Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 2, pp. 201-211 © Pleiades Publishing, Ltd., 2024.

201

Retinoprotective Effect of SkQ1,

Visomitin Eye Drops, Is Associated with Suppression

of P38 MAPK and ERK1/2 Signaling Pathways Activity

Natalia A. Muraleva

1,a

*, Anna A. Zhdankina

Anzhella Zh. Fursova

1,3,4

, and Nataliya G. Kolosova

Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia

Department of Histology, Embryology and Cytology, Siberian State Medical University, 634055 Tomsk, Russia

Department of Ophthalmology, Novosibirsk State Medical University, 630091 Novosibirsk, Russia

State Novosibirsk Regional Clinical Hospital, 630087 Novosibirsk, Russia

e-mail: myraleva@bionet.nsc.ru

Received September 18, 2023

Revised November 20, 2023

Accepted November 22, 2023

Abstract— Visomitin eye drops are the first and, so far, the only drug based on SkQ1 – the mitochondria-tar-

geted antioxidant 10-(6′-plastoquinonyl) decyltriphenylphosphonium, developed in the laboratories of Moscow

State University under the leadership of Academician V. P. Skulachev. SkQ1 is considered as a potential tool to

combat the aging program. We have previously shown that it is able to prevent and/or suppress development of

all manifestations of accelerated senescence in OXYS rats, including retinopathy, similar to the age-related mac-

ular degeneration (AMD). Here, we assessed the effect of Visomitin instillations on progression of the AMD-like

pathology and p38 MAPK and ERK1/2 activity in the OXYS rat retina (from the age of 9 to 12 months). Wistar and

OXYS rats treated with placebo (composition identical to Visomitin with the exception of SkQ1) were used as con-

trols. Ophthalmological examination showed that in the OXYS rats receiving placebo, retinopathy progressed and

severity of clinical manifestations did not differ from the intact OXYS rats. Visomitin suppressed progression of

the AMD-like pathology in the OXYS rats and significantly improved structural and functional parameters of the

retinal pigment epithelium cells and state of microcirculation in the choroid, which, presumably, contributed to

preservation of photoreceptors, associative and ganglion neurons. It was found that the activity of p38 MAPK and

ERK1/2 in the retina of 12-month-old OXYS rats is higher than that of the Wistar rats of the same age, as indicated

by the increased content of phosphorylated forms of p38 MAPK and ERK1/2 and their target protein tau (at posi-

tion T181 and S396). Visomitin decreased phosphorylation of p38 MAPK, ERK1/2, and tau indicating suppression

of activity of these MAPK signaling cascades. Thus, Visomitin eye drops are able to suppress progression of the

AMD-like pathology in the OXYS rats and their effect is associated with the decrease in activity of the MAPK sig-

naling cascades.

DOI: 10.1134/S0006297924020020

Keywords: age-related macular degeneration, mitochondrial antioxidant SkQ1, Visomitin, p38MAPK, ERK1/2, phos-

phorylation, OXYS rats

To whom correspondence should be addressed.

INTRODUCTION

Eye drops Visomitin is the first and, so-far, the

only one pharmaceutical preparation based on SkQ1–

the mitochondria-targeted antioxidant 10-(6′-plastoqui-

nonyl) decyltriphenylphosphonium, developed in the

laboratories of Moscow State University under the

leadership of Academician V. P. Skulachev [1]. Its de-

velopment was initiated by investigation in the frame-

work of the project “Skulachev’s ions” using OXYS

rats, model of accelerated senescence, created in the

Institute of Cytology and Genetics, Siberian Branch of

MURALEVA et al.202

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Russian Academy of Sciences. Accelerated senescence

of the OXYS rats is manifested by the early develop-

ment of a complex of geriatric diseases including cata-

ract and retinopathy according to the clinical, morpho-

logical, and molecular signs of the similar age-related

macular degeneration (AMD) in humans [2, 3]. Several

months of research was sufficient to demonstrate that

administration of SkQ1 with feed is capable of pre-

venting development of the whole complex of phe-

notypic manifestation of accelerated aging in OXYS

rat, which later was confirmed in multiple studies.

During the first discussion of the results of pilot stud-

ies at the V. P. Skulachev seminar, it was concluded that

the most remarkable effect of SkQ1 was its ability to

suppress development of cataract and retinopathy.

