in the serum and brain of CIR animals. Bars in histograms are presented as mean ± SD.
a, IL-1b, IL-6, and factor NF-kB. In addition, exposure to DHQ
for other dihydroquinoline derivatives. Previously, the neuro-
protective properties of a compound belonging to quinoline 1,2-
dihydro derivatives - ethoxyquine [46] were established. It is also
known that 8-hydroxyquinoline derivatives have the ability to
reduce the level of b-amyloid, act as scavengers of free radicals, and
chelates copper ions [47].
immune response is triggered by the damage associated molecular
patterns (DAMP) released from damaged cells. DAMPs are subse-
quently elicited by immune cells with appropriate receptors
recognizing molecules, mediating the activation of intracellular
signaling pathways. Within minutes after brain damage, microglia
are activated, undergo morphological changes and secrete cyto-
kines, which contributes to damage to the parenchyma and cere-
bral vessels. Blood-brain barrier impairment occurs immediately
Oxidative stress can activate inflammation, another key mech-
anism for nerve tissue damage during ischemia/reperfusion. The
138

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 6. The effect of DHQ on apoptotic processes in animals with CIR. DHQ promotes a decrease in the activity of caspase-3 and caspase-8, in the expression of the Aif factor and
reduction of DNA fragmentation in the brain of CIR rats. The electrophoregram shows: 1 e Molecular weight markers; 2 - Control (non-degraded DNA); 3 - Control þ DHQ (non-
degraded DNA); 4 - CIR (apoptotic DNA ladder is visualized); 5 - CIR þ DHQ (apoptotic DNA ladder is less expressed). Data in bars are presented as mean ± SD.
after a stroke and promotes the penetration of peripheral leuko-
cytes into the damaged brain [48]. According to our data, the levels
of transcripts of the proinflammatory cytokines IL-1b, IL-6, TNF-a,
as well as the Ptgs2 gene encoding the enzyme cyclooxygenase-2,
were significantly increased in CIR rats. Cyclooxygenase-2 metab-
olizes arachidonic acid into oxidized fatty acids - prostaglandins,
involved in the inflammatory response [49]. CIR stimulates the
TLR4-mediated signaling pathway, followed by the rapid trans-
Apoptosis is the dominant pathway of cell death in the ischemic
penumbra. Apoptosis also leads to DNA fragmentation, degradation
and cross-linking of cytoskeletal and nuclear proteins, the forma-
tion of apoptotic bodies, the expression of ligands for receptors of
phagocytic cells and, finally, to microglia and astrocyte-mediated
phagocytosis [53]. Key enzymes of apoptosis include the main
effector caspase-3, and initiating caspases, in particular caspase-8
[54]. We have shown an increase in the activity of caspase-3, cas-
pase-8, and DNA fragmentation in CIR rats. An increase in the
expression of the proapoptotic protein AIF was also observed,
which may be associated with the overexpression of Bid and Bax,
forming megapores on the outer mitochondrial membrane,
through which proapoptotic proteins pass into the cytosol [53,55].
CIR þ DHQ rats showed reducing caspase activity, AIF expression,
and DNA fragmentation. Probably, exhibiting an antioxidant effect,
DHQ provided the reducing of ROS-induced apoptosis, inflamma-
tory cytokines expression and subsequent ligand-mediated path-
ways of apoptosis.
Under pathophysiological conditions, antioxidant enzymes are
responsible for regulating the redox balance in neurons. The
functioning of some antioxidant enzymes is associated with the
GSH, an oligopeptide containing a cysteine residue, cleaving unfa-
vorable disulfide bonds of oxidized proteins. Oxidized glutathione
molecules form disulfide bonds, and can be reduced back into GSH
by GR. GSH can also reduce the accumulation of prooxidant xeno-
biotic agents through GT-catalyzed conjugation. GSH also serves as
location of NF-
tion of the expression of inflammatory mediators such as IL-1, TNF-
, and IL-6, which contribute to ischemic brain damage [50]. The
Nfkb2 gene of the p100 precursor subunit of NF- B also activates its
kB from the cytoplasm into the nucleus and activa-
a
k
expression [51]. In addition, our results showed that CIR was
associated with an increase in MPO activity. MPO generates a wide
variety of oxidants such as hypochlorous acid and nitrogen dioxide
radicals [52]. DHQ, in turn, decreased MPO activity, as well as the
expression of Nfkb2 and the IL-1b, IL-6, and TNF-a cytokine genes.
Apparently, these changes could be associated with the antioxidant
effect of DHQ, leading to a decrease in the oxidative stress-induced
inflammation.
Long-living ROS generated by CIR diffuse between organelles
and between cells. They can stimulate both cell survival and pro-
apoptotic signaling cascades. Oxidative damage to the endo-
plasmic reticulum and mitochondrial membrane causes the release
2þ
of Ca
and mitochondrial proapoptotic proteins such as cyto-
chrome c, Smac/DIABLO, AIF, and endonuclease G into the cytosol.
139

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 7. Effect of DHQ on the concentration of non-enzymatic antioxidants in tissues of animals with CIR. DHQ led to a decrease in the concentration of GSH and citrate, an increase in
which was associated with the development of CIR. DHQ also provided the restoration of the -tocopherol pool in animal tissues.
a
a cofactor for the GP enzymes family. All GP enzymes have the
ability to catalyze the degradation of organic hydroperoxides, but
some isoforms are capable of reducing more complex hydroper-
oxides [56]. NADPH is an important metabolic product of the
pentose phosphate pathway (PPP), which is used to maintain
catalase stability and restore oxidized glutathione in the GR reac-
tion. G6PDH is considered a key PPP enzyme, synthesizing NADPH
and protects the brain from ischemic damage [4]. NADP-IDH is
another enzyme generating NADPH, localized primarily in cytosol
and mitochondria [57]. Catalase is a component of cellular perox-
isomes responsible for converting hydrogen peroxide into water
and molecular oxygen. Catalase is ubiquitously expressed by the
neurons and glia of the central nervous system and it is one of the
most efficient enzymes found in nature. Enzymes of the SOD family
are responsible for the conversion of superoxide radical anions into
the
a
-tocopherol pool.
