Galina V. Sukoyan1, Natalya B. Demina2, Nikoloz V. Gongadze3, Galenko-Iaroshevsky P.A.

4,
Edisher T. Tsivtsivadze.1 Nino G. Khvitia1, Veronika V. Golovach1

IMBALANCE IN REDOX-INFLAMMATION AXIS IN AGING SKIN AND

EFFICACY OF PHARMACOLOGICAL AGENTS
1
International Center of Introduction of New Biomedical Technology (assigned of NV Karsanov
Scientific Centre of Introduction of New Biomedical Technology), Tbilisi, Georgia;
2
Department of Pharmacy Technology of Federal State Autonomous Educational Institution of
Higher Education I.M. Sechenov of the Ministry of Health of the Russian Federation (Sechenov
University), Moscow Russia
3
Department of Pharmacotherapy and Pharmacology Tbilisi State Medical University, Tbilisi,
Georgia;
4
Department of Pharmacology Kuban State Medical University, Krasnodar, Russia

Correspondence author: Galina V. Sukoyan, PhD, Department of Molecular and Clinical

Pharmacology, International Centre of Introduction of New Biomedical Technology; Kairskaya str,
19, Tbilisi, 0137, Georgia. Tel.:+995595418030; ORCID: 0000-0002-2259-5901; E-mail:
galinasukoian@mail.ru or galinasukoian@gmail.com

ABSTRACT
Background: Intimal mechanisms of oxidative stress progression and inflammation inextricably
linked and play underpin of most diseases and aging process. The aim of the study was the evaluation
contribution of anti-oxidant potential decreasing and cytokine dysregulation in aging and
comparison of efficacy of various pharmacological agents (antioxidants) as a candidate for include
in treatment algorithm of protective strategy against skin aging.
Methods and findings: 84 female Wistar rats included in the experimental design. Aging model
was reproduced by D-galactose (D-Gal) injection. All animals in main group were randomized for
6 groups: control I – the same aged rats without treatment, control II – animals with skin aging
reproduced model receive saline, III - receive intradermal injection 2% artichoke extracts, IV group
- centella asiatica (CA) extract, 0.1%, V- 0.1% dimethylaminoethanol (DMAE), VI – ascorbic acid,
50 mg/kg, intradermally twice at weeks during 4 weeks. In D-gal-induced skin-aging functioning is
decreased. Artichoke and CA extracts reverse ratio of activities of superoxide dismutase / (catalase
+ glutathione peroxidase) and decreased ratio of pro-/anti-inflammatory cytokines and activity of
nuclear transcription factor kappa B. Efficacy of pharmacological agents to restore the redox
potential O2-/H2O2 and were artichoke extracts > CA> DMAE>ascorbic acid.
Conclusions: Oxidative stress progression and inflamm-ageing are included in age-related
pathogenesis and preventive ageing strategy could include potential multitargets interventions
directed to its cessation for reduce skin age-related diseases and promoting healthy longevity.
Keywords
 2 
 
 

Skin aging, Pharmacological agents, Oxidant defense system, Superoxide anion, Cytokine profile,
Nuclear transcription factor kappa B

