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American Journal of  
Chemistry and Pharmacy (AJCP)

A Study on the Therapeutic Effect of  5-Azacytidine to Attenuate 
the Ramifying Repercussions of  Ischemia Reperfusion Injury on 

Mitochondrial Molecular Machinery 
Vasisht Yegneshwaran1*, Priyanka N Prem1,  Sri Rahavi Boovarahan1, Gino A Kurian2

Volume 1 Issue 2, Year 2022
ISSN: 2834-0116 (Online)

DOI: https://doi.org/10.54536/ajcp.v1i2.1169
https://journals.e-palli.com/home/index.php/ajcp

Article Information ABSTRACT

Received: January 05, 2023

Accepted: January 29, 2023

Published: February 05, 2023

5-Azacytidine is a hypomethylating agent that has for long been used in cancer therapy due 
to its ability to inhibit the protein DNA methyltransferase responsible for hyper-methylating 
DNA strands. Recently, studies involving in vitro, ex vivo, and in vivo experiments have 
assessed the cardioprotective effects of  5-Azacytidine during myocardial ischemia-reperfusion 
injury (IRI). However, the effect of  this compound in restoring the damage induced to 
mitochondrial molecular machinery during IRI has not yet been explored. Understanding 
this would help us analyze the ways through which mito-targeted therapeutics can be used. 
The purpose of  this study is to investigate the therapeutic impact of  5-Azacytidine, as DNA 
methylation is a very common epigenetic modification observed during IRI. Furthermore, 
the protective effect of  the compound in alleviating the damage induced to mitochondria 
during IRI can be identified, as DNA methylation can leave a direct impact on the 
mitochondrial genes as well. An isolated mitochondria model will be used to determine the 
effects of  5-Azacydine on mitochondrial molecular machinery as the capacity to generate 
DNA, RNA, and proteins are preserved in isolated mitochondria. In this study, we focus on 
the mechanisms of  mitochondrial replication, and translation to understand the effect of  
5-Azacytidine on the IRI affected mitochondrial system. Mitochondrial dysfunction is also 
another key turn of  events that happens during IRI. The role of  5-Azacyidine in preserving 
the functionality is also being assessed in our research. The findings of  these experiments 
would help us determine the plasticity the compound imparts on mitochondrial molecular 
mechanism’s integrity and function post-induced IRI.

Keywords
Mitochondria, Ischemia-
Reperfusion Injury, In-Vitro 
Replication, Mitochondrial 
Dysfunction 

1 Vascular Biology Lab, 117, Anusandhan Kendra, SASTRA Deemed University, Tirumalaisamudram, Tamil Nadu, India
2 School of  Chemical and Biotechnology, SASTRA Deemed University, Tirumalaisamudram, Tamil Nadu, India
* Corresponding author’s e-mail: vasisht31@gmail.com

INTRODUCTION
Ischemia, which is caused by the blockage of  blood 
vessels, is a condition that leads to infarction of  tissue. The 
best possible treatment identified till date is reperfusion, 
which involves removing the obstruction to blood flow 
using drugs and partially or completely invasive surgery 
depending on the severity of  the infarction. Reperfusion 
of  tissues affected by ischemia is contraindicated in its 
ability to cause ischemia reperfusion injury, which is 
identified by certain unique pathophysiological hallmarks 
such as the generation of  reactive oxygen species (ROS) 
due to the reestablishment of  circulation, elevated 
inflammation caused by the excessive ROS, Calcium 
overload and mitochondrial dysfunction, which includes 
the abnormal opening of  the MPTPs. (Frank et al., 2012; 
Hausenloy and Yellon, 2013; Sánchez-Hernández et al., 
2020)
Researchers have been trying to understand the 
mechanisms and develop therapeutic measures against 
this condition. Recent studies implicated epigenetic 
modifications in regulating the expression of  genes 
involved in pathways, contributing to IRI. A complete 
understanding of  these epigenetic mechanisms is essential 
to identify an appropriate target to curb the damage 
caused by IRI, as a prophylactic or as a treatment option. 
Since they partially control gene expression patterns, 
these epigenetic mechanisms go beyond genetics. To 
better understand how ischemia reperfusion damage 