a
-tocopherol is the most active antioxidant
-induced lipid
among tocopherols, protecting cells against H
peroxidation by enhancing antioxidant enzyme systems and
reducing apoptosis caused by oxidative stress. In addition to its
2 2
O
antioxidant properties, a-tocopherol acts as a regulator of genes
involved in lipid metabolism, inflammation, and the immune sys-
tem [58]. The accumulation of citrate was observed in the tissues of
rats with the pathology. This finding may be associated with the
inhibition of the main citrate-metabolizing enzyme - AH under
oxidative stress. At the same time, citrate can also possess an
antioxidant effect, due to the presence of three carboxyl groups
capable of chelating metal ions in its structure [59]. The adminis-
tration of DHQ to CIR rats led to a change in all analyzed parameters
towards the control value. Apparently, the antioxidant effect of
DHQ contributed to a decrease of oxidative stress and the mobili-
zation of the antioxidant system decreased. Some enzymes of the
antioxidant system in the CIR þ DHQ group were characterized by
lower values than in the control group. These differences may be
associated with the development of a certain degree of oxidative
stress in animals of the control group that underwent a sham
operation, while the administration of DHQ inhibited oxidative
stress. In addition, a decrease in the serum activity of some en-
zymes may indicate an improvement in the state of the BBB and a
2 2
H O . Since oxidative stress is a pathological sign of neuro-
degeneration, SOD dysfunction is associated with diseases charac-
terized by neuronal loss [56]. As our data showed, animals with CIR
had a significant increase in the activity of SOD, catalase, GP, GR,
and GT in the brain and blood serum. These changes may indicate
the development of a compensatory response to oxidative stress by
the time rats were removed from the experiment. In addition, an-
imals with the pathology were characterized by the depletion of
140

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Fig. 8. The effect of DHQ on the transcriptional regulation of antioxidant defense in CIR. The heat map shows that the administration of DHQ to rats with CIR led to a decrease in the
expression of Cat, Gsr, and Nfe2l2, as well as to an increase in the expression of Sod1, Gpx1, Gsta2, and Foxo1. Data in bars are presented as mean ± SD.
decrease in the release of enzymes from cells into the bloodstream.
The antioxidant system is able to change its functional activity
under stress conditions. One of the most important mechanisms for
mobilizing the activity of the antioxidant system is the transcrip-
tional regulation of their expression. We have shown that CIR is
associated with an increase in the mRNA of antioxidant enzyme
genes. In addition, the expression of Nrf2 and FOXO1 transcription
factors increased in pathology. Under physiological conditions, Nrf2
binds to its natural inhibitor Keap1 in the cytosol. In response to
cellular damage, Nrf2 is released from Keap1, accumulates in the
cytoplasm, translocates to the nucleus, heterodimerizes with bZIP
proteins and recognizes the corresponding ARE sequence [60].
FOXO1 modulates numerous targets such as genes involved in
apoptosis and autophagy, antioxidant enzymes, cell cycle arrest
genes, and metabolic and immune regulators. Oxidative stress,
mediated by the accumulation of ROS, causes a significant increase
in the transcriptional activity of FOXO1, and this has a large effect
on the expression of SOD and catalase. In addition, a low level of
141

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
FOXO1 can cause the decreased transcription of antioxidant en-
zymes in many diseases [61]. As we have shown, the administration
of DHQ to CIR rats was accompanied by a decrease in the expression
of catalase, GR, and Nrf2 genes. At the same time, the expression of
SOD, GP, and GT genes increased. These changes may indicate that
DHQ has the ability to activate some components of the antioxidant
system in CIR. Such stimulation could occur through the FOXO1
factor, the mRNA level of which also increased in rats of the
CIR þ DHQ group. It was shown that this transcription factor
increased the expression of such enzymes as SOD and GP in
Appendix 2. The list of primers used in this study
0
0
0
Hif1a F: 5 - CATGATGGCTCCCTTTTTCA -3 and
0
Hif1a R: 5 - ACATAGTAGGGGCACGGTCA -3
0
0
Nfkb2 F: 5 - GAATTCAGCCCCTCCATTG-3 and
Nfkb2 R: 5 - CTGAAGCCTCGCTGTTTAGG-3
Il1b F: 5 -TGTGATGAAAGACGGCACAC -3 and
Il1b R: 5 -CTTCTTCTTTGGGTATTGTTTGG-3
0
0
0
0
0
0
0
0
Il6 F: 5 -CCTGGAGTTTGTGAAGAACAACT-3 and
0
0
Il6 R: 5 -GGAAGTTGGGGTAGGAAGGA-3
0
0
response to oxidative stress in
b
-cells of the pancreas [62]. Thus, in
Tnf F: 5 -TCTGTGCCTCAGCCTCTTCT-3 and
0 0
Tnf R: 5 -GGCCATGGAACTGATGAGA-3
CIR þ DHQ animals the expression of catalase, GR, and Nrf2
approached the control values at the time of withdrawal from the
experiment, while the expression of SOD, GP, GT, and FOXO1 was
still significantly increased as a result of the enhancement of the
adaptive response by DHQ. At the same time, the mechanisms of
FOXO1 regulation are extremely complex and have not yet been
fully elucidated [61].