INTRODUCTION
An organism is a dynamic tug between the occurrence of damage and the processes of defense,
maintenance and repair organs, tissues and systems. The barrier function, boundary between the
organism’s internal milieu and the external environment, powerful defense system to defend against
injurious insults, carries out the skin, the largest organ of the body. Expansive or prolonged action
of various exogenous and endogenous stressors (trauma, pathogens or irritants, air pollutants,
ionizing and non-ionizing irradiation, toxins, physical damage, etc.) leading to decease the reserve
of skin defense systems ability and increase risk of disorders development which prevalence in the
geriatric age group [1]. These is much important in aging, which is strongly associated with
homeostasis alterations, metabolic or mitochondrial reserve potential and reserve capacity of oxidant
defense and immune systems declined that itself increases the susceptibility to stressors and
development of skin disorders [2-3]. The major agents of oxidative stress, reactive oxygen species
(ROS), which may be both beneficial and deleterious to the skin, in case of its hyperproduction
coupled with the decline in the antioxidant defense system functioning lead to skin injury and
development of dermatological diseases. Oxidative posttranslational modifications form a major
redox-regulated mechanism of subsequent structural and functional consequences, altering signaling
kinase activities and affect downstream signaling pathways in skin ageing [4-6]. From the other
hand, oxidative stress and hyperproduction of reactive oxygen species (ROS) disturbed homeostasis
of immune cells, monocytes, neutrophils, and natural killer cells, as a particularly sensitive to
oxidative stress cells, and normal functioning of which coupled with a higher production of ROS
and higher content of polyunsaturated fatty acids in their plasma membranes [6-9]. Adaptive, at the
initial, as part of the innate immune response, inflammatory response directed to protected
organisms against pathogenic invaders and cleans up damaged cells after injury to prevent further
tissue damage, for re-establishment of tissue homeostasis, become chronic and destructive, and
leading to the development of many diseases and one of the key stimulus of aging [2, 8-9]. Despite
a vast repertoire of pharmacological agents actions studies performed over the past century, the
integral multitargets action to increase reserve ability of an antioxidant defense system and restored
tissue cytokine profile in ageing is very low. For better understanding of skin aging process and to
elaborated rational prevention algorithm we have choice D-Galactose (D-Gal)-induced experimental
model of age-dependent alterations. Expressive administration of D-Gal could induced damage
associate with mitochondrial dysfunction caused by complex I deficiency and can accelerate ageing
 3 
 
 

[10-16]. The aim of this study were elaborated an integral complex evaluation of strengthening of
redox-inflammatory deterioration in aging animals in D-Gal-induced skin aging model in
experimental animals and efficacy of beneficial various pharmacological agents. 
METHODS.
Animals and experimental study design.
All procedures with animals performed in this study were in accordance with the ethical standards
of the institutional research committee and with the 1964 Declaration of Helsinki, and its later
amendments (General Assembly, October, 2013) [Declaration of Helsinki History Website". Ethical
Principles For Medical Research. The JAMA Network. Retrieved 26 July 2015] and European
Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals
used for scientific purposes and was approved by the local Interinstitutional (International Scientific
Centre of Introduction of New Biomedical Technology, Department of Medical Pharmacology and
Pharmacotherapy, Tbilisi State Medical University, Tbilisi) Animal Care and Use Committee. A
total of 84 female Wistar rats, weighing 180-200 g, were housed for at least 5 days under controlled
pathogen free conditions according at constant humidity- and temperature- environment, exposed to
a 12 h light-dark cycle, and allowed drink tap water ad libitum before the experiments. Animals for
ageing experiments were randomized into two groups: control and main. Animals in main group
after randomization received injection with D-Gal (100 mg/kg/day, i.p.), while in control group
received placebo (0.9% saline, 0.5 ml/day, i.p.), for 8 weeks as described by Sukoyan et al [14-15].
At 21 days after injection with D-Gal the 3 cm round tattoo area was prefabricated on each side of
rats previously disinfected hip under sterile condition and general anesthesia with pentobarbital (35
mg/kg). All animals in main group (46 animals) were secondly randomized into 5 groups in
dependence to treatment (twice in week of intradermal injection under general anesthesia) for 5
weeks: control III group animals treated with microinjection of saline (n=10); main I group -
animals received 0.13 mg of 2% lyophilized powder of standardized leaves of the artichoke Cynara
cardunculus L. (Grosso Romanesco) var. Cynara scolymus L., femaly Aesreraceae, preparated in
according with Eur Ph monograph 01/2009:2389 (content of chlorogenic acid =1.95% [14-15]),
salivated in water for injection (equivalent of average intradermal dose for patients 10 mg, n=12)
and main II – animals received 0.1% DMAE (dimethylaminoethanol bitartrate salivated in water for
injection from commercial available form, DMAE, 3%, Szwedo Group s.c., (Polfa), n=12; main III
- animals received 0.1% sterile extracts of leaves of Centella asiatica (CA) in propylene glycol and
water from Bio-Botanica Inc. (USA), n=12 and main IV – animals received conventional
antioxidants ascorbic acid 15 mg/kg in from of mesohyal vitamin C (Mesoestatic Pharma Group,
s.l., Spain). After the experiments, all the rats euthanized by pentobarbital (60 mg/kg i.p.). Body
 4 
 
