ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 956-974 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 1043-1062.
956
Protective Role of Plastoquinone in the Early Stages
of Second-Degree Thermal Skin Burn
Nadezhda I. Pashkevich
1
, Ekaterina S. Pykhova
2
,
Alexander A. Ashikhmin
2
, Daria V. Vetoshkina
2
, Sergey S. Osochuk
1,a
*,
and Maria M. Borisova-Mubarakshina
2,b
*
1
Vitebsk State Order of Peoples’ Friendship Medical University, 210009 Vitebsk, Republic of Belarus
2
Institute of Basic Biological Problems, Russian Academy of Sciences,
142290 Pushchino, Moscow Region, Russia
a
e-mail: oss62@mail.ru
b
e-mail: mubarakshinamm@gmail.com
Received April 22, 2025
Revised July 9, 2025
Accepted July 9, 2025
Abstract— Thermal burns of the skin are associated not only with local tissue alterations but also with the
development of systemic disorders, which promote generalization of inflammatory processes. In particular,
burn injury leads to an overproduction of reactive oxygen species, activation of free-radical oxidation, and
lipid peroxidation. This study investigated the protective role of plastoquinone, a natural plant antioxidant,
on the morphological condition of the skin and on the shape and aggregation of erythrocytes in rats with
second-degree thermal burns. Thermal burn resulted in the decrease in epidermis thickness, increase in the
number of hyperemic vessels, damaged hair follicles and sebaceous glands. Application of plastoquinone, in-
corporated into liposomes, onto the damaged skin had a protective effect on the skin structures; in the case
of liposomes applied without plastoquinone, the protective effect was less pronounced. In addition, thermal
burn altered the state of erythrocytes, leading to their deformation and aggregation. Plastoquinone in lipo-
somes applied topically or administered intravenously showed a protective effect on erythrocytes comparable
to that of ubiquinone, preventing the development of burn-induced erythrocyte shape alterations. However,
only plastoquinone administered intravenously completely prevented erythrocyte aggregation, thus eliminat-
ing negative effects of the burn injury on the functional activity of erythrocytes, indicating the potential of
plant-derived plastoquinone as an effective agent in burn injury management.
DOI: 10.1134/S0006297925601297
Keywords: thermal burn, reactive oxygen species (ROS), liposomes, antioxidants, quinones, plastoquinone
* To whom correspondence should be addressed.
INTRODUCTION
A thermal burn is an injury to the skin and un-
derlying tissues, accompanied by complex pathophys-
iological changes including development of oxidative
stress caused by enhanced generation of reactive ox-
ygen species (ROS) and activation of inflammatory
cascades [1]. ROS include superoxide anion radical
(O
2
•−
) and its protonated form, hydroperoxyl radical
(HO
2
•
), hydrogen peroxide (H
2
O
2
), hydroxyl radical
(HO
•
), singlet oxygen (
1
O
2
), as well as hydroperoxides
and radicals of organic molecules. All the cited ROS
could be produced in animal cells, including
1
O
2
in
the reaction of spontaneous dismutation of O
2
•−
[2].
Under conditions of burn trauma, the balance be-
tween the rate of ROS formation and the activity of
antioxidant system in an organism is disrupted. Due
to their high reactivity, ROS induce lipid peroxida-
tion (LPO), causing damage to cell membranes and
leading to cell dysfunction. ROS also cause oxidative
modifications of amino acid residues in proteins and
are capable of oxidizing carbohydrates and DNA [3].
In addition to local tissue damage, thermal burns neg-
atively affect membranes of blood cells, erythrocytes
membranes in particular. Structural changes have
been reported in the erythrocytes passing through
PROTECTIVE ROLE OF PLASTOQUINONE 957
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
the inflammation region: deformability has been
observed [4] as well as formation of cell aggregates
shaped like a roll of coins (rouleaux) [5] due to mod-
ification of erythrocyte cytoskeleton protein, band
3, which could cause microvascular occlusion and
hypoxia [6]. These changes occur mainly due to the
effects of ROS produced by the damaged tissues, as
well as by neutrophils (see below) [7]. Changes in the
shape and aggregation of erythrocytes decrease their
oxygen releasing capability, i.e. impair their oxygen
transporting function [8].
Changed erythrocytes are mainly subjected to
degradation through hemolysis. Hemoglobin released
during erythrocyte degradation, i.e., extracellular
“free” hemoglobin, is a contributing factor to the devel-
opment of multiple organ dysfunction. Concentration
of free hemoglobin in blood depends on the severity
of skin burn injury [9]. In turn, free hemoglobin is
a factor initiating production of ROS and proinflam-
matory cytokines [10] and, hence, as it circulates sys-
temically in the bloodstream, contributes to second-
ary mitochondrial damage in the kidneys, heart, and
muscle tissue [11], initiating the so-called secondary
production of ROS or secondary oxidative stress [12].
Mitochondrial damage is accompanied by the re-
lease of mitochondrial DNA (mtDNA), which is most
sensitive to the action of ROS due to the absence of
histone proteins in its structure [13]. The presence of
damaged mtDNA is closely associated with the induc-
tion of key proinflammatory cytokines, interleulin-6
(IL-6) in particular, as well as tumor necrosis factor-α
(TNF-α) [14], which contribute to chronic inflamma-
tion and deterioration of tissue regeneration processes
[15, 16]. Furthermore, it is known that mtDNA can
enter the bloodstream, and an increased concentra-
tion of circulating mtDNA is associated with multiple
organ dysfunction syndrome, which is one of the main
causes of death of the patients with severe burn trau-
ma [17, 18]. Thus, protection of erythrocytes against
burn-induced deformation significantly reduces the
risk of multiple organ dysfunction.
Considering destructive role of ROS in the de-
velopment of multiple organ dysfunction in the case
of burns described above, it seems relevant to in-
vestigate the use of antioxidant agents capable of
minimizing the level of ROS and, thus, preventing
primary and secondary tissue damage. Low mo-
lecular weight antioxidants are present in animal
cells for protection against the endogenously formed
ROS including glutathione, reduced pyridine nucleo-
tides, bilirubin, uric acid, ubiquinone (UQ), as well
as antioxidant enzymes: superoxide dismutases, cat-
alase, peroxidases including peroxiredoxins. Some
low molecular weight antioxidants such as ascorbic
acid, tocopherols, flavonoids, and carotenoids are ob-
tained through the diet. The majority of antioxidant
systems are localized in the aqueous compartments
of the cell, and not in the lipid hydrophobic mem-
brane layers, where electron transport chains (in the
inner mitochondrial membrane and in cytoplasmic
membrane) are located, in which ROS could be gen-
erated with higher rates than in the aqueous phase.
Many researchers emphasize the importance of pro-
tection of the cell membranes against the destructive
actions of ROS for treating various diseases [19, 20].