A. M. Olovnikov suggested that it would be very prom-

ising to develop eye drops based on the mitochon-

dria-targeted antioxidant. Already in 2005 we started

investigation of the ability of SkQ1 to affect develop-

ment of cataract and signs of AMD in the OXYS rats not

only during administration with feed, but also admin-

istering the drug in a form of eye drops; results of this

study were published in 2008 [4]. At the same in the

framework of the “Shulachev’s ions” project the work

began on the development of the therapeutic prepara-

tion on its basis followed by clinical trials. Asa result,

in 2012 the Ministry of Health of the Russian Federa-

tion approved the use of Visomitin preparation as a

keratoprotector for prophylactics of dry eye syndrome.

Later it was also approved for prophylactics of early

cataract [5]. The studies with OXYS rats provide hope

that in future Visomitin could be also used for treat-

ing AMD, a multifactorial neurodegenerative disease

of retina, which is the main cause of vision deteriora-

tion and loss in individuals of 60 years and older in

the developed countries [6]. As has been shown in our

studies, retinopathy developing in OXYS rats is similar

to the dry or atrophic type of AMD in humans [3, 7].

Clinical manifestations of retinopathy develop in OXYS

rats by the age of ~3-4 months at the background of

structural and functional changes in the retinal pig-

ment epithelium (RPE) cells and disruption of cho-

roidal microcirculation, which progress with age and

lead to decrease of the thickness of photoreceptor lay-

er and of the outer nuclear layer– events at the basis

of vision loss in AMD [8, 9].

Considering the mitochondria-targeted antioxidant

SkQ1 as a tool in the fight against aging, V. P. Skulachev

explained its effects primarily by its anti-radical activ-

ity, ability to suppress generation of reactive oxygen

species directly in mitochondria [10]. It was shown in

our studies that the accelerated ageing in OXYS rats is

associated with dysfunctions of mitochondria, but we

did not observe any direct associations with oxidative

stress. Nevertheless, SkQ1 is capable to prevent and/

or suppress development of all manifestations of aging

in OXYS rats including signs of AMD [11]. In particular,

it was demonstrated that administration of SkQ1 with

feed starting at the age of 1.5 months prevents devel-

opment of clinical signs of retinopathy in OXYS rats

up to the age of 2 years and is capable to significant-

ly suppress progression of the disease in the animals

with already developed signs of the disease [4, 10]. Ret-

inoprotective effects of this preparation are associated

with normalization of microcirculation in the choroid

vessels, of ultrastructure of RPE and photoreceptors

[12, 13], with its effect on expression of the genes of

angiogenesis regulation– vascular endothelium growth

factor (VEGF) and its antagonist– pigment epithelium-

derived factor (PEDF) [8], as well as with normalization

of autophagy processes in the retina of OXYS rats [14].

Accumulation of toxic amyloid beta (Aβ) in the retina

of OXYS rats is a sign of proteostasis disruption typical

for AMD, which is significantly suppressed on the back-

ground of SkQ1 administration [15]. It seems logical

that administration of SkQ1 with feed provided a sys-

temic effect closely associated with prevention of struc-

tural-functional disruptions of mitochondrial parame-

ters and/or with their restoration [16, 17]. Remarkably,

the long-term administration of SkQ1 suppressed the

mTOR (mammalian target of rapamycin) signaling

pathway [15], activation of which is considered as one

of the main mechanisms of aging and development of

age-related neurodegenerative diseases [18].

We have demonstrated previously that manifesta-

tion and progression of the signs of AMD and Alzhei-

mer’s disease in the OXYS rats occurred on the back-

ground of activation in the retina and brain of the

mitogen-activated protein kinase (MAPK) signalling

pathway [19-23], and the ability of SkQ1 to suppress

their development and progression, including accu-

mulation of toxic Aβ aggregates and hyperphosphor-

ylated tau-protein in the OXYS rat brain was found to

be associated with the decrease of activity of the p38

MAPK and ERK1/2 (extracellular signal-regulated ki-

nase 1 and 2) signaling pathways [20, 21]. Changes in

activity of MAPK have been recognized lately as im-

portant players in pathogenesis of neurodegenerative

diseases, and they are considered as potential targets

for therapeutic interventions [24]. In this study we

evaluated association between the ability of SkQ1

(Visomitin drops) to suppress progression of AMD

manifestations in the OXYS rats and its effect of activi-

ty of p38 MAPK and ERK1/2 in the retina.