0
0
0
Ptgs2 F: 5 -TACACCAGGGCCCTTCCT-3 and
0
Ptgs2 R: 5 -TCCAGAACTTCTTTTGAATCAGG-3
0
0
0
Aifm1 F: 5 - AGTCCTTATTGTGGGCTTATCAAC-3 and
Aifm1 R: 5 - TTGGTCTTCTTTAATAGTCTTGTAGGC-3
0
0
0
Sod1 F: 5 -CCAGCGGATGAAGAGAGG-3 and
0
0
Sod1 R: 5 -GGACACATTGGCCACACC-3
0
0
Cat F: 5 -CAGCGACCAGATGAAGCA-3 and
Cat R: 5 -GGTCAGGACATCGGGTTTC-3
Nfe2l2 F: 5 -GCCTTGTACTTTGAAGACTGTATGC-3 and
Nfe2l2 R: 5 -GCAAGCGACTGAAATGTAGGT-3
0
0
0
0
5
. Conclusions
0
0
0
0
In this study, we demonstrated that DHQ has a neuroprotective
Foxo1 F: 5 -AGATCTACGAGTGGATGGTGAAGAG-3 and
0 0
Foxo1 R: 5 -GGACAGATTGTGGCGAATTGAAT-3
effect in CIR rats, reducing histopathological changes in the brain,
normalizing pyruvate and lactate levels, and decreasing HIF-1
expression. The main mechanisms of the DHQ neuroprotective
action are based on its antioxidant activity, which leads to reduce
oxidative stress-induced inflammation and apoptosis. In addition,
the tested compound modulates the functioning and transcrip-
tional regulation of the antioxidant system, thereby providing an
additional protective effect via the regulation of the antioxidant
defense in experimental animals.
0
0
Gsta2 F: 5 -CGGGAATTTGATGTTTGACC-3 and
0
0
Gsta2 R: 5 -AGAATGGCTCTGGTCTGTGC-3
0
0
0
Gpx1 F: 5 -TTTCCCGTGCAATCAGTTC-3 and
0
Gpx1 R: 5 -GGACATACTTGAGGGAATTCAGA-3
0
0
Gsr F: 5 -TTCCTCATGAGAACCAGATCC-3 and
Gsr R: 5 -CTGAAAGAACCCATCACTGGT-3
Gapdh F: 5 -CCCTCAAGATTGTCAGCAATG-3 and
Gapdh R: 5 -AGTTGTCATGGATGACCTTGG-3
0
0
0
0
0
0
0
0
Actb F: 5 - CCCGCGAGTACAACCTTCT -3 and
0
0
Actb R: 5 - CGTCATCCATGGCGAACT -3
Funding
Appendix 3. Prediction of the main types of biological
activities of DHQ and the most promising candidates. The
probability of the manifestation of biological activity ranges
from zero to one
This work was supported by the Council for grants of President
of Russian Federation for young scientists-candidates of sciences
MK-254.2020.4.
Declaration of competing interest
DHQ
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Biological activity of the compound
Probability of activity manifestation
Reductant
0.888
0.870
0.823
0.791
0.790
0.737
0.707
0.697
0.736
0.725
0.642
0.628
0.645
0.593
0.616
0.578
0.575
0.567
0.565
0.561
0.572
0.571
0.583
Cytochrome P450 stimulant
CYP2C12 substrate
Appendix 1. Synthesis of 6-hydroxy-2,2,4-trimethyl-1,2-
dihydroquinoline by dealkylation of 6-ethoxy-2,2,4-
trimethyl-1,2-dihydroquinoline when exposed to
hydrobromic acid
Progesterone antagonist
Chlordecone reductase inhibitor
Autoimmune disorders treatment
Kidney function stimulant
Lipid peroxidase inhibitor
Membrane integrity agonist
CYP2J substrate
Radiosensitizer
Alkane 1-monooxygenase inhibitor
CYP2J2 substrate
Glucocorticoid antagonist
Lysase inhibitor
Chemosensitizer
Menopausal disorders treatment
Free radical scavenger
Ophthalmic drug
Estrogen receptor beta antagonist
CYP2F1 substrate
MAP kinase stimulant
Anti-inflammatory
142

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Piracetam
(continued)
Biological activity of the compound
Probability of activity
manifestation
Biological activity of the compound
Probability of activity
manifestation
CYP2J2 substrate
UDP-glucuronosyltransferase substrate
MAP kinase stimulant
Ophthalmic drug
Estrogen receptor beta antagonist
0.645
0.612
0.579
0.566
0.561
0.589
0.552
0.556
0.544
0.592
0.612
0.566
0.563
0.554
0.552
0.518
Nootropic
Phobic disorders treatment
Antihypoxic
Calcium channel (voltage-sensitive) activator
Electron-transferring-flavoprotein
dehydrogenase inhibitor
0.876
0.820
0.756
0.739
0.723
5
Hydroxytryptamine release stimulant
Estrogen antagonist
Anticonvulsant
0.721
0.725
0.719
0.714
0.698
Menopausal disorders treatment
Glucocorticoid antagonist
Glutamyl endopeptidase II inhibitor
Membrane permeability inhibitor
Glutathione thiolesterase inhibitor
Thioredoxin inhibitor
HIF1A expression inhibitor
Antiinflammatory
Free radical scavenger
Dimethylargininase inhibitor
NADPH-cytochrome-c2 reductase inhibitor
Antidyskinetic
Protein-disulfide reductase (glutathione)
inhibitor
Neurotransmitter antagonist
Acetylesterase inhibitor
Fatty-acyl-CoA synthase inhibitor
Macrophage colony stimulating factor agonist
0.681
0.679
0.670
0.663
SMILES code: CC1¼CC(C) (C)Nc2cc(O)c(O)cc12.
SMILES code: CC(¼O)C1¼C(N)C2¼CC]CC]C2N]C1C.