In live cells utilization of ROS in the membranes is
realized with participation of tocopherol and UQ [21,
22]. UQ in a reduced form, ubihydroquinone, neutral-
izes ROS responsible for LPO initiation, such as O
2
•−
and HO
2
•
[23]. Moreover, antioxidant function of UQ
in LPO prevention is realized via reactions with LPO
products as well – peroxide-, alkoxyl, and lipid radi-
cals [24-26]. Protective effects of the exogenously ad-
ministered UQ have been demonstrated in many dis-
eases including burns [27-32]. The synthesized UQ has
been actively investigated as a potential therapeutic
agent in such pathologies as heart failure [33], neu-
rodegenerative diseases [27, 28], as well as different
types of cancer [30]. UQ investigations include both
preclinical models and human clinical trials, including
treatment of burns [31, 32]. UQ is also widely used
in cosmetology as an ingredient with antioxidant
properties.
At present, plastoquinone (PQ, component of
the photosynthetic electron transport chain in chlo-
roplasts) attracts more and more attention, together
with other natural and synthetic analogues of PQ (see
reviews [34-41]). This could be due to the fact that
PQ activity in prevention of LPO was shown to be
higher than activity of UQ and even tocopherol [42].
Taking into consideration high antioxidant activity of
PQ, a number of synthetic derivatives of PQ have been
developed and their effectiveness was tested both
in vitro and in vivo. The most effective ones were com-
pounds known as Skulachev ions that are composed
of a synthetic analogue of PQ, decylplastoquinone,
linked to triphenylphosphonium for mitochondria tar-
geting. Effectiveness of Skulachev ions was observed
at very low concentrations due to their high parti-
tion coefficient between the aqueous and hydrophobic
phases, as well as ability of decylplastoquinone to be
reduced to decylplastohydroquinone in the complexes
I and II of the mitochondrial respiratory chain [43].
The developed synthetic derivatives of PQ continue
to be investigated [44]. In addition, information on
other synthetic variants of PQ is available in the lit-
erature, including various derivatives of 1,4-benzo-
quinone, which lack or have a modified isoprenoid
fragment. Such halogenated and non-halogenated
compounds demonstrate pronounced cytotoxic prop-
erties with regard to cancer cells [45, 46] and also ex-
hibit antimicrobial [47, 48] and antifungal effects [49].
PASHKEVICH et al.958
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
At the same time, use of these synthetic derivatives
of PQ in treating burns has not been investigated
in detail.
Neutrophils represent an important source of free
radicals in the early stages of thermal burn damage;
they initiate the so-called respiratory burst due to en-
hanced production of O
2
•−
by NADPH oxidases of the
plasma membrane (see review [1]). In this regard, use
of synthetic derivatives of PQ targeting mitochondria
could be not very reasonable. Considering all the above,
it seems more logical to use PQ without mitochondria
targeting in this case.
However, there is a considerable lack of data in
the literature on protective effects of isolated and pu-
rified plant PQ. In the course of our previous studies
antioxidant properties of PQ in plant leaves have been
investigated (see review [50]). Antioxidant function of
PQ, similar to UQ, is realized by PQ in the fully re duced
form (plastohydroquinone (PQH
2
)); PQH
2
effectively
neutralizes O
2
•−
and HO
2
•
, as well as
1
O
2
(see review
[50]), that protects thylakoid membrane against LPO
and prevents pigment bleaching [51]. Neutralization of
O
2
•−
by the PQH
2
molecules is assumed to be one of the
main reactions demonstrating its antioxidant activity
in a photosynthesizing cell [52]; rate constant of this
second order reaction is estimated to be ~10
7
M
−1
s
−1
[53]. Rate constant of the reaction with
1
O
2
is even
higher – ~10
8
M
−1
s
−1
[54], however, under stress con-
ditions O
2
•−
is generated in leaves with higher rates
than
1
O
2
, hence, the probability of PQH
2
reaction
with O
2
•−
is significantly higher; significant formation
of
1
O
2
is typical only for more severe conditions,
which are not so common in nature (see review [35,
55]). The possibility of PQH
2
reaction with H
2
O
2
has
been also demonstrated [56, 57]. However, as empha-
sized by the authors [56], reaction rate constant in
this case is rather low ~10
4
M
−1
s
−1
. Despite the low-
rate constant, H
2
O
2
effectively oxidizes the PQ pool in
thylakoids in the dark after illumination [58].
Hence, PQ is a promising natural antioxidant
compound with high capability for neutralization
of ROS. Moreover, PQ, similar to UQ, exhibits highly
pronounced membranotropic properties inducing or-
dering of membrane structure [59]. Owing to these
properties, PQ appears to be a promising candidate
protecting cellular structures in burn trauma. This
study aims to evaluate protective properties of PQ
isolated and purified from higher plants under con-
ditions of thermal skin burn damage in laboratory an-
imals. Possible mechanisms of cytoprotective effects
of PQ in burns are considered in the present study
including its effect on inflammatory response and ox-
idative stress.
Traditionally liposomes are employed for deliv-
ery of lipophilic compounds. It is known that ben-
zoquinones with a long side isoprenoid chain, such
as PQ and UQ, are effectively incorporated into lipo-
somes and preserve their antioxidant activity [60-63].
Compositions of membrane lipids and of fatty acids
in cells change in burns, that includes decrease of the
content of cholesterol esters, phospholipids, amount
of essential (ω-3 and ω-6) polyunsaturated fatty ac-
ids (PUFA), as well as oxidation of PUFA [64, 65].
Considering this fact, use of liposomes due to their
composition and size could facilitate replenishment
of the content of essential ω-3 and ω-6 PUFA and
cholesterol. In our previous study on skin thermal
burns in rats, liposomes with varying content of
phosphatidylcholine were used; it was found out that
liposomes, prepared from lecithin with 90% of phos-
phatidylcholine, display more pronounced protective
activity in comparison with the liposomes, prepared
from lecithin with 26% of phosphatidylcholine [66].
Therefore, goal of this study was evaluation of pro-
tective properties of PQ incorporated into liposomes
prepared from cholesterol and lecithin, containing
90% of phosphatidylcholine.
MATERIALS AND METHODS
Enzyme-linked immunosorbent assay of proin-
flammatory cytokines IL-6 and TNF-α content in
human blood. Study was conducted using blood cells
of volunteer donors (males and females) aged 28-38
years. To determine cytokine levels, freshly drawn
venous blood stabilized with heparin was used. For
the experiments, 100 μl of blood was diluted 10-fold
in RPMI-1640 medium (PanEco, Russia) and incubated
in 12-well plates (Nunc, Denmark) at 37°C in a 5%
CO
2
atmosphere for 6 h. Total volume of incubation
mixture was 1ml. Hexa-acetylated lipopolysaccharide
(LPS) from Gram-negative bacterium Escherichia coli
(E. coli) O55:B5 was used as an inducer of TNF-α and
IL-6 [67]. Samples were incubated in the presence of:
100ng/ml of LPS, or 1 µM of PQ, or 10μM PQ, or 1%
ethanol, or without any added compounds (control
group). After incubation plates were centrifuged at
1500g for 15 min using a microplate rotor centrifuge
centrifuge (BioSan, Latvia). The obtained blood plas-
ma was diluted with a RPMI-1640 medium: for TNF-α
assay – 5-fold, for IL-6 – 35-fold. Cytokine production
was evaluated with an enzyme-linked immunosorbent
assay using reagent kits from Vektor-Best (Russia). Op-
tical density was recorded with a STAT FAX 3200 mi-
croplate reader (Awareness Technology Inc., USA) at
450nm. Cytokine concentration was calculated based
on calibration curve constructed with standard sam-
ples provided by the manufacturer.
