Investigation into the potential protective effect of exenatide against dicarbonyl
stress-mediated hepatic inflammation
Siddiqi Fatima
Abbreviations:
μM
NF-κB
IFN-γ
TNF-α
NIK – NFkB inducing kinase
AKT – protein kinase B pathway
Abstract
The prevalence of diabetes is increasing worldwide and hence giving arise to the
vast number of complications associated with chronic hyperglycaemia. This
occurs despite the growing number of hypoglycaemic agents available on the
market. Liver complications have been linked to uncontrolled diabetes and there
is a positive correlation of people with type 2 diabetes and the common liver
disease, non-alcoholic fatty liver disease (NAFLD). The diabetic complications
have more recently been linked to dicarbonyl stress in particular the toxic
metabolite methylglyoxal (MGO). Elevated concentrations of methylglyoxal allow
for conformational changes to the macromolecules of all cells: proteins, lipids and
DNA. Methylglyoxal activates the inflammatory response through NF-κB which
releases an abundance of cytokines including IL-8. IL-8 levels were measured in
this investigation and an increase was reported upon blocking the glyoxalase 1
enzyme, using BBGC, responsible for detoxifying MGO. A loss in cell viability was
also noted when HEPG2 cells were treated with BBGC. Unfortunately, the GLP-1
analogue that has previously been cited as an anti-inflammatory agent had no
protective effect on the HEPG2 cells in this investigation. However, alternate antiinflammatory drugs must be tested in order to deduce a therapeutic option to
prevent the complications of hyperglycaemia on the liver.
Introduction
Diabetes is a metabolic disease that affects insulin resistance; it is increasing in
prevalence worldwide. 90% of diabetes cases are classed as type 2 diabetes
mellitus with the other main type being type 1 diabetes. The sole treatment
available for type 1 diabetes is exogenous insulin due to the condition resulting in
the absence of insulin production due to destruction of the islets in the beta cells.
Type 1 diabetic therapy is limited to exogenous insulin administration however
there is a range of treatments available for type 2 diabetics. GLP-1 analogues are
indicated as an adjunct therapy in combination with other hypoglycaemic agents.
Uncontrolled hyperglycaemia has severe complications; it increases the risk factor
of cardiovascular disease as well as cause nephropathy, retinopathy and
neuropathy. More recently liver complications have been identified as a risk factor
of prolonged hyperglycaemia. Non-alcoholic fatty liver disease (NAFLD) is a
common liver disease associated with diabetes. Other liver complications can
occur as a result of this such as NASH, cirrhosis and hepatocellular carcinoma
(HCC). Hyperglycaemia induced damage caused to proteins, lipids and DNA can
induce non-alcoholic fatty liver disease (NAFLD). The global prevalence of NAFLD
exceeds 15% however there is a much higher incidence in diabetic patients, type
2 diabetic patients are seen to have 80% more fat accumulation in their liver
compared to their non-diabetic match (Mavrogiannaki and Migdalis, 2013). Type
2 diabetics are at an increased risk of developing cirrhosis, hepatocellular
carcinomas (HCC) and liver failure (Mohamed et al., 2016).
NAFLD is characterised by insulin resistance and mitochondrial dysfunction
(Mavrogiannaki and Migdalis, 2013).
Diabetic complications occur due to high glucose levels for prolonged periods of
time. Hyperglycaemia therefore results in an increased risk of cardiovascular
disease due to angiogenesis as a result of glycated red blood cells accumulating in
the blood vessels implicating blood circulation to and from the heart. Recent
evidence suggests hyperglycaemia mediated oxidative stress as the source behind
diabetic complications. The glucose metabolite, methylglyoxal (MGO) plays a
pivotal part in the induction of dicarbonyl stress. Dicarbonyl stress is defined as
an accumulation of dicarbonyl metabolites, these include: glyoxal, methylglyoxal
and 3-deoxyglyucosone. Methylglyoxal, as seen in figure 1, is formed as a result of
glyceroneogenesis hence is a part of normal cell metabolism (Masania et al., 2016).