Biological activity of the compound
Probability of activity
manifestation
Biological activity of the compound
Probability of activity
manifestation
Cytochrome P450 stimulant
Reductant
Progesterone antagonist
0.853
0.845
0.798
Taurine dehydrogenase inhibitor
Gluconate 2-dehydrogenase (acceptor)
inhibitor
0.839
0.744
Testosterone 17beta-dehydrogenase (NADPþ) 0.791
Membrane integrity agonist
Amine dehydrogenase inhibitor
Mucomembranous protector
Complement factor D inhibitor
Thiol oxidase inhibitor
0.731
0.680
0.667
0.602
0.564
inhibitor
Autoimmune disorders treatment
CYP2J substrate
0.748
0.751
0.707
0.737
0.700
0.674
0.663
0.660
0.677
0.705
0.624
0.672
0.619
0.599
0.613
0.644
0.652
0.592
0.557
0.573
0.549
Lipid peroxidase inhibitor
CYP2C12 substrate
Calcium channel (voltage-sensitive) activator 0.577
5
Hydroxytryptamine release stimulant
Endopeptidase So inhibitor
General pump inhibitor
Thioredoxin inhibitor
Leukopoiesis stimulant
Gastrin inhibitor
0.567
0.574
0.569
0.558
0.565
Kidney function stimulant
Radiosensitizer
Thioredoxin inhibitor
CYP2J2 substrate
Ubiquinol-cytochrome-c reductase inhibitor
Glucocorticoid antagonist
Membrane integrity agonist
Aryl-acylamidase inhibitor
Ophthalmic drug
IgA-specific serine endopeptidase inhibitor 0.548
Oxidoreductase inhibitor
Erythropoiesis stimulant
Insulysin inhibitor
0.567
0.543
0.551
Acetylcholine neuromuscular blocking agent 0.555
Alkane 1-monooxygenase inhibitor
Membrane permeability inhibitor
Mucomembranous protector
Antiinflammatory
Platelet aggregation stimulant
Glutamyl endopeptidase II inhibitor
Membrane permeability inhibitor
Nucleotide metabolism regulator
Aspulvinone dimethylallyltransferase
inhibitor
0.553
0.571
0.593
0.524
0.593
Menopausal disorders treatment
Fatty-acyl-CoA synthase inhibitor
Free radical scavenger
SMILES code: CC1¼CC(C) (C)Nc2c(O)cc(O)cc12.
SMILES code: CC(¼O)\C]C\C1¼CC2¼C(C]C1)N(CC3¼CC]CC]C3)C(C) (C)C]C2C.
Biological activity of the compound
Probability of activity
manifestation
Biological activity of the compound
Probability of activity manifestation
Reductant
Cytochrome P450 stimulant
Lipid peroxidase inhibitor
0.811
0.796
0.749
Antiarthritic
Antiallergic
Antiasthmatic
Mucomembranous protector
Antiulcerative
0.771
0.707
0.694
0.646
0.577
0.522
0.502
0.511
0.556
Testosterone 17beta-dehydrogenase (NADPþ) 0.766
inhibitor
CYP2C12 substrate
0.765
0.724
0.710
0.736
0.725
0.730
0.676
0.691
0.661
0.657
0.632
Reductant
Kidney function stimulant
Progesterone antagonist
Membrane integrity agonist
CYP2J substrate
Ubiquinol-cytochrome-c reductase inhibitor
Autoimmune disorders treatment
Antiseborrheic
Antiasthmatic
Alkane 1-monooxygenase inhibitor
Radiosensitizer
Cytochrome P450 stimulant
HIF1A expression inhibitor
CYP2J substrate
(
continued on next page)
143

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
Appendix 4. Effect of DHQ on the activity of antioxidant
enzymes in animals with CIR. The administration of DHQ to
animals with CIR led to a decrease in oxidative stress and a
decrease in the activity of enzymes such as SOD, catalase, GP,
GR, and GT. There was also a decrease in the activity of
enzymes supplying NADPH for the functioning of the
glutathione antioxidant system, NADP-IDH and G6PDH. Data
are presented as mean ± SD. * - p < 0.05 compared to Control;
*
* - p < 0.05 compared to CIR
Groups
Blood serum
Control
Brain
CIR
CIR þ DHQ
Control þ DHQ Control
CIR
CIR þ DHQ
Control þ DHQ
3.05 ± 0.73*
SOD, U/ml serum, U/g brain tissue
0.633 ± 0.15 1.367 ± 0.34* 0.560 ± 0.14**
0.557 ± 0.14*
3.43 ± 0.85
11.80 ± 2.84* 3.12 ± 0.81**
Catalase, U/ml serum, U/g brain tissue 0.109 ± 0.025 0.288 ± 0.071* 0.102 ± 0.034** 0.095 ± 0.031* 0.152 ± 0.036 0.368 ± 0.058* 0.180 ± 0.041** 0.131 ± 0.031*
GP, U/ml serum, U/g brain tissue
GR, U/ml serum, U/g brain tissue
GT, U/ml serum, U/g brain tissue
NADP-IDH, U/ml serum, U/g brain
tissue
0.059 ± 0.014 0.111 ± 0.024* 0.095 ± 0.015** 0.052 ± 0.007
0.073 ± 0.018 0.244 ± 0.058* 0.078 ± 0.026** 0.067 ± 0.021
0.023 ± 0.005 0.079 ± 0.019* 0.043 ± 0.011** 0.020 ± 0.007* 0.034 ± 0.008 0.143 ± 0.034* 0.063 ± 0.021** 0.029 ± 0.07*
0.038 ± 0.009 0.086 ± 0.020* 0.047 ± 0.015** 0.032 ± 0.010* 0.082 ± 0.019 0.474 ± 0.114* 0.100 ± 0.028** 0.073 ± 0.021*