Abbreviations: Aβ, amyloid beta; AMD, age-related macular degeneration; ERK1/2, extracellular signal-regulated kinase 1

and2; MAPK,mitogen-activated protein kinases; mTOR,mammalian target of rapamycin; RPE,retinal pigment epithelium;

SkQ1,10-(6′-plastoquinonyl)decyltriphenylphosphonium; VEGF, vascular endothelial growth factor.

VISOMITIN SUPPRESSES ACTIVITY OF P38 MAPK AND ERK1/2 IN RETINA 203

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

MATERIALS AND METHODS

Animals. Male Wistar and OXYS rats were used

in the study, which were housed under standard lab-

oratory conditions (22 ± 2°C and 12-h light/dark cycle)

in cages (57×36×20 cm) five animal in each with access

to standard feed for rodents (PK-120-1; Laboratorsnab,

Russia) and water ad libitum.

Exposure. OXYS rats from the experimental group

(n = 15) starting from the age of 9 months (age of active

development of retinopathy manifestations) received

daily instillations of one drop of SkQ1-based eye drops

Visomitin (NII Mitoinzhenerii MGU, Russia). OXYS rats

from the control group (n = 15) and Wistar rats (n = 15)

received placebo– preparation with composition iden-

tical to Visomitin, but without SkQ1.

Ophthalmological examinations. At the age of

9 and 12 months (before and after instillations) the

state of fundus of animal eyes was examined with the

help direct ophthalmoscope Beta (HEINE, Germany)

using pupil dilation with 1% tropicamide. Degree of

pathological changes in retina was evaluated using the

commonly accepted clinical classification according to

the Age-Related Eye Disease Study (AREDS) protocol

(https://eyephoto.ophth.wisc.edu): score 0 – no chang-

es; score1– 1st nonexudative state of disease, with dot

hemorrhages, edemas, drusen in the posterior pole of

the eye, defects in RPE, redistribution of pigment, atro-

phy of choriocapillaris layer and RPE; score2– 2nd ex-

udative stage– exudative detachment of RPE, neuroep-

ithelia; score 3 – 3rd stage – exudative-hemorrhagic

detachment of the retinal pigment and/or neuroepi-

thelia, neovascularization, scarring.

Histological examination. Posterior part of an

eye was fixed in a 12% solution of neutral buffered

formalin for 1 day as described previously [2]. Next, a

sample was washed in a running water for two hours,

dehydrated, flatten out, and immersed in paraffin ac-

cording to the standard technique. Slices of the pos-

terior part of an eye with thickness 4-5 µm were pre-

pared with the help of a rotary microtome and stained

with hematoxylin and eosin. Samples were analyzed

using a Carl Zeiss Axiostar plus microscope (Carl Zeiss,

Germany) with the help of Carl Zeiss AxioVision 8.0

program under magnification 10×100. Calculations of

the specific surface area of choroidal vessels (open,

with stasis, with sludge of formed blood elements, or

thrombosis), specific surface area of RPE, of photore-

ceptor and outer nuclear layers, open intraretinal ves-

sels in the retina slice, as well as average surface of cy-

toplasm and nuclei in the RPE were conducted for cells

of each retina (50 samples in each group). Number of

retinal slices and density of cells in the outer nuclear

layer were determined with the help of Avtandilov

ocular grid over the surface of 900 µm

. Five slices of

each retina imaged with magnification 10×100 were

used for analysis. Enumeration of photoreceptors with

nuclear pyknosis were carried out for 1000 photore-

ceptors, pyknomorphic radial gliocytes, neurons of in-

ner nuclear and ganglion layers (200 cells from each

retina).

Western blotting analysis. Retina of Wistar and

OXYS rats exposed to placebo instillations and retina of

the OXYS rats (n = 5) subjected to Visomitin instillations

were homogenized with the help of a RIPA buffer sup-

plemented with protease and phosphatase inhibitors

(Sigma-Aldrich, USA) as described previously [22, 23].