 

weight, skin oedema, water and total collagen (hydrohyproline) content evaluation was investigated
as described by [16]. The skin tissue block, weighed 0.5 g, from the injected area of rats, removed from
subcutaneous fat and other connective tissues and then rinsed using pre-cooled normal saline. Then the
skin tissue was made into 10% pre-cooled normal saline and homogenized. Supernatant of each group
was used for detection of aging-related biomarkers. Determination of activities of enzymatic part of
endogenous antioxidant defense system (superoxide dismutase (total) (SOD), catalase, gluthatione
peroxidase (GSH-Px) and glutathione reductase by velocity of redox NADP+ formation, and redox
glutathione in homogenate) of skin of rats. Preparation of nuclear extracts and determination of NF-
kB [14-15] and cytokine profile in skin homogenate were evaluated using enzyme-linked
immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) according to the
manufacturer’s instructions. The protein concentration was determined with BSA protein assay kit.
Data are expressed as mean ± standard deviation of mean (SD), and differences between groups
were examined for statistical significance with ANCOVA and 2- or 1-tailed Student’s t test. p < 0.05
considered as a significant.
RESULTS
Alteration in body weight, skin oedema and total collagen content in aging animals. Efficacy of
prevention of various pharmacological agents.
The rats in normal control group during the experimental period were active, normal skin
appearance, good appetites and had a steady increase in body weight. D-gal-induced aging group
exhibited hair loss from 2nd weeks and demonstrated evident symptoms of aging: dullness and
decrease activity, dull fur, and a loss of appetite, a unique skin appearance, and had a lower rate of
weight gain than that in the normal control group without changes in morbidity/mortality. The
relative weight of skin markedly decrease in D-Gal model of aging (table 1). Treatment with 2%
Artichoke extract at the doses of 0.13 mg/kg improved body weight of D-Gal-induced aging rats,
restores the water dysbalanced in the aging skin and increased the content of hydroxyproline in aging
skin. Treatment with CA leads to same extent of improvement, DMAE slightly less and efficacy of
vitamin C less.
Comparison of ability of various dermatoprotective drug to maintenance reserve ability of
enzymatic link of antioxidant defense system in aging rats
D-Gal-induced aging in rats cause to significant decreased in total SOD and GPx activities and in
the less extent catalase activities in skin in comparison with control I and control II (table1). At the
same time, the velocity of superoxide anion generation increased by 27% in control II group when
comparing the rate of O2- production in control 1 groups and by 58% in D-Gal group in comparison
with the same old rats (table 1). Treatment with 2% artichoke and 0.1% CA extracts from the 21
 5 
 
 