Laboratory animals. Experimental studies with
laboratory animals were conducted based on collab-
oration agreement between the Vitebsk State Order
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BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
of Peoples’ Friendship Medical University (VSMU) and
Pushchino Scientific Center for Biological Research,
Russian Academy of Sciences, in the research labora-
tory of VSMU.
Seven-month-old white male outbred rats of
the Rattus Muridae line (n = 74 for 3-h experiments,
and n = 78 for 24-h experiments) with mass 190-364 g
were used in the study. Animals were kept under stan-
dard conditions in a vivarium (temperature 22 ± 2°C
and relative humidity 50-65%, 12-hour light cycle,
free access to feed and water). Animals were not fed
during the night before experiment.
Isolation and purification of PQ from higher
plants. PQ was isolated according to the method de-
scribed previously [68-71] with some modifications
to obtain PQ in amount sufficient for conducting ex-
periments. In particular, the step of preliminary chlo-
rophyll removal from the extract using aluminum
oxide was modified. Freshly-cut leaves of amaranth/
spinach/pea with removed stems were homogenized
in a Braun 4184 blender (Braun, Czech Republic) in
a buffer (50 mM Hepes-KOH (pH 7.6), 20 mM NaCl,
5 mM MgCl
2
). Homogenate was filtered through two
layers of nylon fabric and extracted with acetone at a
ratio of 1 : 2 (1 part of filtrate (100-200ml) and 2 parts
of acetone) and 30 ml of hexane followed by 5-min
incubation for layer separation. Next upper hexane
phase was collected and 30ml of hexane were added
followed by extraction and sample collection. Volume
of the hexane mixture was reduced to 1 ml in a ro-
tary evaporator. The concentrate was applied onto an
aluminum oxide chromatography column pre-equili-
brated with hexane. Elution was carried out first with
hexane:acetone 95 : 5, and the PQ-containing fraction
was collected until the appearance of an orange carot-
enoid band. Then, a second eluent (hexane : acetone,
90 : 10) was applied, and the PQ-containing fraction
was further collected. The pooled fraction was con-
centrated in a rotary evaporator and analyzed at
40°C using Shimadzu HPLC system (Shimadzu, Japan)
with a reversed phase C18 column (Waters Spher-
isorb 5 µm ODS2, size 4.6×250 mm, Waters, USA).
Acetonitrile: ethanol mixture 3 : 1 (V/V) was used as
a mobile phase, flow rate was 1,5 ml/min. Retention
time for PQ was 22.4min. PQ concentration was eval-
uated spectrophotometrically based on absorption at
255nm. Extinction coefficient was 18mM
−1
∙cm
−1
[59].
To confirm the compound structure a high-resolution
mass spectrometer Orbitrap Elite ETD (Thermo Sci-
entific, Germany) was used. The obtained spectrum
was compared with theoretical molecular weight of
PQ and literature data confirming identification of the
isolated compound as PQ.
Preparation of liposomes and incorporation of
PQ or UQ into liposomes. Liposomes were prepared
based on lecithin containing 90% of phosphatidyl-
choline (PanReac AppliChem, Spain, Germany) and
cholesterol (Sigma, USA) at a ratio 5 : 1 according to
the method described by Khoshneviszadeh etal. [72].
Lecithin (7.78%) and cholesterol (1.5%) were dissolve-
din chloroform and methanol at a ratio 3 : 1. Ethanol
solution of PQ or UQ (Sigma, USA) to achieve final
concentrations 5 or 50 µM was added to this mix-
ture; in the case of preparation of empty liposomes
same volume of ethanol was added to liposomes, as
in the case of PQ or ubiquinone solutions. Thin lipid
film was formed using vacuum evaporation in a ro-
tary evaporator at 45°C. The formed film was slowly
re-suspended in a 0.1 M potassium-phosphate buffer
with pH 8.0 for topical application, or in isotonic
0,9% NaCl solution for intravenous administration.
Obtained liposome solutions were extruded through
an Avanty 400-nm laboratory mini-extruder (Avanti
Polar Lipids, USA). Liposome size was measured by
dynamic light scattering using with a Zetasizer Nano
(Malvern Instruments, UK). The mean size of lipo-
somes without PQ was 580.23 ± 137.72 nm, and with
PQ – 695.24 ± 167.93 nm (Fig. 1). There were no sta-
tistically significant differences between the sizes of
obtained liposomes (p-value 0.1564 (t)).
Modeling of thermal burn. Animals were sep-
arated into 8 groups, which were examined 3 and
24 h after inducing burn:
1. Control – intact animals, not subjected to any
exposures (for 3-h experiments n = 12, for 24-h exper-
iments n = 12);
2.Sham control – animals subjected to manipula-
tions similar to those in experimental procedures, but
without inducing thermal burn (for 3-h experiments
n = 10, for 24-h experiments n = 10);
3. Thermal burn without treatment — animals
with induced second-degree thermal burns without
following treatment (for 3-h experiments n = 10, for
24-h experiments n = 10);
4. Thermal burn and treatment with empty li-
posomes – animals with thermal burn, which were
treated with liposomes without active agent topical
application (for 3-h experiments n = 8, for 24-h ex-
periments n = 9);
5. Thermal burn and 50 µM PQ, topical applica-
tion – animals with thermal burn treated with topical
application of liposomes with 50 µM PQ (for 3-h exper-
iments n = 8, for 24-h experiments n = 9);
6. Thermal burn and 5 µM PQ, intravenous ad-
ministration – animals with thermal burn treated with
intravenous administration of liposomes with 5 µM
PQ (for 3-h experiments n = 10, for 24-h experiments
n = 10);
7. Thermal burn and 50 µM UQ, topical applica-
tion – animals with thermal burn treated with topical
application of liposomes with 50 µM UQ (for 3-h ex-
periments n = 8, for 24-h experiments n = 9);
PASHKEVICH et al.960
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 1. Size distribution of the obtained liposomes. a) Empty liposomes; b) liposomes with incorporated PQ.
8. Thermal burn and 5 µM UQ, intravenous ad-
ministration – animals with thermal burn treated with
intravenous administration of liposomes with 5 µM
ubiquinone (for 3-h experiments n = 8, for 24-h ex-
periments n = 9).
To model burn, animals were anesthetized by
ketamine according to approved recommendations
[73-75]. Thermal injury was introduced by applying
a metal strip heated to 150°C to the shaved dorsal
skin for 4 min, which morphologically corresponded
to second-degree burn according to The International
Classification of Diseases (ICD), 10th revision (burns
of degree IIIA according the previously used classi-
fication) [76]. An instrument for burn modeling was
constructed in OAO KB Display (Vitebsk, Republic of
Belarus) in the framework of collaboration agreement
with the Vitebsk State Order of Peoples’ Friendship
Medical University. Burn was introduced on a back
of an animal in the preselected region. Burn surface
area was 30% of the total body surface area (TBSA).