Figure 1. The structural diagram of methylglyoxal, a toxic metabolite of glycolysis
involved in hyperglycaemia induced dicarbonyl stress
Exenatide, a GLP-1 analogue, is an antidiabetic agent used in the adjunct treatment
of type 2 diabetes. The therapeutic effect is achieved due to increased glucose
induced insulin secretion and decreased glucagon release. Additionally, there is
increased glucose utilisation in the peripheral tissues. There is an enhanced
metabolic effect as exenatide inhibits gastric emptying and decreases appetite this
results in weight loss and improved insulin sensitivity (Andreozzi et al., 2016).
Exenatide has also been shown to have a secondary anti-inflammatory effect.
Exenatide binds to the GLP-1 receptor blocking PKC and NF-kappaB activation and
the subsequent of TNF-alpha, IL-6, VCAM-1 and IFN-gamma. Exenatide may
provide some protection against increased risk of liver disease therefore the aim
of this study was to determine whether exenatide could inhibit dicarbonyl stress
mediated inflammation.
Once activated the GLP1-R increases intracellular cAMP and induces activation of
the PKA, ERK, PI3K and PKB pathways which exert an anti-inflammatory effect (Andreozzi et al., 2016).
From Lit Review:
The GLP-1 analogue, exenatide, binds to the GLP-1 receptor activating the AMPK
protein. This plays a vital role in mitochondrial biogenesis, fatty acid synthesis and
glucose uptake. Defective AMPK activation is associated with insulin resistance
and T2DM. AMPK activity plays a vital role in the molecular mechanism of action
of this drug. Exenatide increases phosphorylation of the alpha subunit of AMPK at
Thr172. Consequently, there is an increase in the uptake of 2DG and exenatide
induces GLUT-4 translocation to the plasma membrane. As MGO is a model of
chronic glucose toxicity, studies show exenatide is an effective treatment in
dicarbonyl stress because the pathway it utilises is unaffected by this reactive
metabolite (Andreozzi et al., 2016).
Studies show there is a significant decrease in reactive oxygen species generation
as well as NF-kB when subjects are treated with exenatide. The mRNA expression
of TNF-alpha, JNK-1, TLR-2 and TLR-4 inflammatory mediators decreases. TLR-4
and TLR-2 leads to protection from insulin resistance. Exenatide induces the
release of insulin from the beta cells while mediating the suppression of glucagon
release from the alpha cells of the pancreatic islets. It also crosses the blood brain
barrier suppressing diet by allowing slower gastric emptying and causing a
decrease in body weight. Exenatide decreases plasma C-reactive protein (CRP)
concentrations as well as systolic blood pressure. Studies show an increase in Treg cells and IL-10 following exenatide administration. Exenatide has been shown
to suppress oxidative stress and CRP and MCP-1 in type 2 diabetes. This
demonstrates the anti-inflammatory effect exenatide demonstrates. Furthermore,
exenatide significantly suppressed the DNA binding of NF-kB this was associated
with suppression in mRNA expression of TNF-alpha and IL-1beta these are target
genes for NF. Exenatide exerts an anti-inflammatory effect independently of
weight loss(Chaudhuri et al., 2012).
Exenatide significantly inhibits oxidative stress mediated by advanced glycation
end products in diabetic rats by reducing ET-1 and inflammatory cytokine via
ROCK/NF-kB signalling pathways and AMPK activation(Lee and Jun, 2016)-CRP is
a protein produced by the liver in response to inflammation - there is a
significant reduction in CRP and TNF-alpha after exenatide treatment.
GLP-1R agonists bind to the GLP-1R blocking PKC or NF-kB activation and
the subsequent expression of TNF-alpha, IL-6, VCAM-1 and IFN-gamma. The
signalling of GLP-1R activates cAMP/Ca2+ and pAMPK inducing an antiinflammatory effect. GL1-RAs also show a reduction in microvascular events
as there was a reduction in microalbuminuria (Marchand et al., 2021). This
drug is particularly effective in liver disease as most of the oral
hypoglycaemic agents have toxic liver effects due to hepatic metabolism. LP1 is mediated on hepatic steatosis due to the activation of AMP-activated
protein kinase (AMPK) (Shao et al., 2014). GLP-1 proteins in the liver have a
direct effect on lipid metabolism (Stein et al., 2009). GLP-1R agonists
improve β-cell function and survival during endoplasmic reticulum (ER)
stress. Exenatide reduces the expression of the stress marker CHOP (Yusta
et al., 2006). Exenatide decreases oxidative stress by reducing the
concentrations of TG and FFAs stores in hepatocytes(Mells et al., 2012).
