Peroxiredoxin 6 Attenuates Alloxan-Induced Type 1 Diabetes Mellitus in Mice and Cytokine-Induced Cytotoxicity in RIN-m5F Beta Cells

Abstract

Type 1 diabetes is associated with the destruction of pancreatic beta cells, which is mediated via an autoimmune mechanism and consequent inflammatory processes. In this article, we describe a beneficial effect of peroxiredoxin 6 (PRDX6) in a type 1 diabetes mouse model. The main idea of this study was based on the well-known data that oxidative stress plays an important role in pathogenesis of diabetes and its associated complications. We hypothesised that PRDX6, which is well known for its various biological functions, including antioxidant activity, may provide an antidiabetic effect. It was shown that PRDX6 prevented hyperglycemia, lowered the mortality rate, restored the plasma cytokine profile, reversed the splenic cell apoptosis, and reduced the β cell destruction in Langerhans islets in mice with a severe form of alloxan-induced diabetes. In addition, PRDX6 protected rat insulinoma RIN-m5F β cells, cultured with TNF-α and IL-1β, against the cytokine-induced cytotoxicity and reduced the apoptotic cell death and production of ROS. Signal transduction studies showed that PRDX6 prevented the activation of NF-κB and c-Jun N-terminal kinase signaling cascades in RIN-m5F β cells cultured with cytokines. In conclusion, there is a prospect for therapeutic application of PRDX6 to delay or even prevent β cell apoptosis in type 1 diabetes.

Full text

Research Article
Peroxiredoxin 6 Attenuates Alloxan-Induced Type 1 Diabetes
Mellitus in Mice and Cytokine-Induced Cytotoxicity in RIN-m5F
Beta Cells
Elena G. Novoselova , Olga V. Glushkova, Sergey M. Lunin, Maxim O. Khrenov,
Svetlana B. Parfenyuk, Tatyana V. Novoselova, Mars G. Sharapov, Vladimir I. Novoselov,
and Evgeny E. Fesenko
Institute of Cell Biophysics of the Russian Academy of Sciences, PSCBR RAS, Institutskaya Str. 3, 142290 Pushchino,
Moscow Region, Russia
Correspondence should be addressed to Elena G. Novoselova; elenanov_06@mail.ru
Received 21 January 2020; Revised 1 May 2020; Accepted 17 August 2020; Published 27 August 2020
Academic Editor: Fabrizio Barbetti
Copyright © 2020 Elena G. Novoselova et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Type 1 diabetes is associated with the destruction of pancreatic beta cells, which is mediated via an autoimmune mechanism and
consequent inflammatory processes. In this article, we describe a beneficial effect of peroxiredoxin 6 (PRDX6) in a type 1 diabetes
mouse model. The main idea of this study was based on the well-known data that oxidative stress plays an important role in
pathogenesis of diabetes and its associated complications. We hypothesised that PRDX6, which is well known for its various
biological functions, including antioxidant activity, may provide an antidiabetic effect. It was shown that PRDX6 prevented
hyperglycemia, lowered the mortality rate, restored the plasma cytokine profile, reversed the splenic cell apoptosis, and reduced the
βcell destruction in Langerhans islets in mice with a severe form of alloxan-induced diabetes. In addition, PRDX6 protected rat
insulinoma RIN-m5F βcells, cultured with TNF-αand IL-1β, against the cytokine-induced cytotoxicity and reduced the apoptotic
cell death and production of ROS. Signal transduction studies showed that PRDX6 prevented the activation of NF-κB and c-Jun
N-terminal kinase signaling cascades in RIN-m5F βcells cultured with cytokines. In conclusion, there is a prospect for therapeutic
application of PRDX6 to delay or even prevent βcell apoptosis in type 1 diabetes.
1. Introduction
Insulin-dependent diabetes mellitus, or type 1 diabetes
(T1D), is a multifactorial disease, in which autoimmune fac-
tors play a key role. Clinical symptoms of T1D manifest
themselves when most of insulin-producing pancreatic beta
cells have already died because of the activation of autoreac-
tive T lymphocytes. The massive death of insulin-producing
βcells, which is caused by cytotoxic T lymphocytes migrating
into the pancreas, leads to the accumulation of glucose in the
blood, and patients with T1D need regular administration of
insulin for the rest of their lives. Despite the administration
of insulin, T1D causes serious inflammatory complications
in many systems and organs, including the cardiovascular
system [1], kidneys [2], and eyes [3]. It is known that in dia-
betes, large blood vessels are especially severely damaged;
therefore, mortality from stroke and heart attack is three
times higher among patients with diabetes than in the rest
of the population.
Currently, most of the studies on the pathogenesis of
T1D mellitus focus on pancreatic βcells, which are targets
of the autoimmune attack. Meanwhile, the disease is usually
caused by oxidative stress and imbalances in the immune sys-
tem, which are related to autoaggressive clones of T lympho-
cytes [4, 5]. During autoimmune inflammatory reactions,
proinflammatory cytokines, including interleukin- (IL-) 1β,
tumor necrosis factor-alpha (TNF-α), and interferon- (IFN-
)γ, are released into the environment of βcells by activated
T cells and macrophages, causing βcell dysfunction and
death [6, 7]. Usually, a proinflammatory response protects
Hindawi
Journal of Diabetes Research
Volume 2020, Article ID 7523892, 11 pages
https://doi.org/10.1155/2020/7523892

the mammalian organism from foreign pathogens and main-
tains the integrity of tissues and cellular systems. However, a
defective proinflammatory response may cause the opposite
effect, increasing the risk of autoimmune pathologies, which
include T1D [6].