0.036 ± 0.01 0.106 ± 0.031* 0.029 ± 0.087** 0.029 ± 0.075* 0.085 ± 0.021 0.270 ± 0.057* 0.095 ± 0.031** 0.081 ± 0.024
G6PDH, U/ml serum U/g brain tissue 0.018 ± 0.004 0.049 ± 0.013* 0.010 ± 0.0025** 0.010 ± 0.0023* 0.110 ± 0.019 0.390 ± 0.092* 0.164 ± 0.042** 0.105 ± 0.021
Appendix 5. Specific activity of antioxidant enzymes in
experimental animals. Data are presented as mean ± SD. * -
p < 0.05 compared to Control; ** - p < 0.05 compared to CIR
Groups
Blood serum
Control
Brain
CIR
CIR þ DHQ
Control þ DHQ Control
CIR
CIR þ DHQ
Control þ DHQ
0.073 ± 0.019*
SOD, U/mg protein 0.019 ± 0.004
0.041 ± 0.010*
0.015 ± 0.004**
0.015 ± 0.003*
0.103 ± 0.025
0.354 ± 0.088*
0.075 ± 0.021**
Catalase, U/mg
protein
0.0039 ± 0.0010 0.0074 ± 0.0021* 0.0036 ± 0.0010** 0.0033 ± 0.0009* 0.019 ± 0.0046 0.047 ± 0.0110* 0.017 ± 0.0043** 0.015 ± 0.0041*
GP, U/mg protein
GR, U/mg protein
GT, U/mg protein
NADP-IDH, U/mg
protein
0.0015 ± 0.0003 0.0029 ± 0.0007* 0.0026 ± 0.0007** 0.0014 ± 0.0006 0.011 ± 0.003 0.021 ± 0.005* 0.018 ± 0.005** 0.009 ± 0.002*
0.0008 ± 0.0002 0.0022 ± 0.0005* 0.0013 ± 0.0004** 0.0006 ± 0.0002* 0.0047 ± 0.0011 0.0153 ± 0.0037* 0.0063 ± 0.0018** 0.0041 ± 0.0013*
0.0013 ± 0.0003 0.0027 ± 0.0006* 0.0016 ± 0.0005** 0.0011 ± 0.0003* 0.0064 ± 0.015 0.0250 ± 0.006* 0.0066 ± 0.0018** 0.0055 ± 0.0014*
0.0013 ± 0.0003 0.0028 ± 0.0008* 0.0010 ± 0.0003** 0.0010 ± 0.0003* 0.011 ± 0.0028 0.024 ± 0.0054* 0.012 ± 0.0034** 0.010 ± 0.0031
G6PDH, U/mg
protein
0.0010 ± 0.0002 0.0018 ± 0.0004* 0.0006 ± 0.0002** 0.0006 ± 0.0002* 0.013 ± 0.003
0.038 ± 0.007*
0.024 ± 0.0071** 0.012 ± 0.0031
[6] I. Bellezza, I. Giambanco, A. Minelli, R. Donato, Nrf2-Keap1 signaling in
oxidative and reductive stress, Biochim. Biophys. Acta Mol. Cell Res. 1865
(
2018) 721e733, https://doi.org/10.1016/j.bbamcr.2018.02.010.
References
[7] H. Ren, Y. Shao, C. Wu, X. Ma, C. Lv, Q. Wang, Metformin alleviates oxidative
stress and enhances autophagy in diabetic kidney disease via AMPK/SIRT1-
FoxO1 pathway, Mol. Cell. Endocrinol. 500 (2020) 110628, https://doi.org/
10.1016/j.mce.2019.110628.
[8] F. Carlomosti, M. D'Agostino, S. Beji, A. Torcinaro, R. Rizzi, G. Zaccagnini,
B. Maimone, V. Di Stefano, F. De Santa, S. Cordisco, A. Antonini, R. Ciarapica,
E. Dellambra, F. Martelli, D. Avitabile, M.C. Capogrossi, A. Magenta, Oxidative
stress-induced miR-200c disrupts the regulatory loop among SIRT1, FOXO1,
and eNOS, Antioxidants Redox Signal. 27 (2017) 328e344, https://doi.org/
10.1089/ars.2016.6643.
[9] D.L. Bernstein, V. Zuluaga-Ramirez, S. Gajghate, N.L. Reichenbach, B. Polyak,
Y. Persidsky, S. Rom, miR-98 reduces endothelial dysfunction by protecting
bloodebrain barrier (BBB) and improves neurological outcomes in mouse
ischemia/reperfusion stroke model, J. Cerebr. Blood Flow Metabol. 40 (2020)
1953e1965, https://doi.org/10.1177/0271678X19882264.
[
1] T. Peng, Y. Jiang, M. Farhan, P. Lazarovici, L. Chen, W. Zheng, Anti-inflamma-
tory effects of traditional Chinese medicines on preclinical in vivo models of
brain ischemia-reperfusion-injury: prospects for neuroprotective drug dis-
covery and therapy, Front. Pharmacol. 10 (2019) 204, https://doi.org/10.3389/
fphar.2019.00204.
[
[
2] A. Kahl, A. Stepanova, C. Konrad, C. Anderson, G. Manfredi, P. Zhou, C. Iadecola,
A. Galkin, Critical role of flavin and glutathione in complex Iemediated bio-
energetic failure in brain ischemia/reperfusion injury, Stroke 49 (2018)
1223e1231, https://doi.org/10.1161/STROKEAHA.117.019687.
3] T.H. Sanderson, J.M. Wider, I. Lee, C.A. Reynolds, J. Liu, B. Lepore, R. Tousignant,
M.J. Bukowski, H. Johnston, A. Fite, S. Raghunayakula, J. Kamholz,
L.I. Grossman, K. Przyklenk, M. Hüttemann, Inhibitory modulation of cyto-
chrome c oxidase activity with specific near-infrared light wavelengths at-
tenuates brain ischemia/reperfusion injury, Sci. Rep. 8 (2018) 3481, https://
doi.org/10.1038/s41598-018-21869-x.
[10] X. Liu, X. Zhang, F. Wang, X. Liang, Z. Zeng, J. Zhao, H. Zheng, X. Jiang, Y. Zhang,
Improvement in cerebral ischemiaereperfusion injury through the TLR4/NF-
kB pathway after Kudiezi injection in rats, Life Sci. 191 (2017) 132e140,
https://doi.org/10.1016/j.lfs.2017.10.035.