days after D-Gal induced aging in rats leads to increase SOD activity by 46% and 27% and these
accompanied with markedly decreasing in velocity of O2- generation by 33% and 27% respectively.
While treatment with DMAE and vitamin C significantly did not changes both parameters. Exposure
to D-gal did not induced significantly changes in catalase activity in skin (table 1). However, the
production of H2O2 under treatment of D-Gal increased, and exceeds control II level by 195%. In
contrast to D-Gal-induced aging group, artichoke and CA extracts decreased the level of H2O2
production by 42 and 27% and in less extent DMAE and ascorbic acids by 10% and 7% respectively.
Simultaneously, the redox potential, ratio of generation O2-/H2O2which equal in intact group
0.17±0.04 decrease to 0.09±0.01 in D-Gal treated control III group and increase to 0.12±0.2 (p<0.01)
after artichoke treatment. Exposure to D-Gal reduced the GSH content in skin tissue from 1.20±0.13
nmol/mg/protein to 0.74±0.13 nmol/mg/protein (p< 0.01 vs. control III). Due to D-Gal-treatment
observed significantly decreasing of GSH-Px activity, and as a results the ratio SOD/(Catalase +
GSH-Px), which represents equilibrium between formation of hydrogen peroxide from superoxide
dismutation and its utilization by catalase and GSH-Px, increased from and 5.4±0.2 x10-3 in control
II group (5.5±0.3x10-3 in rats at the beginning of the experiments) to 5.8±0.3 x10-3. Treatment with
DMAE and ascorbic acids decreased the ratio SOD/(Catalase + GSH-Px) towards to the
hyperproduction of H2O2, while the treatment of artichoke and CA extracts have ability to
maintenance the balance between formation of H2O2 and its neutralization by specific antioxidant
enzymes. The activity of GSH-Px and gluthathione redox potential were more sensitive to aging: in
D-Gal model it decreased in skin by 37% and increased up to the level in rats at the same age under
treatment with DMAE, artichoke and CA extracts and did not changed in group received ascorbinic
acids. Ratio of activities SOD/(Catalase + GSH-Px) increased to 5.8±0.2 x10-3 in D-Gal induced
aging group and decreased to 5.4±0.2 x10-3 after artichoke and to 5.25±0.18 x10-3 CA extracts
treatments. In contrast, in DMAE and vitamin C treatment group the ratio decrease to 4.7±0.2x10-3
and 4.2±0.2 x10-3 respectively.
Treatment with DMAE, artichoke and CA extracts restored the glutathione redox and it has
reached level in the same aging groups under treatment with DMAE and artichoke extracts (table
1). In the model of D-Gal-induced aging levels of MDA in skin significant elevated, when compared
to the control group (p < 0.001) following 42 days of exposure to D-Gal, but not in aging group
without D-Gal (table). Interestingly, treatment of rats with all pharmacological agents decreased the
MDA content in skin homogenates. There were no correlation between the level of ratio
SOD/(Catalase + GSH-Px), redox potential, ratio of generation O2-/H2O2 and MDA content in skin
(r=0,37, NS and 0.21, NS respectively).
 6 
 
 

Comparison of ability of various dermatoprotective drug to maintenance pro-/anti-inflammatory

cytokines balance in aging rats.
D-Gal induced aging in rats skin associated with disturbances in cytokine profile towards to
proinflammatory TNF-α, IL1β, and IL-6 production. The integral index of pro-anti-inflammatory
cytokine increased about 2.5-folds and accompanied with the increased of level of TNF-α in more
than 3-fold, IL6 by 25% and IL1β by 67%, while level of the anti-inflammatory cytokine, IL-10,
decreased by 33% (table 2). Treatment with DMAE and ascorbic acids in less mainly decreased the
level of IL1β, artichoke extract - TNF-α and IL1β, and extract of CA - IL1β and, is much important,
increased the level of IL-10 (table 2). As a result the integral index of pro-anti-inflammatory (TNF-
α + IL1β + IL6): IL10 in skin aging decreased in DMAE treatment group by 33%, ascorbic acid –
by 19%, 2% artichoke extracts – by 42% and CA extracts – by 39%. The increasing in about in 2.5
time activity of NF-kB (p65) decreased only in case of treatment of artichoke and CA extracts (table
2).
DISCUSSION