It is known that human T1D is sometimes linked with
altered genes providing susceptibility to diabetes [8]. How-
ever, studies on identical twins with familial diabetes showed
that only approximately half of them develop diabetes [9],
confirming an important role of environmental factors, such
as dietary factors during infancy, vaccination, and others
[10], in the risk of development of T1D [11]. T1D susceptibil-
ity involves a complex interplay between genetic and envi-
ronmental factors and has historically been attributed to
adaptive immunity, although there is now increasing support
for a role of innate inflammation [12].
Oxidative stress has been proven to play a key role in the
pathogenesis of diabetes and related complications [13], and
there is evidence that antioxidants, mainly low-molecular-
weight natural and synthetic substances, may be useful for
the treatment of various pathologies associated with diabetes
mellitus [14, 15]. Meanwhile, there are many reasons to
believe that antioxidant enzymes can be more effective in
neutralising reactive oxygen species (ROS) than low-
molecular-weight antioxidants. Previously, we have shown
the therapeutic effects of a recombinant peroxiredoxin 6
(PRDX6) in various pathologies associated with inflamma-
tion and oxidative stress, such as intestinal hypoxia/reperfu-
sion [16]. We believe that PRDX6 may be effective as an
agent that suppresses the level of oxidative stress in diabetes
mellitus. Indeed, it was shown that pancreatic βcells contain
lower levels of antioxidant enzymes, such as SOD, catalase,
and GPX, than do other mammalian tissues [17]. Therefore,
these cells are more sensitive to the damaging effects of ROS.
Because of this deficiency of endogenous antioxidant
enzymes in βcells, there is an increasing interest in the use
of external proteins with antioxidant activities to protect
pancreatic βcells during diabetes.
Increased superoxide production in the development
and progression of diabetes causes the activation of several
signal pathways involved in the pathogenesis of chronic
complications. Oxidative stress activates cellular signaling
pathways and transcription factors, including protein
kinase C (PKC), c-Jun-N-terminal kinase (JNK), p38
mitogen-activated protein kinase (MAPK), and nuclear
factor kappa-B (NF-κB) [18]. Recently, we have shown
that signal transduction systems of immune cells are
involved in the development of diabetes in animals, with
a special role played by the nuclear factor kappa B (NF-
κB) cascade [19]. Thus, we showed that the use of an
inhibitor of the NF-κB signaling pathway, as well as the
thymic hormone thymulin, and a diet with antioxidants
significantly reduced the immune imbalance in cells of
mice with alloxan-induced diabetes [20].
In the present study, the efficacy of PRDX6 for reducing
the damaging effects of alloxan-induced diabetes in mice
was studied for the first time. Furthermore, taking into
account the key role of the pancreatic βcell loss in the devel-
opment of diabetes mellitus, we studied the effects of PRDX6
on the viability and functional activity of the RIN-m5F βcell
line under conditions that simulate diabetes.
2. Materials and Methods
2.1. Animals, Diabetes Model, and Peroxiredoxin 6
Treatment. Six- to eight-week-old male BALB/c mice (22–
25 g) were maintained under standard laboratory conditions
(20–21
°
C, 10–14 h light/dark cycle, and 65% humidity), with
food and water provided ad libitum. Standard food pellets
contained a balanced diet of proteins, vitamins, and minerals
according to the Code of Practice for the Housing and Care
of Animals Used in Scientific Procedures [21]. Experimental
procedures were approved by the Institutional Ethical Com-
mittee (approval #57, 30/12/2011), and the experiments were
performed in accordance with the Guidelines for Ethical
Conduct in the Care and Use of Animals. Mice were sacri-
ficed using cervical dislocation and decapitated using a small
animal guillotine with a sharp blade.
Severe diabetes was modelled using a single intraperito-
neal injection of 500 mg/kg alloxan in 0.2mL of physiological
saline, and physiological saline was injected into the control
mice. Experiments were performed 10 days after the injec-
tion, when the blood glucose was consistently greater than
18 mM, indicating that the mice were diabetic. The mortality
rate of the parallel groups of diabetic mice was observed for
32 days. Blood glucose concentration was measured using a
glucometer (Accu-Chek Performa Nano, Germany) and Test
Strips (Accu-Chek Performa Solo, Germany). A drop of
blood was obtained from the tip of the tail of fasting mice.
PRDX6 (20 mg/kg body weight in 0.1 mL saline) was
applied intravenously directly before the onset of diabetes
on the first day and repeatedly on the eighth day during dia-
betes development. Control mice received intravenously
0.1 mL of physiological saline. Previously, we found that after
intravenous administration, PRDX6 retained the highest
level in the blood for 10 minutes; then, its amount gradually
decreased, but after 6 hours, about 30% of the administered
PRDX6 remained in blood plasma [22]. So, it was proved that
possible PRDX6 effects were caused by its presence in the
animal tissues.
2.2. Isolation and Purification of the PRDX6. Genetic con-
structions encoding (human) PRDX6 enzymes were obtained
and expressed earlier in E. coli BL21(DE3) cells [22]. Recom-
binant proteins harbored His-tag, so the enzymes were puri-
fied by affinity chromatography on Ni-NTA-agarose
(Thermo Fisher Scientific, USA), according to the manufac-
turer’s recommendations. The technique of protein isolation
was described earlier [23]. According to electrophoresis in
12% SDS-PAAG, the purity of the obtained enzymes was at
least 98%. PRDX6 in phosphate buffer (1.7 mM KH
2
PO
4
,
5.2 mM Na
2
HPO
4
, and 150 mM NaCl, pH 7.4) at a concen-
tration of 10 mg/mL were stored at –20
°
C. No reduction of
enzymatic activity was observed following 2 months of
storage.