To calculate TBSA, the Meeh formula (1) was used:
S = k × W
2/3
, (1)
where S – total body surface area, cm
2
; k – Meeh
constant equaling to 9.46 [77]; W – body mass of an
animal, kg.
Immediately after thermal injury, the test com-
pounds were administered either topically or intrave-
nously. Animals in group 4 were treated with 0.45ml
of suspension of empty liposomes per burn area.
Animals in groups 5 and 7 were treated with 0.45ml
of suspension of liposomes with incorporated 50 µM
PQ or UQ, respectively. After application, the liposomal
suspension was polymerized in air, forming a dense
dry film. To prevent interactions and traumatization
of burn surface animals were housed individually in
separate cages. Animals in groups 6 and 8 were im-
mediately after burn initiation treated with lateral tail
vein injection (0.45 ml) of liposomal forms of PQ or
UQ at concentration 5 µM, administered into the lat-
eral tail vein, using isotonic 0.9% NaCl as a diluent.
Animals were euthanized by decapitation using a
laboratory guillotine following induction of ether an-
esthesia. Euthanasia was carried out 3 or 24 h after
modeling thermal burn. Blood was collected into hep-
arinized tubes for further analysis during time period
of preservation of spinal automatism. Biopsy samples
from the burn injury area were obtained using a
sharp instrument and a technique that minimized tis-
sue deformation, for further histological examination.
Assessment of erythrocyte count and mor-
phology. The number and morphological alterations
of erythrocytes were evaluated using in a Goryaev
counting chamber under a Leica DM 2000 microscope
equipped with an 8× objective and a 10× eyepiece
(Leica Microsystems, Germany) [78]. To examine num-
ber and shape of erythrocytes whole blood was used,
PROTECTIVE ROLE OF PLASTOQUINONE 961
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
which was diluted 200-fold in 0.9% NaCl (to prevent
hemolysis in the tube).
Assessment of erythrocyte aggregation. The
number of aggregated erythrocytes was determined
according to established methods using a Goryaev
counting chamber and a Leica DM 2000 microscope
equipped with a 20× objective (Leica Microsystems,
Germany) [79]. Whole blood was used diluted 200-fold
with 0.9% NaCl (to prevent hemolysis in the tube).
Determination of haptoglobin concentration.
Determination of haptoglobin concentration in blood
plasma was carried out using commercial ELISA kit
(cat. no. E-EL-R0473, Elabscience, China) according to
the manufacturer’s instructions. The analysis was per-
formed using theVityaz F300TP immunoassay analyzer
(Republic of Belarus).
Preparation of skin biopsies and morphologi-
cal analysis. Skin samples were fixed in 10% neu-
tral buffered formalin at room temperature prior to
histological processing. After standard dehydration in
a graded series of ethanol solutions with increasing
concentration and paraffin embedding, samples were
washed with running water for several hours, next,
sections with 3-4 µm thickness were prepared from
block samples with a Leica RM 125 rotary microtome.
Histological samples were stained with hematoxylin
and eosin according to the standard technique and ex-
amined in a Leica DM 2500 microscope, 10× eyepiece,
20× and 40× objectives (Leica Microsystems, Germa-
ny), equipped with a Leica DFC 320 digital camera.
The number of damaged elements was counted in 10
fields of view, from which average numbers of skin
elements were calculated.
Measurement of spontaneous chemilumines-
cence. Whole blood samples collected after decapi-
tation with heparin added as an anticoagulant were
examined. An aliquot (0.1 ml) of a heparinized blood
was placed into a cuvette. Spontaneous chemilumines-
cence was recorded over a period of 2 min using a
Lum-100 device (Disoft, Russia). The area under the
chemiluminescence curve was used as the analytical
parameter.
Statistical data processing. Statistical analysis of
the data was carried out with the help of R version
4.0.5 (2021-03-31), and Origin 2021 software packages.
The distribution of the studied variables was assessed
using the Shapiro–Wilk test; in the case of Gaussian
distribution parametric statistical methods were used
for comparison, otherwise non-parametric methods
were used. Pairwise comparisons were carried out
based on the Student’s and Mann–Whitney–Wilcoxon
tests. Multiple comparisons were carried out with the
help of ANOVA (in the case of heterogeneity of disper-
sions of the investigated parameters Welch correction
was used) or Kruskal–Wallis H test. Post-hoc analysis
was carried out using Tukey’s test or Kruskal–Wallis
H test in Dunn modification with correction for mul-
tiple comparisons according to the Benjamini–Yekutieli
or Holm–Bonferroni methods. Differences were consid-
ered statistically significant at p<0.05. When present-
ing data as boxplots: boxes (rectangles) represent the
interquartile range (lower border corresponds to 25th
percentile and the upper one – 75th percentile), hor-
izontal line is a median, square – arithmetic mean,
whiskers – minimum and maximum values not con-
sidering outliers, diamond-shaped symbols – outliers.
RESULTS
Investigation of the effects of PQ addition on
production of proinflammatory cytokines IL-6 and
TNF-α in human blood. It is known that appearance
in blood of even small concentrations of LPS, which
possesses very high proinflammatory activity, could
result in the development of endotoxin shock increas-
ing production of early proinflammatory cytokines
such as TNF-α and IL-6 [67]. Therefore, the level of
TNF-α and IL-6 production serves as a marker of proin-
flammatory activity.
To evaluate content of TNF-α and IL-6 blood
plasma obtained from whole human blood was used.
As can be seen in Fig. 2, the addition of LPS from
E. coli (100 ng/ml) to whole blood resulted in signif-
icant, more than 100-fold, increase of production of
TNF-α. To establish possible proinflammatory effect of
PQ, blood was first incubated with PQ at final concen-
tration 1 or 10 µM. Considering that PQ was added
in ethanol solution (resulting in a final ethanol con-
centration of 1%), in the first step effect of ethanol
addition on production of TNF-α was evaluated; it was
revealed that ethanol in the used concentration did
not exhibit proinflammatory effect. It was established
in the following experiments that PQ in the investi-
gated concentrations also did not induce production
of TNF-α by the cells in whole blood (Fig. 2), which
indicated absence of manifestations of proinflamma-
tory activity of the plant-derived PQ.
In addition, it was shown in our study that ad-
dition of LPS to whole human blood significantly
activated IL-6 synthesis (Fig. 3). The obtained data
confirm the fact that the used LPS indeed has clearly
pronounced proinflammatory activity. Similar to the
case of TNF-α measurements, addition of PQ at both
concentrations did not cause enhanced production of
IL-6; the level of IL-6 production was similar to the
one measured in the control samples and in the sam-
ples with ethanol.