Total protein concentration was determined with the

help of bicinchoninic acid (BCA) (Thermo Fisher Scien-

tific, USA) assay. Proteins were separated using electro-

phoresis in a 12% polyacrylamide gel, transferred onto

a nitrocellulose membrane (Bio-Rad, USA), and blocked

with a 5% bovine serum albumin solution in a phos-

phate buffered saline (10 mM; pH 7.4) for 1 h. Next,

the membrane was incubated overnight at 4°C with

primary antibodies against p38 MAPK, phospho-p38

MAPK (Thr180, Tyr182), ERK1/ERK2, phospho-ERK1/

ERK2 (Thr202, Tyr204) (# 33-1300, # 36-8500, # 36-8800,

# 13-6200, respectively; dilution 1 : 1000; Invitrogen,

USA), Tau, phospho-Tau (S396), phospho-Tau (T181) or

GAPDH (ab80579, ab75679, ab109390, ab8245, respec-

tively; dilution 1 : 1000; Abcam, USA) followed by 1-h

incubation with secondary anti-rabbit and anti-mouse

antibodies (ab6721, ab6808, Abcam; dilution 1 : 5000).

Intensity of luminescence was recorded with the help

of a ChemiDoc MP visualization system (Bio-Rad) and

evaluated with the help of ImageJ software (NIH, USA).

Statistical analysis. Statistical analysis was

carried out with the help of Statistica 10.0 software

package (Statsoft, USA). One-way analysis of variance

(ANOVA) with post hoc comparison of differences in

means (Newman–Keul test) were used. Data are pre-

sented as a median (q1-q3). To estimate significance

of the differences during comparison of mean values

Mann–Whitney test was used. Differences were con-

sidered significant at the value p<0.05.

RESULTS

Visomitin slowed down progression of clinical

signs of retinopathy. Preliminary ophthalmological

examination of 9 months old rats from experimental

and control groups of OXYS rats revealed that there

were no differences in manifestations of pathological

signs in retina (p = 0.74), which were presented by the

first stage of retinopathy. At the age of 9-12 months the

OXYS rats receiving placebo demonstrated significantly

progressing pathological changes, which in 87% of the

cases corresponded to the second stage of the disease,

and in 13% of the cases– to the third most severe stage

of AMD. Manifestations of pathological changes were

MURALEVA et al.204

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

pronounced significantly less in the group of animals

subjected to instillation of Visomitin: in 90% of the cas-

es pathological changes corresponded to the first stage

of AMD, and only 10% – to the second stage. Average

levels of manifestation of pathological changes were

2.14 ± 0.07 and 1.33 ± 0.09, respectively, and were sig-

nificant (according to one-way ANOVA, F

1.59

 = 53.8,

p < 0.0000 and Mann–Whitney test – p = 0.000003).

In the Wistar rats pathological changes were not re-

vealed in both initial and second examinations– place-

bo instillation did not affect the state of retina.

Visomitin suppressed pathological changes in

the retina of OXYS rats. Histological examination of

all retina layers of 12 months old OXYS rats receiving

placebo revealed changes typical of AMD contrary to

the retina of Wistar rats (Fig.1, table). Specific surface

area of open choroidal vessels in OXYS rats was twice

less (p < 0.01), and fraction of the vessels with signs of

occlusion (with stasis, sludge, or thrombosis) – twice

higher (p < 0.01) than in the Wistar rats (table). With re-

gards to the intraretinal vessels in retina of OXYS rats,

microcirculatory changes have been also noted such

as appearance of swelled blood vessels with stasis and

sludge of formed elements, but no statistically signif-

icance of the differences has been revealed. Average

surface area of the RPE cells in the slice of OXYS rat

retina was twice less than in the retina of Wistar rats

(p < 0.01) mainly due to flattening of nuclei and de-

crease of the cytoplasm volume, however, nuclei with

signs of pyknosis were also revealed (Fig.1, table).

Part of photoreceptors in the retina of OXYS rats

was also subjected to destruction: percent of nuclei

with pyknosis was twice as high in comparison with

the Wistar rats (p < 0.01). Cell death was, respectively,

accompanied with the 1.7-fold decrease in the num-

ber of receptors rows (p < 0.01). Fraction of the asso-

ciative neurons with cytoplasm edema and shrinkage

of nucleus as well as of pyknomorphic hyperchro-

mic radial gliocytes in the inner nuclear layer of reti-

na of the OXYS was increased in comparison with the

Wistar rats (1.4- and 1.84-fold, respectively, p < 0.05;

Fig. 2, a and b; table). Percent of neurons with focal

and total chromatolysis and of neurons with signs of

pyknosis increased 1.4- (p < 0.05), 6.4-, and 11.2-fold

(p < 0.01; table), respectively, in the ganglion layer in

the retina of OXYS rats.