The ability of the PRDX6 to reduce hydrogen peroxide
(H
2
O
2
) and tert-butyl-hydroperoxide (t-BOOH) was deter-
mined by Kang et al.’s method [24], with minor
2 Journal of Diabetes Research

modifications. Peroxidase activity of recombinant PRDX6
was 230 nmol/min/mg of protein (measured with H
2
O
2
)
and 100 nmol/min/mg of protein (measured with t-BOOH).
2.3. Blood Plasma. Plasma was isolated from the blood col-
lected during decapitation of the mice. Blood samples were
kept for 3–5 h at 4
°
Сand then centrifuged at 200 × g; the
supernatants were collected for cytokine assays. Splenic lym-
phocytes were isolated in Dulbecco’s modified Eagle’s
medium (DMEM; Sigma, USA) containing 10 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulphonic acid solution,
100 μg/mL streptomycin, and 10% fetal bovine serum. Eryth-
rocytes were lysed in Tris-buffered ammonium chloride
(0.01 M Tris-HCl, with 0.15 M NaCl, and 0.83% NH
4
Cl at
9 : 1, pH 7.2). After being washed, the samples were stored
at a concentration of 1×10
8cells/mL in RPMI 1640 medium
at −20
°
C.
2.4. Cytokine Measurements. Enzyme-linked immunosor-
bent assays (ELISAs) were used to determine concentrations
of cytokines in blood plasma using ELISA development kits
for mouse TNF-α, IL-5, IL-17, and IFN-γ(PeproTech,
USA). Binding was visualised by adding 100 μL of the 2,2′
-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) green
dye (Sigma), dissolved in 0.05 M citrate buffer (pH 4.0) with
0.01% hydrogen peroxide. Absorbance was measured at
405 nm using a Multiscan EX spectro photometer (Thermo
Electron Corporation, Vantaa, Finland).
2.5. Western Blotting Analysis. To prepare specimens, 1×10
8
splenic cells were lysed using a lysis buffer containing 50 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100 , and
5 mM ethylenediaminetetraacetic acid (Alfa Aesar, UK).
The total protein concentration was determined using Brad-
ford’s method [25] (Sigma) following protein precipitation
with acetone in an ice bath for 15 min. Next, proteins were
diluted 1 : 1 with a solution containing 65.8 mM Tris-HCl,
pH 6.8, 2.1% sodium dodecyl sulphate, 26.3% (w/v) glycerol,
and 0.01% bromophenol blue, boiled for 5 min, and stored at
4
°
C. Proteins were resolved by 10% polyacrylamide gel elec-
trophoresis using a protein MW marker (Thermo Scientific,
USA) and then transferred onto a nitrocellulose membrane
(GE Healthcare, Amersham, UK) in a transblot chamber.
After being blocked with 5% (w/v) nonfat dry milk in Tris-
buffered saline/Tween 20, the membranes were exposed for
2 h to the following antibodies raised against mouse proteins:
a phospho-NF-κB p65 (Ser 536) antibody (#3031, Cell Sig-
naling Technology, Danvers, MA, USA), rabbit phosphoin-
hibitor of NF-κB kinase (IKKα/β) antibody II (Ser 176/180)
(Cell Signaling Technology), rabbit phospho-stress-
activated protein kinase/c-Jun N-terminal kinase
(SAPK/JNK) antibody to synthetic phospho-SAPK/JNK pep-
tide (Cell Signaling Technology), and rabbit caspase-3 mono-
clonal antibody (Cell Signaling Technology). After being
washed, the membranes we re incubated for 1 h with an
anti-rabbit biotinylated antibody (Jackson ImmunoResearch,
West Grove, PA, USA), followed by incubation with
peroxidase-conjugated streptavidin for 1 h. As a loading con-
trol, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
was used, which was detected with a rabbit monoclonal anti-
body raised against a synthetic peptide corresponding to res-
idues near the C-terminus of human GAPDH (Cell Signaling
Technology). The ECL Plus chemiluminescent cocktail
(Amersham/GE) was used to develop the blots according to
the manufacturer’s instructions. The developed blots were
photographed using a TFX-35.WL transilluminator (Vilber
Lourmat, France). Protein bands were quantified densitome-
trically using Image Studio Software, version 5.2 (LI-COR,
NE, USA). Two-three independent experiments were per-
formed for each protein using cells from different passages
or splenocytes from individual animals. The obtained data
were normalised to the corresponding loading control
(GAPDH) and expressed in relative units.
2.6. Histology and Immunohistochemistry. The pancreases
from different groups were fixed in 10% formalin, then
embedded in paraffin, and 3 μm sections were prepared and
stained with haematoxylin and eosin (H&E) for histopatho-
logical examination. For the detect ion of insulin, the 3 μm
sections were dewaxed, rehydrated, and incubated with a
peroxidase-blocking reagent (DAKO Cytomation, Fort Col-
lins, CO, USA) to block endogenous peroxidase. Next, the
slides were incubated with phosphate-buffered saline
(PBS)+1% bovine serum albumin to block nonspecific bind-
ing. A rabbit anti-mouse insulin monoclonal antibody (Santa
Cruz Biotechnology, Santa Cruz, CA, USA) was subsequently
applied to the sections, followed by incubation with the
LSAB™system-HRP (DAKO Cytomation). The slides were
stained with diaminobenzidine according to the manufac-
turer’s instructions (DAKO Cytomation).