Exenatide acts on the GLP-1 receptor and prevents apoptosis via a
PKA/PI3K/Akt-dependent pathway(Li et al., 2018)
Exenatide induces the expression of serine protease inhibitor-9 in human islets.
Exenatide attenuates translational down-regulation of insulin and improves
survival of purified rat β-cells and islet cell lines after ER stress induction in vitro
via mechanisms that include enhancement of ATF-4 translation, increased
expression of GADD34, and dephosphorylation of eIF2α (Lee and Jun, 2016).
Methods
Materials
Materials used for the purpose of this investigation include BBGC, MTT and DMSO
purchased from Sigma-Aldrich, IL-8 from BD Biosciences and cell culture and cell
plastic from Thermo Fisher Scientific.
Cell culture
A human hepatic cell line, HEPG2 obtained from European Collection of
Authenticated Cell Cultures (ECACC), was used as the cell culture for this
investigation. The HEPG2 cells were passaged weekly and seeded at a density of
3.5x104 cells per well in 96-well plates for the MTT assay and 14x104 cells per well
in 48-well plates for the measurement of IL-8 release. The cells were cultured in
RPMI-1640 media containing 10% fetal calf serum (FCS), supplemented with 1%
penicillin and streptomycin, for treatment the FCS percentage was reduced to 3%.
The cells were incubated at 37°C in a humidified atmosphere of 5% CO2. Cells were
left for 24h prior to treating for the assessment of cell viability by MTT assay while
for measurement of IL-8 release the cells were grown for 48h prior to treatment.
Experimental protocol
In the first series of experiments the HEPG2 cell line was exposed to the GLO-1
inhibitor BBGC [1-20 μM] for 24 hours before measuring cell viability and
inflammation. In the second set of experiments the experimental procedure
described was conducted with exenatide concentrations: 0, 5, 10, 20, 30nM. In the
final series of experiments HepG2 cells were exposed to a combination of BBGC
[10 or 20 μM] + exenatide [10-30nM] before again measuring cell viability and
inflammation. This was done to determine whether exenatide provides a
protective effect from the damage caused to the hepatocytes by BBGC.
Cell viability
Cell viability was determined by the metabolic assay, MTT. The yellow MTT dye is
converted to insoluble purple crystals by NADPH dependent mitochondrial
enzymes that catalyse the reduction of MTT to formazan in the presence of
metabolically active cells. Following experimental protocol, the media was
removed from the plates and replaced with MTT (0.5mg/mL) the plates were then
incubated at 37°C for 30 minutes. The MTT was then removed and the purple
crystals were solubilised with DMSO before the absorbance was measured at
540nm in a microplate reader. The results were expressed as a % of absorbance
observed in untreated cells and were expressed as mean ± SEM.
Inflammation
Inflammation was measured through IL-8 release; following experimental
protocol the media was frozen, removed and stored at -20°C in order to conduct
the assay. IL-8 was measured using the commercially available ELISA kit and
according to the manufacturer’s instructions. The total protein concentration was
measured using the Bradford assay and results were expressed as pg/μg of
protein.
Statistical analysis
Statistical analysis was carried out using the student’s T-tests, for BBGC and
exenatide separately and one-way analysis of variance with Bonferroni’s
correction for the series of combinations all results are expressed as mean ± SEM
and p<0.05 was considered significant.
Results
Effect of BBGC on HEPG2 cell viability and inflammation
Following 24 hour exposure, BBGC dose dependently decreased cell viability as
seen in figure 2A while increasing inflammation shown in figure 2B E.g 20
micromolar BBGC reduced cell viability by 30.3% whilst increasing inflammation
by 166%.
Figure 2
Effect of BBGC on HEPG2 cell viability (A) and inflammation (B) following 24-hour
exposure. BBGC induced a dose dependent decline in cell viability with the most
prominent decline at its highest concentration of 20μM. A marked increase in release
of IL-8 (B) was seen. Results are expressed as mean ± SEM from n=2-4 (3-6 replicates
per experiments). Statistical analysis was carried out using student’s t–test where
p<0.05 was considered significant. ** Indicate where p<0.05 vs untreated cells.