Thus, the results obtained in investigation of the
level of TNF-α and IL-6 in the samples of whole hu-
man blood provide reliable evidence that PQ does not
exhibit proinflammatory activity and may be further
PASHKEVICH et al.962
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 2. Effect of PQ on production of TNF-α in human blood
plasma. LPS – hexa-acylated lipopolysaccharide of Gram-
negative bacterium E.coli O55:B5. LPS concentration in incu-
bation medium was 100 ng/ml (see “Materials and Methods”
section). Mean values from two independent experiments
are presented, for each experiment blood was obtained from
two donors. Statistically significant differences between the
groups were evaluated with one-way ANOVA followed by
post-hoc comparison according to Holm–Bonferroni meth-
od. Columns designated by different letters (a, b), differ
significantly between each other with p < 0.05.
Fig. 3. Effect of PQ on production of IL-6 in human blood
plasma. LPS – hexa-acylated lipopolysaccharide of Gram-
negative bacterium E. coli O55:B5. LPS concentration in in-
cubation medium 100 ng/ml (see “Materials and Methods”
section). Mean values from two independent experiments
are presented, for each experiment blood was obtained from
two donors. Statistically significant differences between the
groups were evaluated with one-way ANOVA followed by
post-hoc comparison according to Holm–Bonferroni meth-
od. Columns designated by different letters (a, b), differ
significantly between each other with p < 0.05.
investigated as a protective agent in thermal burns
induced in laboratory animals.
Effect of PQ on morphological parameters of
skin in second-degree thermal burns. In the next
step of our study modeling of thermal burns affecting
30% of total body surface was carried out using labo-
ratory animals (rats); and potential protective effect of
PQ on the state of skin was evaluated. Outbred white
rats were used in experiments. For burn modeling the
shaved skin of animals was exposed to heat (4 min,
150°C) using a special device; the resulting injuries
morphologically corresponded to the second-degree
burns (see Materials and Methods section). Immedi-
ately after burn injury the affected skin region was
treated with either empty liposomes or liposomes
with incorporated PQ. One day (24 h) after the burn
injury, morphology of skin biopsies from experimental
animals were examined.
Thermal burn of animals (group 3) resulted in the
statistically significant decrease, almost 2-fold, of the
epidermis thickness, stratum corneum thickness, and
epidermis thickness excluding the stratum corneum,
compared both to the control animals (group 1) and
in comparison with the animals subjected to sham
control (group 2), i.e. animals subjected to all same
manipulations, but without exposure to heat (Table 1).
The revealed changes are associated with capacity of
burns to cause necrosis of epidermis and denaturation
of proteins in epidermal layer [80]. The animals of
group 3 have increased number of hyperemic vessels,
damaged hair follicles, and sebaceous glands in com-
parison with the animals of groups 1 and 2 (Fig. 4,
Table 1).
Liposomes without PQ (group 4) did not display
any protective effect on epidermis thickness, thickness
of the stratum corneum, and thickness of the epider-
mis without stratum corneum (Table 1). However,
numbers of hyperemic vessels, as well as number of
damaged hair follicles were lower after application of
empty liposomes on the skin of animals in comparison
with the animals of group 3. No damaged sebaceous
glands were observed in animals of group 4. The pre-
sented data are in agreement with the data reported
in our previous study obtained during comparison of
the effects of liposomes with different content of phos-
phatidylcholine in treatment of burn injuries, where
similar regularities were revealed for the liposomes
produced based on lecithin containing 90% phospha-
tidylcholine [66].
To investigate effects of PQ on the state of skin
and its associated elements such as hair follicles and
sebaceous glands, as well as on the vessels in dermis
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Fig. 4. Typical skin micro-preparations of the studied animal groups (stained with hematoxylin and eosin; objectives– 20×
and 40×). Group 1 –control (n = 12); Group 2 – sham control (n = 10); Group 3 – thermal burn without treatment (n = 10);
Group 4 – thermal burn and liposomes without active ingredient (n = 9); Group 5 – thermal burn and PQ 50 µM, topical
application (n = 9). One typical micro-preparation each is presented for Group1 and Group2. Labels: 1–epidermis, 2–der-
mis, 3 – hair follicles, 4 – sebaceous glands, 5 – hyperemic blood vessel.
during thermal burn, PQ purified from plant leaves
biomass was incorporated into the lipid phase of li-
posomes as described in the Materials and Methods
section. As can be seen from the results presented in
Table 1, topical application of liposomes with 50 µM
PQ (group 5) decreased the degree of damage of
skin layers after thermal burn: decrease of the total
thickness of epidermis was less pronounced than in
groups 3 and 4. The thickness of epidermis without
the stratum corneum in the group 5 was the same
as in groups 1 and 2. A positive, but less pronounced
effect was observed for the stratum corneum. More-
over, use of liposomes with incorporated PQ protected
hair follicles from damage: no damaged hair follicles
were observed in animals of group 5 as in the cases
of groups 1 and 2 (Table 1).
Hence, thermal burn of skin layers caused statisti-
cally significant changes in all investigated parameters.
Liposomes without PQ, by replenishing the PUFA defi-
cit, decreased the number of hyperemic vessels and
prevented damage to the sebaceous glands. The use
of liposomes with incorporated PQ facilitated preser-
vation of dermis structure, and completely prevented
damage to hair follicles.
Effect of PQ on the state of erythrocytes and
oxidative destruction in the blood of experimen-
tal animals with second-degree thermal burn. In
the next stage of the study the effect of skin burn on
the shape and aggregation of erythrocytes was inves-
tigated (Fig. 5), as well as the level of spontaneous
chemiluminescence in the blood of experimental
animals.
Skin thermal burn significantly increased num-
ber of erythrocytes with morphological alterations
(group 3) both in comparison with the group 1 and
the group 2 (Fig.6,a,b); at the same time, no changes
PASHKEVICH et al.964
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Table 1. Effect of the second-degree thermal burn on morphological parameters of the skin of animals 24 h
after thermal burn injury, and evaluation of protective effect of empty liposomes or liposomes with PQ on
the investigated parameters (details on group composition are presented in “Materials and Methods section”)
Group 1 Group 2 Group 3 Group 4 Group 5
Epidermis thickness, µm
29.42 ± 2.75 a 29.83 ± 3.33 a 12.33 ± 4.83 b 12.25 ± 4.03 b 23.08 ± 4,10 c
Thickness of the stratum corneum of the epidermis, µm
14.00 ± 1.65 a 15.92 ± 2.47 a 5.92 ± 2.84 b 5.00 ± 2.95 b 10.50 ± 2.65 ab
Thickness of the epidermis (excluding stratum corneum), µm
15.42 ± 2.07 a 13.92 ± 1.68 a 6.42 ± 2.27 b 7.25 ± 1.71 b 12.58 ± 1.78 a
Number of hyperemic vessels
0.00 ± 0.00 a 0.00 ± 0.00 a 14.50 ± 4.72 b 7.75 ± 1.22 c 6.42 ± 2.78 c
Number of damaged hair follicles
0.00 ± 0.00 a 0.00 ± 0.00 a 6.58 ± 1.51 b 3.42 ± 0.90 c 0.00 ± 0.00 a
Number of damaged sebaceous glands
0.00 ± 0.00 a 0.00 ± 0.00 a 6.42 ± 1.00 b 0.00 ± 0.00 a 0.00 ± 0.00 a
Note. Group 1 – control (n = 12); Group 2 – sham control (n = 10); Group 3 – thermal burn without treatment (n = 10);
Group 4 – thermal burn and liposomes without active ingredient (n = 9); Group 5 – thermal burn and PQ 50 µM, topical
application (n = 9). Values are presented as an arithmetic mean ± standard deviation of the mean (M ± SD). Statistical
processing was performed using non-parametric method (Kruskal–Wallis H test in Dunn modification with correction for
multiple comparisons according Benjamini–Yekutieli). Values denoted with different letters (a, b, c) are statistically signifi-
cantly different with p < 0.001. Designation of ab type indicate a group, which does not differ significantly either from a,
or from b at p = 0.001.