2.7. Quantification of βCell Mass. Images of the sections were
analysed using the ImageJ software (National Institutes of
Health, USA, http://rsb.info.nih.gov/ij/) to measure insulin-
positive areas and the total pancreas area. The βcell mass
(mg) per pancreas was calculated by multiplying the relative
insulin-positive area (the percentage of insulin-positive area
over total pancreas area) by the pancreas weight as was
reported earlier [26].
2.8. Culture of RIN-m5F Cells. Rat insulinoma RIN-m5F cells
(Vertebrate Cell Collection, St. Petersburg, Russia) were
grown in culture flasks in a medium, with a low glucose con-
tent (8.0 mM), consisting of a mixture of RPMI 1640 med-
ium/DMEM (1 : 1), supplemented with 10% fetal calf serum
(FCS), 100 μg/mL penicillin, 100 μg/mL streptomycin, and
50 μg/mL gentamicin, at 37
°
C and 5% CO
2
. Cells were only
used between passages 3 and 7 and were cultured for 24 h
and washed. To induce apoptosis, 30 ng/mL TNF-α(Recom-
binant Murine TNF-α, PeproTech)+15 ng/mL IL-1β
(Recombinant Murine IL-1β, PeproTech) were added.
Recombinant PRDX6 (150 μg/mL) was added 30 min before
the addition of the cytokines. Cells were cultured for 24 h
and washed before measuring the viability and signal pro-
teins. In each independent experiment performed using sep-
arate passage, the measurements were provided for 9–12
replicates. The average values from four independent exper-
iments were used to determine the significance of differences
3Journal of Diabetes Research

between groups. Cells incubated without the cytokines and
PRDX6 were used as controls.
2.9. Measurement of ROS Using Carboxy-2′,7′
-Dichlorodihydrofluorescein Diacetate (H
2
DCFDA). RIN-
m5F cells were cultured for 24 h in a 96-well plate
(2:5·×10
4cells per well in 100 μL) in DMEM and then
washed with PBS. The cells were then incubated with car-
boxy-H
2
DCFDA (Invitrogen, USA; freshly prepared in ster-
ile DMSO) at a final concentration of 2.5 μM in the
medium supplemented with 2% depleted FCS in the darkness
for 1 h; then, PRDX6 and the cytokines were added, and the
cells were incubated for another 1 h. Cells incubated in the
absence of PRDX6 and cytokines were used as a control.
The fluorescence was measured at an excitation of 485 nm
and an emission of 535 nm using an Infinite 200 plate reader
(Tecan, Austria), as described earlier [27].
2.10. Statistical Analysis. Statistical analysis was performed
using the Statistica/Win 6.0 software (Tulsa, OK, USA).
One-way analysis of variance, followed by Tukey’s post hoc
test, was performed to determine the significance of differ-
ences among groups. Values of p≤0:05 were considered
significant.
3. Results
3.1. Effects of PRDX6 on Mortality Rate and Plasma Glucose
Levels in Diabetic Mice. Three groups of mice were used
(alloxan-treated, alloxan plus PRDX6-treated, and untreated
age-matched controls), and each group consisted of 10 mice.
It was revealed that the mortality rate following administra-
tion of a high dose of alloxan (500 mg/kg) achieved about
80% on the 32nd day after alloxan treatment (Figure 1(a)).
The mortality rate of diabetic mice pretreated with PRDX6
was markedly lower compared to diabetic mice that did not
receive PRDX6.
Another three mouse groups consisting of 10 mice per
group (alloxan-treated, alloxan plus PRDX6-treated, and
untreated age-matched controls) were observed for blood
glucose. Blood glucose levels were measured for 9 days after
the administration of alloxan (Figure 1(b)). It was demon-
strated that on day 5 after the administration of alloxan, the
average glucose level in the blood of the alloxan-treated mice
exceeded 20 mM, while that in the mice treated with PRDX6
decreased to almost the control level (Figure 1(b)). Thus, the
administration of PRDX6 inhibited the glycemia raise in
mice with alloxan-induced diabetes. So, pathophysiological
manifestations in mice treated with alloxan were significantly
lowered by PRDX6 application.
3.2. Effects of PRDX6 on Spleen Cell Apoptosis in Diabetes
Mice. The level of apoptosis in splenocytes was assessed
based on the ratio of activated/nonactivated caspase-3. A
very sharp increase in the level of splenocytic apoptosis was
observed in alloxan-induced diabetes (Figure 2). However,
administration of PRDX6 to the diabetic mice completely
normalised the ratio of activated/nonactivated caspase-3 in
splenocytes, indicating a protective effect of PRDX6 in devel-
oped diabetes.
3.3. Effects of PRDX6 on Cytokine Levels in the Blood of
Diabetic Mice. A study of the cytokine response demon-
strated that in the mice with developed alloxan-induced dia-
betes, the concentrations of all measured cytokines in the
plasma increased on the 10th day after the administration
of alloxan, with the most marked increase observed in
TNF-αand IL-5. The concentrations of IFN-γand IL-17 also
increased, although these changes were relatively small
(Figure 3). The administration of PRDX6 reduced the peaks
of TNF-αand IL-5 in the blood of diabetic mice. It is impor-
tant to emphasise that the use of PRDX6 had no effect on the
level of IL-17 in the blood of diabetic mice.