Effect of Exenatide on HEPG2 cell viability and inflammation
Following 24 hours exposure to exenatide there was no effect seen on both cell
viability (figure 3A) and inflammation (figure 3B).
Figure 3
Effect of exenatide on HEPG2 cell viability (A) and inflammation (B) following 24hour exposure. Exenatide had no significant effect on cell viability (A) or
inflammation (B). Results are expressed as mean ± SEM from n=2-4 (3-6 replicates
per experiments). Statistical analysis was carried out using student’s t–test where
p<0.05 was considered significant. ** Indicate where p<0.05 vs untreated cells.
Effect of exenatide on BBGC-mediated loss of cell viability and inflammation
BBGC [20 μM] was used in combination with increasing concentrations of
exenatide. There is no protective effect of exenatide on HEPG2 cells following
exposure to BBGC.
All concentrations of the combinations of BBGC and exenatide resulted in
statistically significant increase in IL-8 release, furthermore, 20micromolar BBGC
increased inflammation to a greater extend in comparison to its lesser
concentration 10micromolar. 30nM exenatide + 10micromolar BBGC resulted in
117% increase in IL-8 release this can be compared to the 20micromolar BBGC
combination which showed a 172% increase.
Figure 4
Effect of BBGC and exenatide combination on HEPG2 cell viability, A, and
inflammation, B. Loss in cell viability was seen across all concentrations of exenatide,
however the % loss decreased as the exenatide concentration increased. B –
inflammation increased across all concentrations of the combinations with a greater
release at 20micromolar concentration of BBGC. Results are expressed as mean ±
SEM from n=2-4 (3-6 replicates per experiments). Statistical analysis was carried
out using ANOVA where p<0.05 was considered significant. **p<0.05 vs. untreated
cells.
Discussion
Section 1: For the first time we have shown that inhibition of GLO 1 resulted in a
loss of hepatocyte cell viability and an increase in inflammation mimicking what
has been showed in studies where MGO is applied directly therefore our results
suggest the effects are due to increased dicarbonyl stress. Following treatment of
HEPG2 cells with the GLO-1 inhibitor BBGC, a dose dependent decrease in cell
viability was observed. There is a marked loss of cell viability at 20μM
concentration of BBGC. This is due to the inhibition of methylglyoxal to its nontoxic intermediate via GLO-1. Hence, there is an accumulation of MGO in the
HEPG2 cells. MGO induces inflammation intracellularly through its activation on
inflammatory transcription factor NF-κB. This has not been shown in HEPG2 cells
therefore this research provides insightful results. Unfortunately, exenatide failed
to protect hepatocytes against both the loss of cell viability and inflammation
induced by dicarbonyl stress.
Find a study where they have shown the effect of methylglyoxal applied directly
to cells then include this study with BBGC and explain how inhibiting GLO-1
provides a more physiological effect. Studies have shown the cytotoxic effect of
BBGC on other cell types such as HL60 cells where a concentration-dependent
decline in cell viability as well as an increase in cytotoxicity was reported
(Thornalley et al., 1996). In diabetes methylglyoxal levels have been found to be
as high as 8 micromol/L. Glyoxalase is the main detoxifying system that maintains
intracellular homeostasis in the presence of glutathione. Glyoxalase 1 (GLO-1) is
the enzyme that converts methylglyoxal to the inactive intermediate, S-DLactoylglutathione. In our investigation we used the GLO-1 inhibitor, BBGC, to
induce dicarbonyl stress in the hepatocytes. BBGC, as seen in figure 5, is a diester
and a cell permeable competitive inhibitor of GLO-1(Sakamoto et al., 2000). The
loss of cell viability induced by BBGC is likely due to activation of JNK1 and MAPK
resulting in caspase activation and hence inducing loss of cell viability in
hepatocytes (Sakamoto et al., 2001). Inhibition of GLO-1 is a more physiological
induction of oxidative stress and mimics diabetic pathophysiology.