Skin aging as a one of the link in the natural human aging, which provides first obvious marks, is a
complex biological process triggered by endogenous and intrinsic factors leading and associated
with reducing in overall collagen content per unit of skin area approximately 1% /year [16]. Besides
that aging is multifactorial process, it was suggested that uncontrolled progressive accumulation
oxidative stress (imbalance between the prooxidant and antioxidant levels in favor of prooxidants)
in the cells, is one of the key link in aging process, which ultimately determines the lifespan of an
organism and associated with physiological decline. Thus, the reliable animal model reproduced by
chronic (6-8weeks) administration of D-Gal, which characterized by blocking of glycometabolism,
decreasing the activity of antioxidant enzymes and accumulation of ROS-derived increased level of
MDA (an end-product generated by decomposition of arachidonic acid and larger polyunsaturated
fatty acids) response the “mitochondrial free radical theory of aging” and could use in
gerontological research [10-12]. In present work on the D-Gal-treated animals model of aging (have
a shortened life span and exhibit symptoms similar to those of natural aging) we have shown that
more sensitive in the antioxidant enzyme defense system in skin is SOD and GSH-Px, increased
content of O2- and H2O2. Moreover, the dysbalance between production of ROS and activity of
antioxidant defense system strongly correlated with shift in cytokine profile to proinflammatory
components and decrease activity of NF-kB (table 2): the positive linear correlation was observed
between increasing of ratio SOD/(catalase+GSH-Px) and [(TNF-α +IL6+IL1β): IL10], r=0.96,
p<0.001. Extract of artichoke, which rich in phenolic and flavonoids and gives a powerful
antioxidant activity [15, 17-22], has ability to increased hydroxyproline and collagen type 1
 7 
 
 

expression [14, 17], improved action of antioxidant defense system [15, 17-20, 22-25] and through
decreasing the activity of NF-kB reverse the shifting to proinflammatory cytokine production (table
2). Early it was shown that treatment with artichoke (40 mg/kg body weight) per os in D-Gal-
induced skin aging model daily for 36 days increased activity of SOD in brain and liver, GSH-Px
in brain [13], and catalase activity in liver [11]. Interesting, that pure constituents of artichoke
extracts shown to produce less inhibitory activity on free radical production than the extract itself
[17, 21-23, 25]. The skin GSH/GSSG ratio decreased in controls receiving D-Gal and gradually
returned to near normal levels with DMAE>artichoke extract>CA extract>ascorbic acid (Table 2).
The immune imbalance and the cytokine dysregulation, an inability to fine-control systemic
inflammation, is believed to play a key role in the remodeling of the immune system at older age
and a marker of related physiological functional declines and aging-accompanying chronic diseases
development. Aging-associated inflammation and increased proinflammatory cytokine responses
may be caused by a declining level of IL-10, an essential cytokine, mainly produced by
macrophages, and is responsible for suppressing proinflammatory response in various tissues,
including skin, during aging. IL-10 prevents inflammation by suppressing the activation of
macrophages and blocking the antigen presentation, as well as the release and activity of
inflammatory cytokines, such as IL-6, TNF-α, and IL-1β [26]. Thus, available data suggest that ratio
between the anti-inflammatory cytokine IL-10 and the mainly proinflammatory cytokines IL-6, IL-
1β and TNF-α could play important roles in the association between inflammation and aging.
Interestingly, that artichoke is favors that synthesis of coenzymes NAD((NADH2)) and
NADP(NADPH2)) and mainly of the NADP(NADPH2) pair, which take key plays in the regulation
of antioxidant/prooxidant status and primary decreasing of TNF-α in aging skin. DMAE is a
naturally-occurring substance, a acetylcholine precursory, small amounts of which produced
naturally in the human brain, with significant anti-inflammatory, anti-aging properties. DMAE
occurs a building block of the neurotransmitter acetylcholine, and acts as an antioxidant by
stabilizing cell membranes, helping to protect them from free radical damage [27-29]. Numerous
studies have shown that topical application of DMAE increases the appearance of firmness, tone
and lift to the skin. DMAE also improves the appearance of skin elasticity and luminosity, helping
to decrease the look of fine lines and aging skin’s appearance. The primary constituents of CA is the
triterpenic fractions which showed wide range of defensive and therapeutic effects, helps to prevent
the breakdown of acetylcholine, most prominently influencing of collagen production and
deposition in wound healing. Ultrasonographic data suggest that collagen synthesis increased
significantly after topical vitamin C therapy [30-31]. Titrated extract of CA is used to treat several
microcirculatory problems, skin inflammation (eczema, atopic dermatitis, leprosy, varicose ulcers,
 8 
 