3.4. Effects of PRDX6 on the Pancreatic Islet Structure, Insulin
Expression, and βCell Mass in Diabetic Mice. To elucidate
PRDX6 effects on the pancreas in diabetic mice, immuno-
staining for insulin was performed. Immunohistochemical
examination of the pancreas revealed a reduction in the islet
density in diabetic mice, and the residual βcells were severely
disorganised (Figure 4). PRDX6 injections substantially
restored the islet density in diabetic mice. Using sections
from the control and diabetic pancreases, we quantitatively
evaluated the βcell mass. As shown in Figure 4, the number
of βcells in the diabetic pancreas was significantly smaller
than that in the control pancreas. Treatment with PRDX6
markedly increased the βcell numbers, thereby protecting
the islet structure. This finding was consistent with the effects
of PRDX6 on the cytokine profile and cell apoptosis. The data
demonstrated the expected destruction of pancreatic βcells
in advanced diabetes and indicated that PRDX6 markedly
increased the βcell mass, thus supporting a protective func-
tion of the antioxidant enzyme. Moreover, immunostaining
confirmed the protective effect of PRDX6 on the immunity
of mice with alloxan-induced diabetes.
3.5. Effects of Cytokines and PRDX6 on RIN-m5F βCells.
RIN-m5F βcells were cultured under adverse conditions
(in the presence of proinflammatory cytokines), and the via-
bility of these cells was studied with or without the PRDX6
addition. To elucidate the molecular mechanisms of the pro-
tective effects of PRDX6, the level of apoptosis and the activ-
ity of the NF-κB and SAPK/JNK signaling cascades were
determined in RIN-m5F βcells. In addition, the PRDX6 anti-
oxidant activity was tested using a carboxy-H
2
DCFDA
probe.
The level of apoptosis in RIN-m5F βcells was assessed by
measuring the ratio of activated to nonactivated form of
caspase-3 (Figure 5). It was demonstrated that the addition
of the mixture of cytokines (TNF-αand IL-1β) to the cell cul-
ture medium led to a significant increase in the level of apo-
ptosis. At the same time, the presence of PRDX6 in the
medium completely eliminated the toxic effect of the cyto-
kines, reducing the ratio of the activated to nonactivated
form of caspase-3.
The activity of the NF-κB signaling cascade was assessed
based on the level of RelA/p65 protein phosphorylation at the
Ser 536 residue (Figure 5) and IKKα/βactivation. It was
shown that in the cells incubated with the proinflammatory
cytokines, the phosphorylation of the RelA/p65 protein at
4 Journal of Diabetes Research

Ser 536 increased more than twofold. It is important to note
that the addition of PRDX6 under these conditions had
almost no effect on the phosphorylation of RelA/p65. On
the other hand, incubation of RIN-m5F cells with the cyto-
kines led to increased phosphorylation of IKKα/β. At the
same time, PRDX6 completely alleviated the cytokine-
induced IKKα/βactivation. Thus, PRDX6 decreased the acti-
vation of the canonical NF-κB pathway in RIN-m5F βcells
under conditions of oxidative stress caused by proinflamma-
tory cytokines.
An even more profound protective effect of PRDX6 was
revealed by studying the JNK activity in RIN-m5F cells incu-
bated with the proinflammatory cytokines. Indeed, the addi-
tion of the cytokines to the RIN-m5F cell culture medium led
to the activation of the JNK signaling cascade, while the addi-
tion of PRDX6 completely blocked the cytokine-induced
JNK activation in βcells.
Furthermore, the antioxidant efficiency of PRDX6 was
also tested in vitro using RIN-m5F cells. As expected, the
addition of the proinflammatory cytokine mixture to the cul-
tural medium produced a sharp increase in the ROS content
in these cells. The results also showed that the addition of
PRDX6 significantly reduced the level of ROS in RIN-m5F
cells cultured in the presence of TNF-αand IL-1β(Figure 6).
Thus, we obtained evidence that the protective effects of
PRDX6 in T1D are related to its antioxidant activity. Inter-
estingly, using another model of oxidative stress in vivo,we
showed that the preliminary PRDX6 treatment reduced the
level of malonic aldehyde in the ischemia-reperfusion kidney
[22].
4. Discussion
Reduced antioxidant activity and increased oxidative stress
are among the intrinsic characteristics of T1D mellitus, both
in patients and in animal diabetes models. T1D is provoked
by the destruction of pancreatic βcells, which is mediated
via an autoimmune mechanism and consequent inflamma-
tory processes. Numerous inflammatory cytokines and
ROS, which are produced during development of diabetes,
have been proposed to play an important role in βcell
destruction. For example, ROS can penetrate through the cell
membrane and cause damage to βcells of the pancreas [28].
Possible causes of T1D are genetic or involve chemical,
1 4 7 10 14 18 21 25 32
120
100
80
60
40
20
0
Control
PRDX6 + diabetes
Diabetes
Surviving mice (percent to control)
Days aer alloxan treatment
⁎
⁎
⁎⁎⁎⁎
⁎⁎⁎
####
(a)
0
5
10
15
20
25
3579
PRDX6 + diabetes
Diabetes
Days aer alloxan treatment
Blood glucose (mM)
⁎
⁎
⁎
⁎
⁎
#
#
#
Control
(b)
Figure 1: Effects of PRDX6 on the mortality rate of mice and on blood glucose in diabetic animals. (a) Time course of mortality in different
groups of mice (mean ± standard deviation ðSDÞof 10 mice). (b) Time course of changes in plasma glucose in different groups of mice
(mean ± standard deviation ðSDÞof 10 mice). ∗p<0:05 vs. control;
#
p<0:05 vs. the diabetic group.