[11] L. Gong, Y. Tang, R. An, M. Lin, L. Chen, J. Du, RTN1-C mediates cerebral
ischemia/reperfusion injury via ER stress and mitochondria-associated
[
[
4] L. Cao, D. Zhang, J. Chen, Y.Y. Qin, R. Sheng, X. Feng, Z. Chen, Y. Ding, M. Li,
Z.H. Qin, G6PD plays a neuroprotective role in brain ischemia through pro-
moting pentose phosphate pathway, Free Radic. Biol. Med. 112 (2017)
433e444, https://doi.org/10.1016/j.freeradbiomed.2017.08.011.
5] B.A. Freeman, J.D. Crapo, Biology of disease: free radicals and tissue injury, Lab.
apoptosis pathways, Cell Death Dis.
10.1038/cddis.2017.465.
8 (2017), e3080, https://doi.org/
Invest. 47 (1982) 412e426.
144

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
[
12] S. Merel, J. Regueiro, M.H.G. Berntssen, R. Hannisdal, R. Ørnsrud, N. Negreira,
Identification of ethoxyquin and its transformation products in salmon after
controlled dietary exposure via fish feed, Food Chem. 289 (2019) 259e268,
https://doi.org/10.1016/j.foodchem.2019.03.054.
[37] J. Zhang, Z. Deng, J. Liao, C. Song, C. Liang, H. Xue, L. Wang, K. Zhang, G. Yan,
Leptin attenuates cerebral ischemia injury through the promotion of energy
3
metabolism via the PI K/Akt pathway, J. Cerebr. Blood Flow Metabol. 33
(2013) 567e574, https://doi.org/10.1038/jcbfm.2012.202.
[
13] YuA. Ivanov, N.L. Zaichenko, S.V. Rykov, O.Ya Grinberg, A.A. Dubinsky,
S.D. Pirozhkov, E.G. Rozantsev, I.E. Pokrovskaya, A.B. Shapiro, Synthesis of oxy-
[38] O. Vetrovoy, E. Rybnikova, Neuroprotective action of PHD inhibitors is pre-
dominantly HIF-1-independent, J. Neurochem. 150 (2019) 645e647, https://
doi.org/10.1111/jnc.14822.
,
acyl-, oxo-, N-oxides of oxo- and morpholillox- derived hydrogenated
quinolines and study of their radical analogues by EPR, Izv, USSR Acad. Sci.
Ser. chem. 8 (1979) 1800e1807.
14] PASS Online. http://www.pharmaexpert.ru/passonline/index.php (accessed
[39] S. Page, S. Raut, A. Al-Ahmad, Oxygen-glucose deprivation/reoxygenation-
induced barrier disruption at the human bloodebrain barrier is partially
mediated through the HIF-1 pathway, NeuroMolecular Med. 21 (2019)
414e431, https://doi.org/10.1007/s12017-019-08531-z.
[
28 February 2021).
[
15] S.O. Rotimi, O.A. Rotimi, I.B. Adelani, C. Onuzulu, P. Obi, R. Okungbaye, Ste-
vioside modulates oxidative damage in the liver and kidney of high fat/low
streptozocin diabetic rats, Heliyon 4 (2018), e00640, https://doi.org/10.1016/
j.heliyon.2018.e00640.
[40] M. Xia, Q. Ding, Z. Zhang, Q. Feng, Remote limb ischemic preconditioning
protects rats against cerebral ischemia via HIF-1a/AMPK/HSP70 pathway, Cell.
Mol. Neurobiol. 37 (2017) 1105e1114, https://doi.org/10.1007/s10571-016-
0444-2.
[
16] S.T. Shah, W.A. Yehya, O. Saad, K. Simarani, Z. Chowdhury, A.A. Alhadi, L.A. Al-
Ani, Surface Functionalization of Iron Oxide Nanoparticles with Gallic Acid as
Potential Antioxidant and Antimicrobial Agents, 7, 2017, p. 306, https://
doi.org/10.3390/nano7100306.
17] M. Roman, D.L. Roman, V. Ostafe, A. Ciorsac, A. Isvoran, Computational
assessment of pharmacokinetics and biological effects of some anabolic and
androgen steroids, Pharmaceut. Res. 35 (2018) 41, https://doi.org/10.1007/
s11095-018-2353-1.
[41] A. Kanavaki, K. Spengos, M. Moraki, P. Delaporta, C. Kariyannis,
I. Papassotiriou, A. Kattamis, Serum levels of S100b and NSE proteins in pa-
tients with non-transfusion-dependent thalassemia as biomarkers of brain
ischemia and cerebral vasculopathy, Int. J. Mol. Sci. 18 (2017) 2724, https://
doi.org/10.3390/ijms18122724.
[
[42] Z. Lou, A.P. Wang, X.M. Duan, G.H. Hu, G.L. Song, M.L. Zuo, Z.B. Yang, Upre-
gulation of NOX2 and NOX4 mediated by TGF-b signaling pathway exacer-
bates cerebral ischemia/reperfusion oxidative stress injury, Cell. Physiol.
Biochem. 46 (2018) 2103e2113, https://doi.org/10.1159/000489450.
[43] L. Wu, X. Xiong, X. Wu, Y. Ye, Z. Jian, Z. Zhi, Li Gu, Targeting oxidative stress
and inflammation to prevent ischemia-reperfusion injury, Front. Mol. Neu-
rosci. 13 (2020) 28, https://doi.org/10.3389/fnmol.2020.00028.
[44] A. Janyou, P. Wicha, J. Jittiwat, A. Suksamrarn, C. Tocharus, J. Tocharus,
Dihydrocapsaicin attenuates blood brain barrier and cerebral damage in focal
cerebral ischemia/reperfusion via oxidative stress and inflammatory, Sci. Rep.
7 (2017) 10556, https://doi.org/10.1038/s41598-017-11181-5.