Daily instillations of Visomitin slowed down pro-

gression of retinopathy in the 9-12 months old OXYS

rats. Visomitin administration led to significant im-

provement in the state of microcirculation in the cho-

roidal vessels: specific surface area of the vessels with

signs of occlusion decreased 2-fold in the OXYS rats

(p < 0.01), and surface area of the open vessel increased

1.6-fold (p < 0.05) in comparison with the animals re-

ceiving placebo. Analysis of the RPE state revealed ac-

tivation of the compensatory-adaptive processes, which

was manifested in OXYS rat as the increase of average

surface area of nuclei in the RPE cells by 7.5% (p < 0.01),

thus offsetting the differences in this parameter in com-

parison with the Wistar rats (table).

Visomitin revealed clearly pronounced neuropro-

tective and glia-protective effects protecting neurons

and radial gliocytes in the retina of OXYS rats from

damage. As a result, the percent of pyknotic nuclei

in photoreceptors of the retina of OXYS rats treat-

ed with Visomitin was not higher than the level of

Fig. 1. Choroid complex in retina of Wistar(a) and OXYS(b) rats receiving placebo and of OXYS rats receiving Visomitin(c) at

the age 9-12 months. Designations: Ch,choroid; PL,photoreceptor layer; ONL,outer nuclear layer. Black arrows,choriocapillaris

with open lumen; white arrows,choriocapillaris blocked due to thrombosis; red arrows,pyknosis of RPE nucleus.

VISOMITIN SUPPRESSES ACTIVITY OF P38 MAPK AND ERK1/2 IN RETINA 205

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Effect of Visomitin on morphometric parameters of retina in rats

Parameters Wistar OXYS OXYS+ Visomitin

Specific surface area of open vessels, % 19.50 ± 1.93

9.67 ± 1.06*

p < 0.01

15.04 ± 1.43

p < 0.05

Specific surface area of vessels with stasis

or thrombosis, %

6.10 ± 0.44

11.36 ± 0.90*

p < 0.01

5.71 ± 0.83

p < 0.01

Specific surface area of pigment epithelium

in the retinal slice (%)

5.77 ± 0.41

2.64 ± 0.19*

p < 0.01

2.47 ± 0.19*

p < 0.01

Average surface area of RPE cell, µm

247.49 ± 3.96

183.65 ± 2.82*

p < 0.01

207.98 ± 3.52*

p < 0.01

Average surface area of the nucleus of RPE cell, µm

53.71 ± 0.82

48.11 ± 0.59*

p < 0.01

51.72 ± 0.90

p < 0.01

Number of photoreceptor rows 11.0 ± 0.42

6.62 ± 0.35*

p < 0.01

7.57 ± 0.29*

p < 0.01

Pyknosis of photoreceptor nuclei, % 0.37 ± 0.09

0.72 ± 0.06*

p < 0.01

0.57 ± 0.07

Pyknomorphic associative neurons, % 0.54 ± 0.06

0.75 ± 0.06*

p < 0.05

0.48 ± 0.05

p < 0.01

Pyknomorphic radial gliocytes, (%) 3.61 ± 0.78

6.67 ± 0.65*

p < 0.01

3.40 ± 0.59

p < 0.01

Ganglion neurons with focal chromatolysis, % 3.25 ± 0.31

4.41 ± 0.30*

p < 0.05

4.54 ± 0.16*

p < 0.01

Ganglion neurons with total chromatolysis, % 0.71 ± 0.19

4.53 ± 0.59*

p < 0.01

0.85 ± 0.08

Ganglion pyknomorphic neurons, (%) 0.28 ± 0.05

3.13 ± 0.38*

p < 0.01

0.44 ± 0.05

Specific surface area of intraretinal vessels, % 0.97 ± 0.10 1.13 ± 0.23 0.76 ± 0.13

Note. Data are presented as a mean ± SEM.

* Statistically significant differences between the OXYS and Wistar rats receiving placebo.

 Statistically significant effect of Visomitin instillations. Differences were considered statistically significant at the value p<0.05.