⁎
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Cleaved caspase-3/caspase-3
ratio
Caspase-3
caspase-3
Cleaved
Control Diabetes PRDX6+
diabetes
GAPDH
Figure 2: Expression of caspase-3 and cleaved caspase-3,
determined using western blot analysis. Representative blots from
three independent experiments are shown. Histograms show the
protein levels normalised to those of GAPDH, used as a loading
control, and to total forms of relative proteins and represent the
average densitometry values for the blots from two experiments
(mean ± SD). ∗p<0:05 vs. control.
5Journal of Diabetes Research

immune, or virus-associated damage to insulin-producing β
cells. Regardless of the cause, a common pathway may exist
that leads to the destruction of βcells.
Animal models play an important role in the develop-
ment of present concepts concerning T1D pathogenesis and
therapeutic approaches. In the present study, we modelled a
severe and rapidly developing form of T1D using a large dose
of alloxan. Indeed, worse blood glucose control in patients
with diabetes is generally associated with a more rapidly pro-
gressing disease and a wider range of complications. Using
0.3
0.25
0.2
0.15
0.1
0.05
0
Concentration (ng/ml)Concentration (ng/ml)
Concentration (ng/ml)Concentration (ng/ml)
IL-5
⁎
0.4
0.3
0.2
0.1
0
0.6
0.5
0.4
0.3
0.2
0.1
0
Control Diabetes PRDX6+
diabetes
Control Diabetes PRDX6+
diabetes
IL-17 IFN-𝛾
⁎⁎
⁎
#
1
0.8
0.6
0.4
0.2
0
TNF-𝛼
⁎
#
Figure 3: Effects of PRDX6 on plasma cytokine concentrations in diabetic mice. Each value is the mean ± SD for three mice; six
measurements were performed for each individual mouse. ∗p<0:05 vs. control;
#
p<0:05 vs. the diabetic group.
1.5
H&E
Ins
Control Diabetes PRDX6+
diabetes
Beta cell mass (mg/pancreas)
1
0.5
0
⁎
#
100 𝜇m
100 𝜇m
100 𝜇m
100 𝜇m
100 𝜇m
100 𝜇m
Figure 4: Effects of PRDX6 on the pancreas structure and βcell mass in diabetic mice. Representative images show islet histology using H&E
staining and islet immunostaining for insulin in the pancreas of control and diabetic mice. The βcell mass is shown as the mean ± SD for 15
sections/pancreas from three individual mice. ∗p<0:05 vs. control;
#
p<0:05 vs. the diabetic group.
6 Journal of Diabetes Research

this model, we demonstrated the beneficial effect of PRDX6
on the T1D pathology in terms of the plasma glucose level,
plasma cytokine profile, and splenic cell apoptosis. Moreover,
using histological and immunohistochemical assays, we stud-
ied the pancreatic islet structure and insulin expression in β
cells after treatment of diabetic mice with PRDX6.
In addition, to elucidate the molecular mechanisms of
PRDX6 protective activity, rat insulinoma RIN-m5F сells
were cultured with cytokines (TNF-αand IL-1β) and used
as an in vitro diabetic model to measure the ROS secretion,
βcell apoptosis, and the activity of the NF-κB and JNK sig-
naling pathways.
Among proinflammatory plasma cytokines, TNF-α, IFN-
γ, IL-5, and IL-17 were shown to increase in alloxan-induced
diabetic mice, which may indicate the activation of T helper 1
(Th1) cells, producing TNF-αand IFN-γ; Th2 cells, produc-
ing IL-5; and Th17 cells, secreting IL-17 [29]. The treatment
with PRDX6 reduced the plasma TNF-αand IL-5 levels but
showed no significant effects on the plasma concentrations
of IFN-γand IL-17 in diabetic mice. Apparently, this can
be explained by the fact that the use of various drugs to
reduce the pathological effects of diabetes can be more or less
effective, depending on the stage and severity of the disease.
Indeed, it has earlier been demonstrated that antidiabetic
approaches are more effective in prediabetic mice than in
mice with advanced diabetes [20]. In addition, IFN-γis
known to inhibit Th17 cells, and the role of Th17 cells in
T1D remains largely unknown [30].
More clear evidence of the protective activity of PRDX6
was obtained by the measurement of the blood glucose level,
as well as by the assessment of the level of apoptosis of sple-
nocytes. Indeed, administration of a large dose of alloxan
caused a sharp increase in the blood glucose level in diabetic
mice within 5 days after the administration, and glucose
remained at a high level throughout the experiment. In the
mice treated with PRDX6, a slight increase in plasma glucose
was observed on day 5, but by the end of the experiment, the
plasma glucose level was fully normalised. These observa-
tions are in agreement with the data of our recent study
showing the protective effects of PRDX6 on RIN-m5F сells
cultured with high glucose concentrations [31]. It should be
emphasised that according to our previous study, PRDX6
itself did not cause changes in either the activity of the NF-
κB and JNK signaling cascades or the level of apoptosis of β
cells, estimated by the ratio of activated to nonactivated form
of caspase-3 [31]. Thus, preliminary results indicate the non-
toxicity of the PRDX6 recombinant protein.