Fig. 5. Typical images demonstrating the condition of erythrocytes in experimental animals 24 h after burn injury,
and evaluation of the protective effect of empty liposomes, liposomes with PQ or UQ on the studied parameters. Im-
ages were obtained using a Leica DM 2000 microscope. Group 1 – control (n = 12); Group 2 – sham control (n = 10);
Group 3 – thermal burn without treatment (n = 10); Group 4 – thermal burn and liposomes without active ingredient
(n = 9); Group 5 – thermal burn and 50 µM PQ, topical application (n = 9); Group 6 – thermal burn and 5 µM PQ, intrave-
nous (n = 10); Group 7 – thermal burn and 50 µM UQ, topical application (n = 9); Group 8 – thermal burn and 5 µM UQ,
intravenous (n = 9).
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Fig. 6. Relative numbers of erythrocytes with morphological alterations in the rat blood 3 h (a) and 24 h (b) after ther-
mal burn injury. Statistical significance of the differences between the groups were evaluated by one-way ANOVA with
post-hoc comparison using Holm–Bonferroni method. Group 1 – control (for 3-h n = 12, for 24-h n = 12); Group 2 – sham
control (for 3-h n = 10, for 24-h n = 10); Group 3 – thermal burn without treatment (for 3-h n = 10, for 24-h n = 10);
Group 4 – thermal burn and liposomes without active ingredient (for 3-h n = 8, for 24-h n = 9); Group 5 – thermal burn
and 50 µM PQ, topical application (for 3-h n = 8, for 24-h n = 9); Group 6 – thermal burn and 5 µM PQ, intravenous (for
3-h n = 10, for 24-h n = 10); Group 7 – thermal burn and 50 µM UQ, topical application (for 3-h n = 8, for 24-h n = 9);
Group 8 – thermal burn and 5 µM UQ, intravenous (for 3-h n = 8, for 24-h n = 9). Columns denoted with different letters
(a, b, c) differ significantly with p < 0.05.
in the total number of erythrocytes were observed
(data not shown). The deformed erythrocytes were
observed both 3 and 24 h after the burn injury
(Fig. 6, a, b); furthermore, their amounts were com-
parable. Application of empty liposomes (group 4) re-
duced the number of erythrocytes with morphological
alterations. In the case of the group 5, this parameter
practically was the same as in groups 1 and 2, i.e.
no any erythrocytes with morphological alterations
were detected. In the case of intravenous adminis-
tration of physiological solution containing liposomes
with 5µM PQ, erythrocytes with morphological alter-
ations were also not observed. The obtained results
indicate the protective effect of PQ on erythrocytes
in thermal burns.
Similar data were obtained in our previous study
modeling ultraviolet-induced skin burn in the animals
with burn area of 30% of the total body surface. Ap-
plication immediately after burn of liposomes with
incorporated PQ (10 and 100 µM, topical application)
or intravenous administration of liposomes with 5 µM
PQ prevented development of morphological chang-
es in the erythrocytes observed 24 h after ultraviolet
burn injury (Patent of the RF (11) 2 819 761) [81].
Moreover, in our experiments we compared the
effects of PQ with the effects of UQ which is a na-
tive component of animal cells, and is widely used at
present as an antioxidant agent. It was revealed that
topical application of UQ in liposomes (group 7) or
its intravenous administration (group 8) at the same
acting concentrations as in the case of PQ also com-
pletely prevented erythrocyte deformation already 3 h
after thermal burn (Fig. 6a).
The obtained data indicate that thermal burn fa-
cilitates emergence of erythrocytes with morphological
alterations. Empty liposomes are capable of reducing
the number of deformed erythrocytes, and use of lipo-
somes with antioxidants can completely prevent eryth-
rocyte deformation in the second-degree thermal burn.
As noted in the Introduction, changes in erythro-
cyte morphology occur due to enhanced formation of
ROS in the inflammation area and initiation of oxida-
tive destruction, including activation of LPO. Mecha-
nisms of these processes involve free-radical oxidation
and are accompanied by an increase in spontaneous
chemiluminescence of blood [82]. Therefore, intensi-
ty of spontaneous chemiluminescence in the blood of
experimental animals was assessed in all investigat-
ed groups. Three hours after burn injury, i.e., under
conditions, when a large number of deformed eryth-
rocytes was observed (see above), increase of spon-
taneous chemiluminescence of the blood in animals
was detected (Fig. 7), which is in agreement with the
literature data [82].
Topical application of liposomes without antiox-
idants (group 4) decreased the level of spontaneous
PASHKEVICH et al.966
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 7. The level of spontaneous chemiluminescence in the
blood of animals 3 h after burn injury. Data are present-
ed as the arithmetic mean (denoted by bars with stan-
dard error). Statistically significant differences between
groups were assessed using one-way analysis of variance
(ANOVA) followed by Holm–Bonferroni post-hoc compar-
ison. Group 1 – control (n = 12); Group 2 – sham control
(n = 10); Group3– thermal burn without treatment (n = 10);
Group 4 – thermal burn and liposomes without active in-
gredient (n = 8); Group 5 – thermal burn and 50 µM PQ,
topical application (n = 8); Group 6 – thermal burn and
5µM PQ, intravenous (n = 10); Group 7– thermal burn and
50 µM PQ, topical application (n = 8); Group 8 – thermal
burn and 5 µM UQ, intravenous (n = 8). Bars denoted by
different letters (a, b) are significantly different from each
other at p < 0.05. The designation ab indicates an interme-
diate group that is not significantly different from either
group a or b (p > 0.05).
chemiluminescence in blood (Fig. 7). Topical appli-
cation and intravenous administration of liposomes
with PQ (groups 5 and 6) or UQ (groups 7 and 8)
provided the level of spontaneous chemiluminescence
already 3 h after burn injury, which was compara-
ble with the level observed for the groups 1 and 2.
The obtained data indicate that both PQ and UQ
suppress oxidative destruction caused by the second-
degree burn.
In addition to affecting shape of erythrocytes,
burns also initiate erythrocyte aggregation [83]. Ex-
perimental facts have been presented in the literature
indicating that the ability of erythrocytes for aggrega-
tion is determined by their ζ-potential, which quanti-
tatively characterizes the surface negative charge of
erythrocytes determining their resistance to aggre-
gation [84]. Decrease of the ζ-potential level facili-
tates erythrocyte aggregation and their assembly into
rouleaux, which interferes with oxygen release from
erythrocytes causing tissue hypoxia [8]. Analysis of the
level of erythrocyte aggregates demonstrated (Fig. 8),
that after 3h number of aggregates increased in com-
parison with the control animals and animals from
the group subjected to sham control and remained at
the increased level even 24 h after inducing thermal
burn, that indirectly implies decrease of their ζ-poten-
tial and worsening of oxygen transport function for at
least one day after burn.