Figure 5 The structural diagram of BBGC, a competitive inhibitor of glyoxalase
1, the prominent metabolic pathway in detoxifying MGO
Don’t know If I need anything further from this ref - BBGC induces apoptosis https://www.jbc.org/article/S0021-9258(19)46693-3/fulltext - Miller 2006
Dicarbonyl stress exerts its effects due to the accumulation of methylglyoxal. The
glyoxalase system plays a role in detoxifying methylglyoxal. MGO is metabolised
by the glutathione (GSH) dependent enzyme glyoxalase 1 (GLO-1) to an
antioxidant intermediate, S-D-lactoylglutathione (SLG). SLG is further
metabolised to non-toxic D-lactate by glyoxalase 2. This protective cellular
mechanism plays a key role in maintaining cell homeostasis and ensuring the
concentration of MGO remains in range, 50-150nM (Rabbani et al., 2016). The
levels of MGO have been seen to be markedly increased, 8micromol/L in
diabetes. The protective mechanisms become overwhelmed as a result of
hyperglycaemia. Metabolic states such as diabetes induce the increased
metabolism of nutrients resulting in additional stress on the mitochondria and
the release of reactive oxygen species (ROS). ROS cause a loss of the
mitochondrial membrane potential resulting in mitochondrial dysfunction due to
a loss in cellular ATP production and therefore induces apoptosis.
Methylglyoxal depletes glutathione levels therefore causes a disruption in the
detoxifying mechanism of the glyoxalase system. MGO decreases the activity of
the antioxidant enzyme SOD, catalase and Glutathione S Transferase (GST) in the
liver (Seo et al., 2014).
MGO inactivates glutathione reductase via an NADPH dependent mechanism
(Vander Jagt et al., 1997).
Previous studies show that MGO causes apoptosis in HEPG2 cells - (Seo et al.,
2014)
Methylglyoxal binds irreversibly with proteins, nucleic acids and lipids causing
structural and functional changes. The binding and glycation of MGO to proteins
triggers endocytosis through the presentation of cell surface receptors and
activation of macrophages (Thornalley et al., 1996).
NF-κB is responsible for the regulation and release of inflammatory cytokines
resulting in chronic inflammation and loss of cell viability. MGO increases the
expression of pro-inflammatory cytokines IFN-γ and TNF-α these cytokines
induce cell death.
Methylglyoxal interacts with DNA causing single strand breaks as well as DNAprotein cross links and cytotoxicity (Sakamoto et al., 2001)
The effect of MGO on proteins, nucleic acids and lipids results in the formation of
ROS - (Hollenbach et al., 2021)
DNA strand breaks result in the activation of PARP – (Rolo and Palmeira, 2006) PARP additionally activates pro-inflammatory pathways
MGO modified proteins may also produce inactivation of enzymes such as
membrane ATPases resulting in membrane deformation – this modification
provides a signalling for protein degradation - (Lo et al., 1994)
Methylglyoxal induces apoptosis – (Thornalley, 1996) – Proteins modified by MGO
undergo receptor- mediated endocytosis and lysosomal degradation is macrophages
and monocytes and induce cytokine synthesis and secretion
H2O2 is catalysed to water by the enzymes glutathione peroxidase and catalase
MGO inactivates glutathione peroxidase and glutathione reductase Activation of NFkB involves degredation of IkBa (this is the inhibitory protein for
the transcription factor) https://www.ahajournals.org/doi/10.1161/hy0302.105207?url_ver=Z39.882003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
Aldehyde reductase is a major hepatic enzyme that detoxifies MGO – inactivation
of this enzyme by glycation results in apoptosis - (Okado et al., 1996)
Caspase 3 is a major protease in apoptosis due to oxidative stress - (Kim et al.,
2004)- the activation of NFkB is known to be regulated by ROS – studies suggest
that NFkB has a pro-apoptotic role in MGO-induced apoptosis mediated by ROS
Towards the end of section 4:
Studies show that the rough endoplasmic reticulum is reduced in diabetic
patients, this gives rise to the endoplasmic reticulum (ER) stress that occurs as a
result of oxidative damage to proteins. (Mohamed et al., 2016)
Build-up of fatty acids disrupts the beta-oxidation in the hepatic mitochondria
resulting in further accumulation of fats in the liver (Mohamed et al., 2016).
Liver damage can result in high levels of ferritin which is the protein that carries
iron.
MGO induced oxidative stress causes a loss of cell viability by the damage it causes
to proteins, lipids and DNA.
MGO impairs insulin signalling by inhibition of phosphorylation of IRS1 (insulin
receptor substrate 1) and activation of PI3K (phosphoinositide 3-kinase) - (Seo et
al., 2014)
Altering mitochondrial protein function through the electron transport chain
increases inflammatory protein expression resulting in apoptosis (Ahmed, 2005).