It is generally known that the pathogenic effect of hyper-
glycemia is mediated, to a significant extent, via increased
production of ROS. Therefore, it seems quite possible that
PRDX6 may actually reduce the oxidative stress in diabetic
mice, as was shown in the present in vitro study using RIN-
5mF cells. In addition, alloxan-induced diabetes is a rapidly
developing severe form of diabetes, which may be more
dependent on oxidative stress than other forms. Consistent
with our results, another study demonstrated that acute ele-
vation of glucose resulted in a more specific triggering effect
on oxidative stress than did chronic sustained hyperglycemia
[32].
0
⁎
200
400
600
800
1000
1200
1400
Relative units
0
⁎
400
200
600
800
1000
1200
1400
1600
Relative units
0
⁎
⁎
200
100
300
400
500
600
800
700
900
Relative units
0
0.2
0.1
0.3
0.4
0.5
0.6
0.8
Control Cytokines PRDX6+
cytokines
0.7
Caspase/cleaved caspase
ratio
GAPDH
Caspase-3
Cleaved
caspase-3
phRelA
phIKK
phJNK
Figure 5: Effects of PRDX6 on the activation of caspase-3, NF-κB,
IKK, and JNK pathways in RIN-m5F βcells cultured with
proinflammatory cytokines. Blot images are shown from a single
representative experiment. Histograms show the protein levels (in
relative units) normalised to those of GAPDH, used as a loading
control, and to total forms of relative proteins, representing the
average densitometry values for blots from two-three experiments
with cells from different passages (mean ± SD). ∗p<0:05 vs. control.
7Journal of Diabetes Research

It is commonly known that βcell death in T1D involves
necrosis and apoptosis [33]. One of the immunocytochemi-
cal markers for apoptosis is cleaved caspase-3. The caspase-
3 protein is a member of the cysteine-aspartic acid protease
(caspase) family and plays a central role in the execution
phase of cell apoptosis [34]. The ubiquitously distributed
caspase-3 is the main effector of the apoptotic cascade within
cells and is activated through cleavage [35].
In the present study, we demonstrated that both βcells
in vitro and splenic cells in vivo underwent apoptosis in situ-
ations modelling T1D, and the treatment with PRDX6 sub-
stantially reduced the diabetogenic apoptosis, thus
indicating a protective effect of PRDX6. In addition, the loss
of the βcell mass, observed in mice with alloxan-induced dia-
betes and mediated by the activation of proapoptotic signal-
ing events, is increasingly recognised as the causal and
committed stage in the development of T1D mellitus. Thus,
our data suggest that this stage may also be alleviated by
PRDX6 administration.
The recombinant PRDX6 can affect the level of ROS in
animals, inhibiting the development of oxidative stress and
normalising the redox status of cells. However, the question
arises: how does recombinant PRDX6 located in the extracel-
lular space neutralise ROS in cells into which it does not pen-
etrate? It is known that hydroperoxides, in addition to
passive diffusion through the cell membrane, are actively
transported to the intercellular space using aquaporins [36].
Probably, being in the extracellular space, recombinant
PRDX6 can participate in the elimination of peroxides
formed in the intercellular space and released from the cells
by the aquaporins.
Earlier, it was shown that PRDX6 can affect the level of
NF-κB via the TLR4/NF-κB signaling pathway [37]. The
authors showed that during ischemic damage of the brain,
PRDX6 released during the destruction of cells can act as
an endogenous ligand for the TLR4 receptor. The interaction
of PRDX6 with TLR4 triggers a cascade of processes in which
NF-κB plays a major role, resulting in an emergency cell
repair and suppression of apoptosis [38]. It is possible that
intravenous application of the recombinant PRDX6 before
exposure to alloxan can lead to a preconditioning effect,
inducing the mechanisms of repair and antioxidant response
through stimulation of the TLR4/NF-κB signaling pathway
in the cells. Therefore, exposure to alloxan does not lead to
a synergistic increase in NF-κB expression.
In addition to the peroxidase activity of PRDX6, a Ca
2+
-
dependent phospholipase A2 activity (aiPLA2) of PRDX6
was also shown, which normally manifests itself only under
acidic conditions and plays an important role in the metabo-
lism of phospholipids and the transmission of intracellular/-
intercellular signals [39]. Interestingly, with an excess of
ROS, peroxidase cysteine center of PRDX6 is oxidized, which
leads to a significant increase in the Ca
2+
-dependent phos-
pholipase A2 activity (aiPLA2) [39]. In addition, regardless
of the oxidation of the peroxidase center, an increase in the
phospholipase activity (more than 10 times) of PRDX6 is
observed after specific phosphorylation of the Thr177 residue
by mitogen-activated protein kinases (MAPKs) (ERK2, p38γ,
and p38δ) [40]. Accordingly, with the induction of the
aiPLA2-activity of PRDX6, there is an increase in the level
of lysophospholipids and fatty acids, which serve as second-
ary messengers both in normal and in pathologies. It has
been shown that the phospholipase activity of PRDX6 stimu-
lates signaling pathways (p38, PI3K/Akt) and also promotes
the formation of arachidonic acid, which, in turn, affects
the activity of Src (SFK) kinases, stimulating cell growth
and division [41].