Topical application of empty liposomes without
antioxidants decreased significantly 3 and 24 h after
burn the number of erythrocyte aggregates. Topical
application of PQ (group 5) and UQ (group 7) at con-
centration 50 µM resulted in the significant decrease
of the number of aggregated erythrocytes, however,
this level did not differ statistically significantly from
the level observed in the group 4 and remained high-
er than in groups 1 and 2. In the case of intravenous
administration of 5 µM UQ (group 8) aggregation of
erythrocytes decreased significantly only after 24 h
in comparison with the group 4, but aggregation did
not cease completely. Only intravenous administration
of PQ (5 µM) (group 6) completely prevented eryth-
rocyte aggregation: aggregated erythrocytes were ob-
served neither after 3 h nor after 24 h.
The obtained results allow confirming capabili-
ty of PQ and UQ to reduce erythrocyte aggregation
likely due to preservation of their ζ-potential. Data
presented in Fig. 8 demonstrate that intravenous-
ly administered PQ exhibits the highest efficacy in
preventing erythrocyte aggregation, which suggests
potential for the use of PQ in the complex therapy
of burns.
Considering that the changes in the shape and ag-
gregation of erythrocytes are accompanied with the
increase of hemolysis and release of free hemoglo-
bin as a factor of generalization of burn injury and
the role of PQ in preventing the pathogenetic events,
haptoglobin concentration in plasma was additionally
measured by ELISA in the animals with burns treated
with PQ (groups 5 and 6). It is known that haptoglobin
binds free hemoglobin [85], hence, decrease of its con-
centration indicates presence of hemolysis. Amount of
haptoglobin was significantly higher in animals of the
group 5, and was insignificantly higher in the case of
group 6 in comparison with the animals after burn
(group 3): in the group of animals with burns this
parameter was 71.3 ± 12.5g/L (n = 10, p = 0.0260), and
in the animals treated with liposomal PQ by topical
application – 104.6 ± 27.4 g/L (n = 12, p = 0.0304), and
in the animals with intravenous administration –
88.2 ± 25.7 g/L (n = 15, p = 0.3508). The obtained data
demonstrate less intensive hemolysis in the animals
treated with liposomal PQ administered on skin. These
results were obtained with the blood samples collect-
ed 24 h after burn and do not reflect dynamics of
the changes of haptoglobin/hemoglobin concentration
in the blood of animals, which requires further in-
vestigation.
PROTECTIVE ROLE OF PLASTOQUINONE 967
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Fig. 8. Changes in the number of aggregated erythrocytes in the blood of animals 3h (a) and 24 h (b) after thermal burn
injury. Statistically significant differences between groups were assessed using one-way analysis of variance (ANOVA) fol-
lowed by Holm–Bonferroni post-hoc comparison (for details on group composition, see the legend to Fig.6). Columns denoted
with different letters (a, b, c, d) differ significantly with p < 0.05. The designation bc indicates an intermediate group that
is not significantly different from either group b or c (p > 0.05).
DISCUSSION
Protective effects of PQ isolated from plant cells
in treatment of burns in animals was demonstrated
for the first time in this work. For this purpose, the
method of isolation and purification of PQ from leaves
of higher plants was optimized to obtain quantities
sufficient for conducting tests (see “Materials and
Methods” section). Thermal burn causes changes in
morphological structure of skin layers: thickness of
epidermis is decreased including thickness of basal
layer and epidermis thickness without basal layer, as
well as hair follicles and sebaceous glands are dam-
aged (Table 1). Empty liposomes without PQ facilitate
preservation of hair follicle structure, which mainly
is, most likely, due to phospholipid composition of the
liposomal particles [86], in particular due to the high
content of fatty acids in phosphatidylcholine includ-
ing lineic, palmitic, and oleic acids. Lecithin contain-
ing 90% of phosphatidylcholine used for preparation
of liposomes in our study contained fatty acids (% of
total content of fatty acids): C16:0 – 13.63; C18:0 – 3.68;
C18:1n9c – 11.36; C18:2n6c – 62.88; C18:3n3 – 6.12 [66].
Data are available in the literature that linoleic and
oleic acids are capable of decreasing intensity of acute
inflammation due to inhibition of synthesis and activi-
ty of proinflammatory cytokines [87, 88], and palmitic
acid displays high stability and resistance against oxi-
dation, and also could be used as an alternative source
of energy under conditions of energy deficit [89].
The noticed in our study decrease of the num-
ber of hyperemic vessels after the use of empty li-
posomes could be also associated with replenishing
of the PUFA deficit. Hence, liposomes prepared from
lecithin, containing 90% phosphatidylcholine, decrease
the manifestation of the inflammatory reaction in the
zone of burn probably due to stabilization of cellular
membranes, which is in agreement with the data re-
ported in our previous study [66].
Use of PQ incorporated into lipid phase of the
liposomes results in much less pronounced damages
of skin layers after thermal burns: epidermis in the
group of animals threated with these liposomes was
less damaged in comparison with the epidermis of
animals subjected to burns and animals with burns
treated with empty liposomes, numbers of damaged
hair follicles decreased (Table 1). It is possible that
the antioxidant and membrane-stabilizing properties
of PQ (see “Introduction” section) underlie the de-
crease of damages of skin layers and skin-associated
elements, such as hair follicles.
Considering that free-radical oxidation also in-
volves PUFA, it is possible that PQ is capable of po-
tentiating activity of liposomes associated with com-
pensation of PUFA deficit thus protecting the newly
replenished PUFA against further oxidation decreas-
ing activity of production of proinflammatory li-
poxins from PUFA of ω-9 series [90]. In the process
PQ itself does not exhibit anti-inflammatory effects,
because its use in the whole blood samples is not
PASHKEVICH et al.968
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
accompanied by the increase of production of TNF-α
and IL-6 (Figs. 2 and 3), that indicates lack of acti-
vation of classic proinflammatory signaling pathways
when this antioxidant is used.
PQ affected positively not only skin layers during
burns, but also state of blood cells, shape of erythro-
cytes, in particular. Despite its plant origin, PQ demon-
strated protective effect comparable with the effect of
UQ, preventing development of burn-induced changes
of erythrocyte shape (Fig. 6). It must be mentioned
that liposomes without PQ decreased the number of
deformed erythrocytes, however, the liposomes with
PQ and UQ maintained this number at the level typ-
ical for the control animals. Deformation of erythro-
cytes is also associated with activation of free-radical
oxidation and accompanying reactions [65, 91, 92].