RPE,retinal pigment epithelium.

this parameter in the Wistar rats, fraction of the py-

knomorphic associative neurons decreased by 36%

(p < 0.01) and of radial gliocytes– by 51% (p < 0.01) in

comparison with the OXYS rats receiving placebo, thus

reaching the levels typical for the Wistar rats (Fig. 2;

table). Fractions of the cells with signs of destruction

in the population ganglion neurons were comparable

in the OXYS rats receiving Visomitin and Wistar rats

receiving placebo (table).

Visomitin decreased the level of phosphoryla-

tion of p38 MAPK and ERK1/2 in the retina of OXYS

rats. At the age of 12 months content of the p38 MAPK

protein and its phosphorylated form (p-p38MAPK) in

the retina of OXYS rats receiving placebo was higher

than in the Wistar rats (p < 0.005 and p < 0.001, respec-

tively; Fig. 3, a-c). The level of phosphorylated p38

MAPK (evaluated based on the ratio of p-p38MAPK to

p38MAPK) in the OXYS rats was higher than in the Wis-

tar rats (p < 0.017; Fig.3d). Instillations of Visomitin did

not affect the content of p38 MAPK, but there was a de-

creasing trend observed in the content of p-p38 MAPK

(p = 0.054; Fig.3,b andc). The p-p38 MAPK to p38 MAPK

ratio in this group was significantly lower than in the

OXYS rats from the placebo group (p < 0.050), but was

still higher than in the Wistar rats (Fig.3d).

Content of ERK1/2 in the retina of 12 months old

OXYS rats receiving placebo did not differ from the

content in the Wistar rat retina (Fig.3e). As expected,

MURALEVA et al.206

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 2. Retina of Wistar (a) and OXYS(b) rats receiving placebo, and of OXYS rats receiving Visomitin(c). Black arrows– associa-

tive neurons with edema; red arrows– hyperchromic pyknomorphic associative neurons; dashed arrows associative neurons–

pyknomorphic ganglion neurons. Designations: ONL,outer nuclear layer; INL, inner nuclear layer; ORL, inner retinal layer;

GL,ganglion layer. Staining with hematoxylin and eosin.

Fig. 3. Effects of Visomitin instillations on the content of p38 MAPK, p-p38 MAPK, ERK1/2, and p-ERK1/2 in the retina of OXYS

rats. Representative images of blots(a), content of p-38 MAPK(b), p-p38 MAPK(c), ratio p-p38 MAPK to p38 MAPK(d), ERK1/2(e),

p-ERK1/2(f), and ratio p-ERK1/2 to ERK1/2(g) in the retina of Wistar and OXYS rats receiving placebo, and of OXYS rats receiving

Visomitin. GAPDH was used as a control. Data are presented as a median (q1-q3). *Statistically significant differences between

the OXYS and Wistar rats receiving placebo; #Statistically significant effect of Visomitin instillations. Differences were consid-

ered significant at the level of p<0.05.

content of the phosphorylated form (p-ERK1/2) in the

OXYS rats was higher than in the Wistar rats (p < 0.005;

Fig. 3f). In agreement with these data, the level of

phosphorylation of ERK1/2 in the OXYS rats receiving

placebo was higher than in the Wistar rats (p < 0.005;

Fig. 3g), which indicates activation of the ERK1/2 sig-

naling pathway in the retina of these animals. Despite

the fact that Visomitin instillations did not affect the

VISOMITIN SUPPRESSES ACTIVITY OF P38 MAPK AND ERK1/2 IN RETINA 207

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 4. Effect of Visomitin of content of tau, p-tauT181, and p-tauS396 in the retina of OXYS rats. Representative images ofblots(a),

content of tau(b), p-tauT181(c), ratio p-tauT181/tau(d), p-tauS396(e), ratio p-tauS396/tau(f) in the retina of Wistar and OXYS

rats receiving placebo, and of OXYS rats receiving Visomitin. GAPDH content was used as a control. Data are presented asa me-

dian (q1-q3). *Significant differences between the OXYS and Wistar rats receiving placebo. #Statistically significant effect ofthe

preparation. Differences were considered significant at the level of p<0.05.

content of ERK1/2 in the retina of OXYS rats, there was

a decreasing trend observed in the content of p-ERK1/2

(p = 0.069; Fig. 3, e and f). It is important to note that

Visomitin administration significantly decreased the

ratio of p-ERK1/2 to ERK1/2 in the OXYS (p < 0.040;

Fig.3g) to the level observed in the Wistar rats.