Apoptosis was induced by BBGC in HL60 cells therefore similar studies in HEPG2
cells should be carried out to see if the same effect occurs (Thornalley et al., 1996).
– this can be in the future work bit
GAPDH activity is inhibited resulting in an increase in the upstream intermediates
involved in the glycolytic pathway hence activating the AGE pathway due to an
increased production of MGO following an increase in G3P. MGO causes increased
expression of RAGE and the ligands it uses for activation: S100 calgranulins and
HMGB1.
AGEs bind to the transmembrane receptor RAGE and activate NFkB resulting in
the production of pro-inflammatory cytokines - (Hollenbach et al., 2021)
P50 and p65 are NFkB dimers - (Romeo et al., 2002)
Dicarbonyl stress induced inflammation in hepatocytes results in liver disease.
MGO increases pro-inflammatory cytokine production. This occurs as a result of
MGO modifying the mitochondrial proteins resulting in an increase in reactive
oxygen species (ROS) and oxidative stress (Masania et al., 2016). When exposed
to MGO, NF-kappaB translocates to the nucleus (Cha et al., 2019). MGO activates
NF-kappaB through the receptor for advanced glycation end products (RAGE). A
positive feedback loop is established through the activation of NF-kappaB and its
upregulation on RAGE. Mitochondrial dysfunction in hepatocytes results in proapoptotic proteins, cytochrome c, released in to the cytosol (Paradies et al., 2014).
Increased MGO in the liver is seen as a mediator of insulin resistance hence
predisposes an individual to diabetes and NAFLD. Fatty acids are substrates and
inducers of CYP2E1 and CYP4A these are microsomal lipoxygenases that produce
free oxygen radicals and induce lipid peroxidation of hepatocyte membranes. ROS
trigger lipid peroxidation causing cell death via the release of the toxic byproducts
malondialedehyde (MDA) and 4-hydroxynonenal (HNE). MDA and HNE activate
stellate cells increasing collagen production in the liver. HNE also increases the
inflammatory response by activating neutrophils. ROS induce the production of
cytokines TNF-alpha and IL-8 (Mavrogiannaki and Migdalis, 2013). As
inflammation is the cause of progression to liver disease, an anti-inflammatory
adjuvant may help to reduce the incidence of diabetic complications.
Ceramide and diacylglycerol (DAG) activate the IKK/NF-kappaB pathway
In hepatocytes ceramides interact with TNFalpha promoting the release of ROS by
hepatic mitochondria resulting in apoptosis and worse hepatic inflammation
(Mota et al., 2016).
TNF-alpha and Il-6 are the major inflammatory mediators in NAFLD.
MGO modified proteins contribute to the development of diabetic complications
and oxidative stress - (Thornalley, 1996)
Mutations in mtDNA have been linked to the pathogenesis of T2DM
The antidiabetic agent exenatide, a GLP-1 analogue, has been shown to have antiinflammatory effects. Exenatide binds to the GLP-1 receptor blocking PKC and NFkappaB activation and the subsequent of TNF-alpha, IL-6, VCAM-1 and IFNgamma. The activation of the GLP1- receptor induces cAMP/Ca2+ and pAMPK
inducing an anti-inflammatory effect (Marchand et al., 2021). Exenatide may
provide some protection against increased risk of liver disease therefore the aim
of this study was to determine whether exenatide could inhibit dicarbonyl stress
mediated inflammation.
Conclusion
This study indicates the effect of BBGC in inducing dicarbonyl stress in HEPG2
cells. The GLO-1 inhibitor is a more physiological source of inducing dicarbonyl
stress compared to MGO itself as the effects are apparent intracellularly and
previously decreased levels of GLO-1 have been identified in advanced liver
disease and cirrhosis. Moreover, inhibition of this detoxifying enzyme has
consequently been linked to liver disease due to the intracellular increase in
methylglyoxal. The GLP-1 analogue exenatide did not provide a protective antiinflammatory effect in this investigation. However, other anti-inflammatory drugs
should be tested as potential therapeutic options in the attempt to decrease
diabetic complications arisen by dicarbonyl stress and chronic inflammation.
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