may be treated or prevented, the exact definition of  
epigenetics has been employed and controlled with the 
aid of  inhibitors to alter gene expression patterns.
Epigenetics is the mechanism that influences heritable 
changes in gene expression and function without altering 
the genome’s sequence. These epigenetic pathways are 
influenced by external environmental elements as well. 
These epigenetic mechanisms control various mediators 
which results in Ischemia Reperfusion Injury. Some of  
mediators of  reperfusion injury are oxygen free radicals 
(ROS), endothelial dysfunction and microvascular 
injury, alterations in calcium level and altered myocardial 
metabolism. Yet these concepts, key events and the 
complete order of  how these mechanisms can be 
understood is still a work in progress. 
Many studies believe that targeting abnormal DNA 
methylation in Ischemia Reperfusion injury is a key 
technique for the prevention and treatment of  the disease. 
Methylation of  DNA at cytosine phosphate-guanine 
(CpG) dinucleotides is a typical epigenetic alteration 
that serves as a link between the genotype and the 
environment. The methyl group (-CH3) is added to the 
5th position of  cytosine residues in Cytosine-phospho-
guanine (CpG) dinucleotides during DNA methylation, 
resulting in chromatin condensation and gene expression 
changes. Enzymes known as DNA methyltransferases 
(DNMTs) catalyse this process, which is reversed by 
enzymes known as Ten-Eleven-Translocation protein 

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1 (TET1), which converts 5-methyl cytosine (5mc) to 
5-hydroxymethyl cytosine (5hmc). (Moore et al., 2012)
DNA methyltransferase inhibitors (DNMTi) have also 
been demonstrated to be useful in the treatment of  
ischemia-related illnesses. 5-Azacytidine, a DNMTi 
is a medication licensed by the US Food and Drug 
Administration (FDA) for the treatment of  acute myeloid 
leukaemia (AML) and myelodysplastic syndrome (MDS), 
with an inhibitory impact on DNA methylation as the 
underlying mechanism. 5-Azacytidine has a long history 
of  clinical use in cancer therapy. Moreover, several 
studies have been conducted to demonstrate the role of  
5-azacytidine as a cardioprotective drug in the treatment 
of  ischemia/reperfusion (I/R) damage. It is done in the 
animal model of  rat. (Boovarahan & Kurian, 2021)
The discovery that 5-azacytidine was integrated into 
DNA and that it blocked DNA methylation when present 
in DNA led to its widespread use to show the relationship 
between the loss of  methylation in particular gene areas 
and the activation of  the related genes. (Christman, 
2002) Since methylation works on DNA directly, it may 
work on nuclear and mitochondrial DNA. It also allows 
for the observation of  both direct and indirect effects 
on epigenetic modulations on various components 
involved in IRI. An extensive, broad-spectrum target like 
methylation pattern may provide for the development of  
therapeutics for ischemic conditioning in the future. The 
main focus of  this paper, will therefore be with respect 
to 5-Azacytidine and its therapeutic  effects on ischemia 
reperfusion injury. 

MATERIALS AND METHODS
Animals
The guidelines from the Committee for the Purpose of  
Control and Supervision of  Experiments on Animals 
(CPCSEA), Government of  India were strictly adhered 
to throughout all the animal experimental procedures 
involved in the study. All the rights and terms has been 
approved by the Institutional Animal Ethical Committee 
(IAEC) at SASTRA Deemed to be University, Thanjavur, 
India for the conduct of  experiments. Male Wistar Rats 
of  weight between 250-300 grams inbred in the Central 
Animal Facility at SASTRA Deemed to be University, 
Thanjavur, India was used in the study. The rats were 
all housed in a well-ventilated polycarbonate cage in a 
temperature-controlled room at (22 ± 2°C) with a relative 
humidity of  (60 ± 5%). The animals were exposed to 12 
hours of  light and dark cycle with ad libitum supply of  
water and food.

Isolation of  Mitochondria
The organelle mitochondria was isolated using density 
gradient differential centrifugation of  the organ 
homogenate following the guidelines from Palmer, et al. 
A 10% organ homogenate was prepared momentarily 
before the process of  density gradient differential 
centrifugation using Isolation Buffer (220 mM mannitol, 
70 mM sucrose, 5 mM MOPS, 2 mM EDTA, and 0.2% 