Effect of Visomitin on the content of tau,

p-tauT181, and p-tauS396 in the retina of OXYS rats.

In order to confirm inhibitory effect of Visomitin instil-

lations of activity of p38 MAPK and ERK1/2 in the ret-

ina, the level of site-specific phosphorylation of their

target, tau protein, was evaluated. Total content of tau

protein in the retinas of 12 months old Wistar and

OXYS rats receiving placebo did not differ, and Visomi-

tin did not affect its content in the retina of OXYS rats

(Fig. 4, a and b). Increased content of the p-tauT181

and p-tauS396 in the retina of OXYS rats in compari-

son with the Wistar rats was observed (p < 0.025 and

p < 0.001, respectively). The content of p-tauT181 and

p-tauS396 was lower in the retina of OXYS rats receiv-

ing Visomitin in comparison with the control OXYS

rats (p < 0.017 and p < 0.001, respectively), as well as

the ratios of p-tauT181/tau and p-tauS396/tau (p < 0.05

and p < 0.05, respectively; Fig.4,c andd).

DISCUSSION

It has been shown previously in our study devot-

ed to investigation of the efficiency of SkQ1 instillation

(250 nM aqueous solution) that this form of prepara-

tion is capable of not only preventing development

and progression of AMD, but, similar to the case of

administration with feed, also of decreasing to a cer-

tain degree manifestation of the changes in the retina

of OXYS rats [4, 10, 25]. In this study we for the first

time present the results of evaluation of the effects of

the SkQ1-based pharmaceutical preparation, Visomitin

eye drops, on the development of retinopathy and ac-

tivity of MAPK in the retina of OXYS rats.

As can be seen from the results of ophthalmo-

logic examination, Visomitin instillations that start-

ed at the age of 9 months suppressed progression of

AMD signs in the OXYS rats: at the age of 12 months

manifestations of pathological changes in the animals

receiving preparation decreased 1.6-fold in compari-

son with the control animals. The preparation signifi-

cantly improved structural and functional parame-

ters of RPE, photoreceptors, neurons, and radial glia.

It could be suggested that neuroprotective effect of

MURALEVA et al.208

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

the preparation is associated with its ability to restore

microcirculation in the choroidal vessels– administra-

tion of this preparation led to significant decrease of

the number of vessels with disrupted blood flow.

AMD pathogenesis is closely associated with in-

flammation, oxidative stress, and progressive disrup-

tions in proteostasis, which are the processes regulat-

ed via the MAPK signaling cascades mediating signal

transduction from the cell membrane to the nucle-

us [26]. Sequential phosphorylation of downstream

kinases eventually causes phosphorylation and acti-

vation of the target proteins and nuclear transcription

factors. This event serves as an indicator of both effi-

ciency of signal transduction, and activity of the sig-

nal pathway as a whole. Suppression of activity of the

MAPK signaling pathway is considered as a promising

approach for treatment of AMD, despite the fact that

information on their state during the development of

AMD is very limited and is based on the data obtained

using the RPE cell cultures [24].

We have shown previously that manifestation

and progression of AMD signs in OXYS rats occur on

the background of activation of the p38 MAPK and

ERK1/2 signaling cascades in the retina [22, 23]. This

was confirmed in the current study: activity of the p38

MAPK and ERK1/2 signaling cascades in the retina of

12 months old OXYS rats was higher than in the Wis-

tar rats. Visomitin instillations affected activity of the

p38 MAPK and ERK1/2 signaling cascades in the retina

of OXYS rats. In the process, the preparation decreased

the content of p-p38MAPK and p-ERK1/2 only at the lev-

el of trend, but the ratio of phosphorylated forms of the

kinases to their total content decreased significantly.