[45] M. Gu, M.M. Iravani, J.M. Cooper, D. King, P. Jenner, A.H. Schapira, Pramipexole
protects against apoptotic cell death by non-dopaminergic mechanisms,
J. Neurochem. 91 (2004) 1075e1081, https://doi.org/10.1111/j.1471-
4159.2004.02804.x.
[
18] ProTox-II - prediction of toxicity of Chemicals. http://tox.charite.de/protox_II/
index.php?site¼home (accessed 30 January 2020).
[
19] S. Chacko, S. Samanta, A novel approach towards design, synthesis and
evaluation of some Schiff base analogues of 2-aminopyridine and 2-
aminobezothiazole against hepatocellular carcinoma, Biomed. Pharmac-
other. 89 (2017) 162e176, https://doi.org/10.1016/j.biopha.2017.01.108.
20] L. Mendes, S. Ang eꢀ lica, P. Schiavon, H. Milani, R. Maria, W. Oliveira, Cognitive
[
[
impairment and persistent anxiety-related responses following bilateral
common carotid artery occlusion in mice, Behav. Brain Res. 249 (2013) 28e37,
https://doi.org/10.1016/j.bbr.2013.04.010.
21] I.M. Piskarev, S.V. Trofimova, I.P. Ivanova, O.E. Burkhina, Investigation of the
level of free-radical processes in substrates and biological samples using
induced chemiluminescence, Biophysics 60 (2015) 400e408, https://doi.org/
[46] J. Zhu, V.A. Carozzi, N. Reed, R. Mi, P. Marmiroli, G. Cavaletti, A. Hoke,
Ethoxyquin provides neuroprotection against cisplatin-induced neurotoxicity,
Sci. Rep. 6 (2016) 28861, https://doi.org/10.1038/srep28861.
10.1134/S0006350915030148.
[
[
[
[
22] A.Z. Reznick, L. Packer, Oxidative damage to proteins: spectrophotometric
method for carbonyl assay, Methods Enzymol. 233 (1994) 357e363, https://
doi.org/10.1016/S0076-6879(94)33041-7.
23] R.O. Recknagel, A.K. Ghoshal, Lipoperoxidation of rat liver microsomal lipids
induced by carbon tetrachloride, Nature 210 (1966) 1162e1163, https://
doi.org/10.1038/2101162a0.
[47] M.I. Fern aꢀ ndez-Bachiller, C. P eꢀ rez, G.C. Gonz ꢀa lez-Mu n~ oz, S. Conde, M.G. L oꢀ pez,
M. Villarroya, A.G. García, M.I. Rodríguez-Franco, Novel tacrineꢁ8-
hydroxyquinoline hybrids as multifunctional agents for the treatment of
Alzheimer's disease, with neuroprotective, cholinergic, antioxidant, and
copper-complexing properties, J. Med. Chem. 53 (2010) 4927e4937, https://
doi.org/10.1021/jm100329q.
24] K. Suzuki, H. Ota, S. Sasagawa, T. Sakatani, T. Fujikura, Assay method for
myeloperoxidase in human polymorphonuclear leukocytes, Anal. Biochem.
[48] K. Shi, D.C. Tian, Z.G. Li, A.F. Ducruet, M.T. Lawton, F.D. Shi, Global brain
inflammation in stroke, Lancet Neurol. 18 (2019) 1058e1066, https://doi.org/
10.1016/S1474-4422(19)30078-X.
132 (1983) 345e352, https://doi.org/10.1016/0003-2697(83)90019-2.
25] M. Nishikimi, N.A. Rao, K. Yagi, The occurrence of superoxide anion in the
reaction of reduced phenazine methosulphate and molecular oxygen, Bio-
chem. Biophys. Res. Co 46 (1972) 849e864, https://doi.org/10.1016/s0006-
[49] D.E. L oꢀ pez, S.J. Ballaz, The role of brain cyclooxygenase-2 (Cox-2) beyond
neuroinflammation: neuronal homeostasis in memory and anxiety, Mol.
Neurobiol. 57 (2020) 5167e5176, https://doi.org/10.1007/s12035-020-
02087-x.
291x(72)80218-3.
[
[
[
[
26] L. G oꢀ th, A simple method for determination of serum catalase activity and
revision of reference range, Clin. Chim. Acta 196 (1991) 143e151, https://
doi.org/10.1016/0009-8981(91)90067-m.
[50] X. Li, L. Su, X. Zhang, C. Zhang, L. Wang, Y. Li, Y. Zhang, T. He, X. Zhu, L. Cui,
Ulinastatin downregulates TLR4 and NF-kB expression and protects mouse
brains against ischemia/reperfusion injury, Neurol. Res. 39 (2017) 367e373,
https://doi.org/10.1080/01616412.2017.1286541.
[51] H.Y. Wei, X. Ma, Tamoxifen reduces infiltration of inflammatory cells,
apoptosis and inhibits the IKK/NF-kB pathway after spinal cord injury in rats,
Neurol. Sci. 35 (2014) 1763e1768, https://doi.org/10.1007/s10072-014-1828-
z.
[52] G. Yu, Y. Liang, S. Zheng, H. Zhang, Inhibition of myeloperoxidase by N-acetyl
lysyltyrosylcysteine amide reduces oxidative stressemediated inflammation,
neuronal damage, and neural stem cell injury in a murine model of stroke,
J. Pharmacol. Exp. Therapeut. 364 (2018) 311e322, https://doi.org/10.1124/
jpet.117.245688.
27] D.E. Paglia, W.N. Valentine, Studies on the quantitative and qualitative char-
acterization of erythrocyte glutathione peroxidase, Lab. Clin. Med. 70 (1967)
158e169.
28] G. Zanetti, Rabbit liver glutathione reductase. Purification and properties,
Arch. Biochem. Biophys. 198 (1979) 241e246, https://doi.org/10.1016/0003-
9861(79)90415-6.