BSA) with a pH of  7.4. The organ homogenate was then 
centrifuged at lower speed of  800g for 10 minutes at 4°C 
to pellet the nuclear fraction. The supernatant was then 
transferred into a new Eppendorf  tube. The supernatant 
was then centrifuged at 8000g for 10 minutes at 4°C to 
remove any cellular debris. The supernatant was removed 
and the pellet was briefly resuspended in Isolation Buffer. 
This suspension was subjected to high centrifugation 
of  12,000g for 10 minutes at 4°C to pellet a pure 
fraction of  mitochondria (Graham J. M. et al. 2002). 
The mitochondria was resuspended in Storage Buffer 
(100mM KCL, 100mM Tris-HCl, 75mM Sorbitol, 25mM 
Sucrose, 10mM K2HPO4 , 5mM MgCl2 , 0.05mM EDTA, 
0.2% BSA) at a pH of  7.4 and their protein concentration 
was determined with the use of  Bradfords reagents (Bio-
Rad). A Bovine Serum Albumin standard was used to 
determine the concentration. After the process of  protein 
estimation, the isolated mitochondria were randomly 
divided in to groups and subjected to Normoxia and 
Hypoxia Reperfusion using Respiratory Buffer (300mM 
Mannitol, 100mM KCl, 20mM HEPES, 10mM KH2PO4, 
5mM MgCl2 , 1mM EGTA, 0.2% BSA) at pH 7.1 and 
Hypoxia Buffer purged with N2 (75mM NaCl, 25mM 
HEPES, 20mM Lactate, 16mM KCl, 10mM NaHCO3 
, 5mM Deoxy-d-glucose, 1.2mM MgCl2 , 1.2mM CaCl2, 
1mM KH2PO4) at pH6.8.
Experimental Groups
Isolated mitochondria from rat hearts after normalisation 
were randomly divided into six groups and details of  each 
experimental groups are as follows:
1. Normal - After the process of  mitochondria isolation, 
the organelle was subjected to equilibration using 
Respiratory Buffer for 1 hour 15 minutes to maintain 
normal respiration.
2. Normal + 5-Azacytidine - After the process of  
mitochondria isolation, the organelle was subjected to 
equilibration using Respiratory Buffer for 15 minutes 
followed by 0.5 μM 5-Azacytidine pre-treatment for 
another 15 minutes. At the end of  drug pre-treatment, 
the mitochondria were centrifuged at high speed of  
12,000g for 10 minutes at 4°C to pellet a pure fraction 
of  mitochondria. Then, the mitochondrial pellet was 
resuspended in Respiration Buffer for 45 minutes to 
maintain normal respiration.
3. Normal + DMSO - After the process of  mitochondria 
isolation, the organelle was subjected to equilibration 
using Respiratory Buffer for 15 minutes followed by 0.5 
μM DMSO pre-treatment for another 15 minutes. At 
the end of  drug pre-treatment, the mitochondria were 
centrifuged at high speed of  12,000g for 10 minutes at 
4°C to pellet a pure fraction of  mitochondria. Then, 
the mitochondrial pellet was resuspended in Respiration 
Buffer for 45 minutes to maintain normal respiration.
4. IR - After the process of  mitochondria isolation, 
the organelle was subjected to equilibration using 
Respiratory Buffer for 30 minutes followed by highspeed 
centrifugation at 12,000g for 10 minutes at 4°C to pellet 
a pure fraction of  mitochondria. Then, the mitochondrial 

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pellet was resuspended in Hypoxia Buffer for 15 minutes 
to hypoxia followed by a 30 minutes incubation in 
Respiration Buffer to facilitate reperfusion.
5. IR + 5-Azacytidine - After the process of  mitochondria 
isolation, the organelle was subjected to equilibration 
using Respiratory Buffer for 15 minutes followed by 0.5 
μM 5-Azacytidine pre-treatment for another 15 minutes. 
At the end of  drug pre-treatment, the mitochondria were 
centrifuged at high speed of  12,000g for 10 minutes at 
4°C to pellet a pure fraction of  mitochondria. Then, the 
mitochondrial pellet was resuspended in Hypoxia Buffer 
for 15 minutes to hypoxia followed by a 30 minutes 
incubation in Respiration Buffer to facilitate reperfusion.
6. IR + DMSO - After the process of  mitochondria 
isolation, the organelle was subjected to equilibration 
using Respiratory Buffer for 15 minutes followed by 0.5 
μM DMSO pre-treatment for another 15 minutes. At 
the end of  drug pre-treatment, the mitochondria were 
centrifuged at high speed of  12,000g for 10 minutes at 
4°C to pellet a pure fraction of  mitochondria. Then, the 
mitochondrial pellet was resuspended in Hypoxia Buffer 
for 15 minutes to hypoxia followed by a 30 minutes 
incubation in Respiration Buffer to facilitate reperfusion.
Evaluation of  Mitochondrial Function using 
Mitochondrial Electron Transport Chain Complex 
Activity
After the estimation of  protein and normalisation of  
mitochondria, the mitochondrial electron transport chain 
activity was measured spectrophotometrically by employing 
a specific donor-acceptor oxidoreductase in a 0.1M 
phosphate buffer (Frazier et al. 2012). For determination 
of  mitochondrial Complex1 activity a Rotenone sensitive 
NADH oxidoreductase was used. A Succinate decyl 
ubiquinone 2,6-dichlorophenolindophenol (DCPIP) 
reductase was used determine the mitochondrial Complex 
II activity. To determine the mitochondrial Complex III 
and IV activity cytochrome C reductase and cytochrome 
c oxidase was used as previously described by Ansari et al.