The obtained result could be due to the combined ac-
tion of the used liposomes and antioxidants allowing
not only to replenish PUFA and cholesterol deficit in
the damaged tissues, but also to effectively neutral-
ize ROS, which is in agreement with the results ob-
tained during investigation of spontaneous chemilu-
minescence (Fig. 7). Moreover, the revealed increase
of haptoglobin content after topical application of PQ
indicates decrease of degree of hemolysis [92], which
allows suggesting decrease of the rate of generaliza-
tion of the disorder caused by burns. Lack of the effect
on the content of haptoglobin in the case of intra-
venous administration of PQ likely indicates that the
effective prevention of hemolysis in burn injury could
be achieved only in the case of existence of protective
effect in the skin layers.
Therefore, it was proved in our study that the
use of liposomes with antioxidants, PQ or UQ, in par-
ticular, administered either by topical application or
intravenously, could completely prevent changes in
erythrocyte shape after second-degree burns (Fig. 6)
and prevent disruption of their oxygen transport
function decreasing the degree of hemolysis and re-
lease of hemoglobin as a factor facilitating generaliza-
tion of inflammatory process and multiorgan failure.
It could be suggested that prevention of hemolysis,
as one of the early and critical stages in burn patho-
genesis, could significantly reduce negative effects of
burn injury, however, in order to prove that preven-
tion of hemolysis is a vital step in the interruption of
the chain of pathogenetic events in skin burns, further
investigations are required.
Another manifestation of negative influence of
thermal burn on blood parameters is erythrocyte
aggregation, which, similar to the change of their
shape also negatively affects their oxygen transport
function [8]. The results of our investigation demon-
strate that the plant-derived PQ has clearly pro-
nounced protective properties exceeding protective
effects of ubiquinone; intravenous administration
of PQ incorporated into liposomes completely pro-
tects erythrocytes against aggregation in burn injury
(Fig. 8), which, likely, allows to maintain erythrocyte
functions at the level typical for healthy animals.
Based on the information presented above, it could
be suggested that for maximum protection in treating
burns combination of intravenous administration and
topical application of preparations could be recom-
mended.
Due to its lipophilic nature, PQ is capable of in-
tegration into the membrane lipid bilayer of the dam-
aged cells. Furthermore, incorporation of PQ into com-
position of liposomes enriched with PUFA facilitates
more efficient delivery of antioxidant to the damaged
skin areas. Through integration into the membrane
redox system, PQ, likely, could be reduced to PQH
2
effectively neutralizing such ROS as O
2
•−
, HO
•
,
1
O
2
, and
others (see “Introduction” section), thus preventing
development of LPO and attenuating consequences of
the second-degree thermal burn. Despite the absence
of direct data on the involvement of PQ in mitochon-
drial chain in animals, such effects seem plausible
based on the known properties of quinones [93, 94].
The level of reduced UQ in plasma membrane could be
maintained by a number of enzymes: DT-diaphorase
[95], cytochrome b5 reductase [96], NADPH:quinone
reductase [63], glutathione reductase [97], lipoamide
dehydrogenase [98], and thioredoxin reductase [99].
Majority of these enzymes are NADH- and NADPH-
dependent, and it was shown that incubation of the
isolated liver cell membrane with NADH increased the
fraction of reduced UQ in them [100]. Hence, exog-
enous UQ and PQ are capable of being reduced by
the native membrane components effectively neu-
tralizing ROS in plasma membrane. The obtained
results help to define direction of further research
in the use of PQ in composition of liposomes as an
agent for early intervention in treatment of thermal
burns preventing hemolysis and release of free he-
moglobin, which mediates generalization of inflam-
mation process. The possibility of using PQ at the
later stages of burn injury requires further inves-
tigation.
Synthetic PQ analogues, as is well-known, are
used as therapeutic agents exhibiting anti-inflamma-
tory, antibacterial, anticancer, and other properties;
however, as a rule, synthetic PQ analogues have short-
er side chain, which limits their solubility in mem-
branes and manifestation of antioxidant properties
in prevention of LPO in membranes [35]. Considering
lack of proinflammatory effect of PQ added to whole
blood observed in our study (Figs.2 and3), as well as
its clear protective effect under condition of oxidative
stress, PQ could be used in treatment of pathological
states associated with the increased concentrations
of ROS.
PROTECTIVE ROLE OF PLASTOQUINONE 969
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CONCLUSION
Use of liposomes in composition of burn-treat-
ment mediations results in reduction of negative
consequences of thermal skin injury, and use of the
plant-derived PQ incorporated into liposomes almost
completely eliminates consequences of the second-de-
gree burns. PQ does not induce a proinflammatory
response in vitro and effectively prevents morpholog-
ical changes in the skin layers, as well as change of
the shape and aggregation of erythrocytes in the sec-
ond-degree burn injury. Therefore, application of PQ
incorporated into liposomes could be considered as a
method for protection of erythrocytes against defor-
mation and hemolysis. In addition, it ensures the pres-
ervation of the thickness of the epidermis and pre-
vents damage to hair follicles and sebaceous glands.
Abbreviations. IL-6, interleukin 6; LPO, lipid
peroxidation; LPS, lipopolysaccharide; mtDNA, mito-
chondrial DNA; PQ, plastoquinone; ROS, reactive ox-
ygen species; TNF-α, tumor necrosis factor α; UQ, ubi-
quinone.
Acknowledgments. The authors express their
gratitude to D. V. Vilyanen (Institute of Basic Problems
of Biology, Russian Academy of Sciences) for helpful
discussions of the study results, to I. R. Prokhorenko
(Institute of Basic Problems of Biology, Russian Acade-
my of Sciences) for providing LPS, to A. V. Krupyanko
(Institute of Basic Problems of Biology, Russian Acad-
emy of Sciences) for help in isolation of PQ, and to
A. F. Martsinkevich (Vitebsk State Medical University)
for help with statistical data processing. Equipment
of the Center for Collective Use of the Pushchino Sci-
entific Center for Biological Research (Russian Acade-
my of Sciences) was used in this study (https://www.
ckp-rf.ru/ckp/670266/ [in Russian]).
Contributions. M. M. Borisova-Mubarakshina,
S. S. Osochuk – setting goals and objectives of the
experimental study; N. I. Pashkevich, E. S. Pykhova,
A. A. Ashikhmin, D. V. Vetoshkina – conducting experi-
ments; N. I. Pashkevich, E. S. Pykhova, D. V. Vetoshkina,
S. S. Osochuk, M. M. Borisova-Mubarakshina – dis-
cussion of the obtained results; N. I. Pashkevich,
E. S. Pykhova – writing draft of the paper; N. I. Pash-
kevich, E. S. Pykhova, D. V. Vetoshkina, M. M. Borisova-
Mubarakshina – editing text of the paper.
Funding. This work was financially supported by
the Ministry of Science and Higher Education of the
Russian Federation (State Scientific Program, grant
no. 125051305922-5) and by the Belarusian Republic
Foundation for Fundamental research (grant from
02.05.2023).
Ethics approval and consent to participate. All
applicable international, national, and/or institutional
guidelines for the care and use of animals were fol-
lowed. For the enzyme-linked immunoassay of IL-6
and TNF-α in human blood, the blood of voluntary
donors was used.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
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