We have shown earlier that in slowly developing diabetes,
induced by a small dose of alloxan, the most significant acti-
vation of several signaling cascades, including the interferon
regulatory factor 3, Toll-like receptor 4, and NF-κB path-
ways, as well as an increase in the expression of heat shock
protein 70 in splenic cells, was observed only at the prediabe-
tes stage but not in advanced diabetes [20]. However, the JNK
pathway was an exception in this regard, being activated
more significantly at the stage of advanced diabetes. In the
present study, we demonstrated that the JNK pathway was
profoundly activated in βcells by proinflammatory cytokines
and oxidative stress. This does not contradict the findings
that demonstrated the role of both NF-κB and JNK signaling
220
200
160
180
120
140
80
100
0 20 40 60 80 100 120 140
Fluorescence intensity (percent to control)
Time (min)
Cytokines
Control
PRDX6
+cytokines
⁎
⁎
⁎#
⁎⁎⁎⁎⁎
#####
#
Figure 6: Effect of PRDX6 on the ROS levels in RIN-m5F cells in the presence of proinflammatory cytokines. Cells were incubated for 1 h in
the presence of the fluorescent dye carboxy-H
2
DCFDA, with or without cytokines (TNF-α+IL-1β) and PRDX6 (150 μg/mL). Each value
presents the mean green fluorescence intensity from 9–12 repeats (as a percentage of control). ∗p<0:01 vs. control;
#
p<0:05 vs. the
cytokine treatment group.
8 Journal of Diabetes Research

in rat βcell death that is induced by proinflammatory cyto-
kines [42]. Interestingly, PRDX6 downregulated the JNK
activity in βcells exposed to TNF-αand IL-1β, suggesting
that oxidative stress and subsequent activation of the JNK
pathway could be involved in the pathogenesis of T1D. This
study investigated whether the NF-κB and JNK pathways are
involved in the protective effect of exogenous PRX6 against β
cell destruction induced by alloxan treatment in mice or by
proinflammatory cytokines in RIN-m5F cells. We demon-
strated that the decrease in the ROS levels in RIN-m5F cells
was accompanied by improvement of activity of caspase-3,
IKK, and JNK pathways in RIN-m5F βcells cultured with
proinflammatory cytokines. In addition, PRDX6 adminis-
trated to diabetic mice partially improved the plasma cyto-
kine profile and tended to restore the pancreas structure
and βcell mass in diabetic mice.
The importance of the NF-κB pathway was demonstrated
in both T1D and T2D, due to its role in inflammatory
responses [43]. We have previously shown involvement of
NF-κB, namely, RelA/p65, in the protection of pancreatic β
cells in alloxan-induced diabetic mice, using an inhibitor of
this cascade (IKK inhibitor XII) [20]. It was demonstrated
that knockdown of PRDX6 increased susceptibility of RIN-
m5F cells to the deleterious effects of proinflammatory cyto-
kines and to oxidative stress [44]. These results show that
among the PRDXs significantly expressed in RIN-m5F cells,
only PRDX6 is modulated by the proinflammatory cytokines,
and the PRDX6 downregulation depends on the calpain, pro-
teasome systems, and JNK signaling [44]. Moreover, the link
between PRX6 and NF-κB, which is one of the most promi-
nent redox-regulated proinflammatory regulators, has been
previously observed in hypoxic mouse hippocampal cells
[45]. Similarly, PRDX6 expression is inversely correlated
with NF-κB during Clonorchis sinensis infection [46]. Inter-
estingly, NF-κB activation is crucial for maturation and acti-
vation of immune cells, but in βcells, it has mostly a
deleterious effect [47]. So, understanding the innate inflam-
mation and mechanisms by which βcell susceptibility to pro-
inflammatory cytokines is potentiated or mitigated offers
important insight into T1D progression and avenues for
therapeutic intervention [12].
In conclusion, PRDX6 treatment reduced cell apoptosis
in both in vitro and in vivo models of T1D. Thus, PRDX6
protected RIN-5mF cells against cytokine-induced cytotoxic-
ity in vitro, and it also prevented T1D, normalised blood glu-
cose, lowered mortality rate, and restored the pancreatic islet
structure in vivo. Moreover, PRDX6 normalised the activity
of the NF-κB and JNK pathways in βcells cultured with pro-
inflammatory cytokines and protected these cells from ROS.
Therefore, there is a prospect for therapeutic application of
PRDX6 to alleviate or even prevent βcell apoptosis in T1D.
5. Conclusion
The study demonstrates that administration of recombinant,
exogenous PRDX6 reduces impact of the alloxan toxicity on
pancreatic βcells in mice, improving animal mortality and
lowering the serum glucose level. The beneficial effects of
recombinant PRDX6 treatment are associated with reduced
levels of inflammatory cytokines in vivo. Meanwhile, in the
rat insulinoma cell line, the recombinant PRDX6 attenuated
effect of exogenous proinflammatory cytokines on the ROS
production and activation of NF-κB and JNK signaling path-
ways. In summary, the manuscript provides novel observa-
tions, which have clear implications in understanding the
cytotoxic mechanism underlying death of βcells in DM1
and in forming potential therapeutic approaches.
Data Availability
The data used to support the findings of this study are
included within the article.
Disclosure
The funding sponsors had no role in the design of the study;
in the collection, analyses, or interpretation of data; in the
writing of the manuscript; and in the decision to publish
the results.
Conflicts of Interest
The authors report no conflict of interest.
Acknowledgments
The work was supported by the Russian Foundation for Basic
Research, project Nos. 18-04-00091 and 20-015-00216, and
by the Program of Russian Academy of Sciences 1.18 “Molec-
ular and Cellular Biology and Post-Genomic Technologies.”
The authors thank Editage for help with language editing.
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