Retina cells have unique morphology, lifespan,

and functional organization, which to a large degree

depend on structural and functional preservation of

proteins. Dysfunction of mitochondria during AMD de-

velopment triggers disruption of proteostasis– accumu-

lation of pathological aggregates, which are detected in

the retina of the patients with AMD before their vision

is affected [27, 28]. Such aggregates formed from lipo-

fuscin, toxic Aβ, and hyperphosphorylated tau-protein

are capable of damaging neurons. The MAPK signal-

ing pathways participate in regulation of proteosta-

sis via phosphorylation of target proteins, including

tau. We have shown previously that at the age of

18months, when the changes in the retina of OXYS rats

are clearly pronounced, the content of Aβ and of tau

protein phosphorylated at the MAPK-specific sites is

significantly increased [3, 23]. In the current study we

demonstrated that phosphorylation of the tau protein

in the retina of OXYS rat is increased already at the age

of 12 months, and Visomitin instillations decreased it

due to suppression of the MAPK-dependent phosphor-

ylation. Similar results were obtained during investiga-

tion of the effects of administration of SkQ1 with feed

on the dynamics of accumulation of the phosphorylated

tau and Aβ in the brain and retina of OXYS rats [15, 21].

Accumulation of pathological aggregates is facili-

tated by the decrease of activity of autophagy process-

es (cellular system controlling protein quality) occur-

ring with aging. Dysfunction of autophagy results in

disruption of homeostasis, accumulation of damaged

organelles and toxic proteins, which, in turn, initiate

disruption of mitochondrial functions and oxidative

stress [29]. Enhanced accumulation of lipofuscin and

Aβ in the retina on the background of disruption of

the autophagy process is typical for both the OXYS

rats with pronounced signs of AMD [30, 31], as well

as for the patients at the late stage of the disease [32].

Administration of SkQ1 with feed facilitated activation

of autophagy in the retina of OXYS rats [14]. The data

available in the literature allow suggesting existence

of direct association between the cellular systems of

quality control, changes in mitochondria dynamics,

and MAPK signaling pathways. We have shown previ-

ously that the mechanisms of neuroprotective effects

of SkQ1 both in the course of prophylactic administra-

tion with feed starting at early age, and in the case of

its administration to the OXYS rats with pronounced

neurodegenerative changes are associated with resto-

ration of structural and functional parameters of mito-

chondria in the brain [33,34].

Decrease in the activity of MAPK signaling cas-

cades by antioxidants has been demonstrated previ-

ously in different models of the human RPE damage.

In particular, protective effect of resveratrol has been

associated with inhibition of ERK 1/2 activity in the

RPE cells [35]. Blueberry extract increased the level of

endogenous antioxidant enzymes with simultaneous

decrease of reactive oxygen species and MAPK inhi-

bition in the model of oxidative damage in vitro [36].

Itmust be mentioned that the abovementioned effects

of antioxidants were associated not only with partial

inhibition of MAPK, but also with inhibition of other

signaling pathways. This occurs due to existence of re-

lationships between the MARK signal transmission and

such signaling pathways such as VEGF, mTOR, autoph-

agy, and others. It is known that p38 MAPK and ERK1/2

facilitate increase of expression of VEGF [37, 38], which

is the main proangiogenic factor involved in AMD

pathogenesis [39]. The eye drops with SkQ1 (its aque-

ous solution) decreased expression of VEGF in the ret-

ina of OXYS rats both at the level of mRNA and at the

protein level [25].

CONCLUSIONS

In this work we demonstrated that the eye drops

Visomitin are capable of suppressing progression

of retinopathy in OXYS rat and could, potentially,

VISOMITIN SUPPRESSES ACTIVITY OF P38 MAPK AND ERK1/2 IN RETINA 209

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

berecommended for treating AMD in humans. The re-

sults obtained in our previous studies and results of

the current study indicate that the mechanisms of

therapeutic effects of SkQ1– active compound in Viso-

mitin eye drops– are multifaceted and are associated

with its effect on redox-dependent signaling pathway,

including decrease of activity of the MAPK-dependent

signaling pathways.

Contributions. N.A.M. concept of the study, con-

ducting experiments, discussion of the results of the

study; A.Zh.F. ophthalmological examination of ani-

mals; A.A.Zh. histomorphometric analysis of retina

samples; A.Zh.F., A.A.Zh., N.A.M., and N.G.K. prepara-

tion and editing of the paper text.

Funding. This work was financially supported

by the Russian Foundation of Basic Research, grant

no.22-25-00224.

Ethics declarations. All applicable international,

national, and/or institutional guidelines for the care

and use of animals were followed. The authors of this

work declare that they have no conflicts of interest.

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