29] M. Warholm, C. Guthenberg, C. Von Bahr, B. Mannervik, Glutathione trans-
ferases from human liver, Methods Enzymol. 113 (1985) 499e504, https://
doi.org/10.1016/s0076-6879(85)13065-x.
[30] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959)
70e77, https://doi.org/10.1016/0003-9861(59)90090-6.
[
31] J.M. Valdez, A.F.M. Johnstone, J.E. Richards, J.E. Schmid, J.E. Royland,
P.R.S. Kodavanti, Interaction of diet and ozone exposure on oxidative stress
parameters within specific brain regions of male brown Norway rats, Int. J.
Mol. Sci. 20 (2018) 11, https://doi.org/10.3390/ijms20010011.
[53] A.B. Uzdensky, Apoptosis regulation in the penumbra after ischemic stroke:
expression of pro- and antiapoptotic proteins, Apoptosis 24 (2019) 687e702,
https://doi.org/10.1007/s10495-019-01556-6.
[54] F. Yin, H. Zhou, Y. Fang, C. Li, Y. He, L. Yu, H. Wan, J. Yang, Astragaloside IV
alleviates ischemia reperfusion-induced apoptosis by inhibiting the activation
of key factors in death receptor pathway and mitochondrial pathway,
[32] I.D. Desai, F.E. Martinez, Bilirubin interference in the colorimetric assay of
plasma vitamin E, Clin. Chim. Acta 154 (1986) 247e250, https://doi.org/
10.1016/0009-8981(86)90040-9.
J.
Ethnopharmacol.
248
(2020)
112319,
https://doi.org/10.1016/
[
33] The department of organic Chemistry of Voronezh state University. http://
www.chem.vsu.ru/files/upload/files/vgu_scrining.db, accessed: 11 April 2021.
34] E.D. Kryl'skii, T.N. Popova, O.A. Safonova, A.O. Stolyarova, G.A. Razuvaev,
M.A. Pinheiro de Carvalho, Transcriptional regulation of antioxidant enzymes
activity and modulation of oxidative stress by melatonin in rats under cere-
bral ischemia/reperfusion conditions, Neuroscience 406 (2019) 653e666,
https://doi.org/10.1016/j.neuroscience.2019.01.046.
j.jep.2019.112319.
[55] D. Coimbra-Costa, N. Alva, M. Duran, T. Carbonell, R. Rama, Oxidative stress
and apoptosis after acute respiratory hypoxia and reoxygenation in rat brain,
Redox Biol. 12 (2017) 216e225, https://doi.org/10.1016/j.redox.2017.02.014.
[56] S.M. Davis, K.R. Pennypacker, Targeting antioxidant enzyme expression as a
therapeutic strategy for ischemic stroke, Neurochem. Int. 107 (2017) 23e32,
https://doi.org/10.1016/j.neuint.2016.12.007.
[
þ
[
35] A.V. Makeeva, T.N. Popova, Effects of thioctic acid on the glutathione redox
cycle after cerebral ischemia-reperfusion in rats, Neurochem. J. 3 (2009)
[57] H.J. Cho, H.Y. Cho, J.W. Park, O.S. Kwon, H.S. Lee, T.L. Huh, B.S. Kang, NADP -
dependent cytosolic isocitrate dehydrogenase provides NADPH in the pres-
ence of cadmium due to the moderate chelating effect of glutathione, JBIC J.
Biol. Inorgan. Chem. 23 (2018) 849e860, https://doi.org/10.1007/s00775-018-
1581-5.
110e117, https://doi.org/10.1134/S1819712409020068.
[
36] A.V. Makeeva, T.N. Popova, O.V. Sukhoveeva, L.F. Panchenko, Effects of gua-
nidine derivatives on the activities of superoxide dismutase and catalase
during postischemic reperfusion in the rat brain, Neurochem. J. 4 (2010)
[58] M. Wallerta, M. Zieglera, X. Wang, A. Maluenda, X. Xu, M.L. Yap, R. Witte,
217e221, https://doi.org/10.1134/S1819712410030098.
C. Giles, S. Kluge, M. Hortmann, J. Zhang, P. Meikle, S. Lorkowski, K. Peter, a-
145

E.D. Kryl'skii, E.E. Chupandina, T.N. Popova et al.
Biochimie 186 (2021) 130e146
54 (2017) 6006e6017, https://doi.org/10.1007/s12035-016-0111-0.
[61] Y. Xing, A. Li, Y. Yang, X. Li, L. Zhang, H. Guo, The regulation of FOXO1 and its
role in disease progression, Life Sci. 193 (2018) 124e131, https://doi.org/
10.1016/j.lfs.2017.11.030.
Tocopherol preserves cardiac function by reducing oxidative stress and
inflammation in ischemia/reperfusion injury, Redox Biol. 26 (2019) 101292,
https://doi.org/10.1016/j.redox.2019.101292.
[
[
59] X. Wu, H. Dai, L. Liu, C. Xu, Y. Yin, J. Yi, M.D. Bielec, Y. Hana, S. Li, Citrate
reduced oxidative damage in stem cells by regulating cellular redox signaling
pathways and represent a potential treatment for oxidative stress-induced
diseases, Redox Biol. 21 (2019) 101057, https://doi.org/10.1016/
j.redox.2018.11.015.
[62] T. Zhang, D.H. Kim, X. Xiao, S. Lee, Z. Gong, R. Muzumdar, V. Calabuig-Navarro,
J. Yamauchi, H. Harashima, R. Wang, R. Bottino, J.C. Alvarez-Perez, A. Garcia-
Oca n~ a, G. Gittes, H.H. Dong, FoxO1 plays an important role in regulating
b-cell
compensation for insulin resistance in male mice, Endocrinology 157 (2016)
1055e1070, https://doi.org/10.1210/en.2015-1852.
60] R. Zhang, M. Xu, Y. Wang, F. Xie, G. Zhang, X. Qin, Nrf2da promising thera-
peutic target for defensing against oxidative stress in stroke, Mol. Neurobiol.
146