 

etc.). Ethanolic extract of CA at dose 100mg/kg of body weight showed anti-inflammatory activity
in rats similar to standard Ibuprofen [32-34], possible throughout inhibition of enzymes
cyclooxygenase-2 activities and expression of IL-6, IL-1β, TNF-α through the down-regulation of
NF-κB activation [35]. Administration of aqueous extracts of CA showed to counteract lead-
induced oxidative stress male rats and treatment with water extract of CA significantly increased
proliferation and the production of IL-2 and TNF-α in human peripheral blood mononuclear cells,
but ethanol extract had inhibitory effect [36-37]. An abnormal regulation of these signaling pathways
during both the early and chronic phases of intestinal inflammation could result in a persistent
inflammatory process. Intradermal treatment of 2% artichoke extract and in less extent 0.1% CA
could improve level of NF-kB(p65), while DMAE (a acetylcholine precursore) and ascorbic acid
influence only on the production of some proinflammatory cytokines. As a result the ratio between
pro-and anti-inflammatory cytokines in D-gal induced skin aging model reverse only under
treatment of artichoke and CA extracts, but intimal mechanisms of action of these two extracts may
be different, because CA extracts more primary improved production of IL-10 and artichoke extracts
decreased TNF-α.
Free radical and mitochondrial theories of aging supported by estimation of positive relation
between the sings of aging and progression of imbalance of free radical metabolism and oxidative
damage and associated with the close relationships between oxidative stress and inflamm-aging.
Influence of artichoke and CA extracts, DMAE and ascorbinic acids restored skin relative weight
and leads to an increase hydroxyproline content in skin in D-Gal model of aging. Intradermal therapy
with DMAE attenuated aging changes in skin and interfered in action of antioxidant defense system
and cytokine dysregulation, but did not decreased the activity of NF-kB (p65), increased SOD
activity and restore the redox potential O2-/H2O2. Concomitant use of 2% artichoke and 0.1% CA
extracts improve reserve ability of antioxidant defense system, restored pro-/anti-inflammatory
cytokine balance and exert antiaging action in this model of skin aging in experimental animals. The
increased reserve ability of intrinsic antioxidant defense system and improvement in cytokine profile
under treatment with artichoke  CA extracts > DMAE > ascorbic acid gave based for application
of these pharmacological agents in cosmetology and its beneficial effects for anti-aging skin care.

Acknowledgments
Authors are grateful to the Chief animal’s clinic Kamkamudze R. and his staff for animal housing
and Associate Professor Bobkova Natalia V. of the Pharmaceutical Natural Sciences Department
Institute of Pharmacy of Sechenov First Moscow State Medical University (Sechenov University)
 9 
 
 

for authentication of artichoke leaves used in this study and Tsivtsivadze E, PhD, as a director of
“Biotechpharm GE” in part of artichoke extracts preparations.
Funding
The work has not funding.

Competing and connflicting of interest

The authors declare that they have no conflict of interests regarding the publication of this paper.
The authors alone are responsible for the content and writing of this article. On behalf of all
authors, the corresponding author states that there is no conflict of interest.

Abbreviations
CA- centella asiatica; D-Gal – D-galactose; DMAE – dimethylaminoethanol; IL-interleukin, MDA
– Malone aldehyde; NF-kB – nuclear transcription factor kappa B; ROS- reactive oxygen species;
SOD - superoxide dismutase; GSH-Px - gluthatione peroxidase; TNF- tumor necrosis factor

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