Oxidative Stress Assessment
The antioxidant profile of  the isolated mitochondria 
homogenate was evaluated using Catalase activity, SOD 
activity and GSH:GSSG ratio. All the experiments used 
a multimode spectrophotometric plate reader to measure 
kinetic absorbance as well as endpoint absorbance. 
A reaction buffer containing 0.1 M sodium phosphate 
buffer, pH 7.2, 4 mM H2O2 , and 5 N H2SO4 was added 
to the isolated mitochondrial samples. The reaction 
was initiated by the addition of  0.005M KMnO4 to the 
reaction buffer containing samples. The change in optical 
density was measured kinetically measured at 515 nm to 
assess the catalase activity (Goldblith et al. 1950). 
A reaction buffer containing 45mM Tris, 1mM EDTA was 
added to the isolated mitochondria samples. The reaction 
was initiated by the addition of  2.5mM Pyrogallol to the 
reaction buffer containing samples. The change in optical 
density was measured kinetically at 420 nm to assess the 
superoxide dismutase activity (Nandi et al. 1988). 

A reaction buffer containing 0.25m sodium phosphate 
and 5% Trichloroacetic acid was added to the isolated 
mitochondria samples. The reaction was initiated by 
the addition of  Ellman’s reagent (5,5’-dithiobis-2-
nitro-benzoic acid) to the reaction buffer containing 
samples. The change in colour due to formation of  
thionitrobenzoate was measured at 412 nm to assess the 
GSH activity (Sedlak et al. 1968).
A reaction buffer containing 0.25m sodium phosphate 
buffer, 0.5mM EDTA, 4mM Oxidised glutathione and 
0.2mM NADPH was added to the isolated mitochondria 
samples. The reaction was initiated by the addition of  
Ellman’s reagent (5,5’-dithiobis-2-nitro-benzoic acid) 
to the reaction buffer containing samples. The change 
in optical density due to oxidation of  NADPH was 
measured at 340 nm to assess the GSSG activity (Sedlak 
et al. 1968). 

In vitro Mitochondrial Protein Synthesis
The experiment in vitro mitochondrial protein synthesis 
was carried out according to the procedures earlier by 
Fernandez-Silva et al. The isolated mitochondria were 
briefly suspended in MAITE Buffer (75mM Sorbitol, 
25mM Sucrose, 10mM KCl, 10mM K2HPO4 , 0.05mM 
Tris-HCl) at pH 7.4 containing 10mM Glutamate, 10mM 
Succinate, 2.5mM Malate, 1mM ADP and 1mg/ml of  
BSA. The process of  translation was initiated by adding 
100 μg/ml emetine, 100 μg/ml cycloheximide, and 10 
μM of  the 20 L-amino acids to the medium followed by 
an incubation for 25 minutes in gentle shaker. At the end 
of  incubation process, mitochondria were pelleted by a 
high-speed centrifugation at 12000g for 10mins at 4°C. 
The pelleted mitochondria were then suspended in Lysis 
buffer (137mM NaCl, 20mM Tris-HCl, 50mM EDTA, 
1% NP40) with protease inhibitors (2mM Na2VO4, 
2mM NaF, 0.1 PMSF[Phenylmethylsulfonylfluoride]) 
followed by Sodium dodecyl sulphate polyacrylamide gel 
electrophoresis (SDS PAGE) (Garrido et al., 2008).
A 12% Resolving gel was used for SDS PAGE. After the 
process of  electrophoresis, the gels were stained using 
Coomassie Brilliant Blue Stain for a period of  4 hours. 
At the end of  4 hours, a de-staining solution was used to 
remove the excess stain for a period of  2 hours. The gels 
were then visualised using Quantity One Software (Bio-
Rad, California, USA). The band intensity was quantified 
using ImageJ software.

In vitro Mitochondrial DNA Synthesis
The experiment in vitro mitochondrial DNA synthesis 
was carried out according to the procedures earlier by 
Fernandez-Silva et al. The isolated mitochondria were 
briefly suspended in MAITE Buffer (75mM Sorbitol, 
25mM Sucrose, 10mM KCl, 10mM K2HPO4, 0.05mM 
Tris-HCl) at pH 7.4 containing 10mM Glutamate, 
10mM Succinate, 2.5mM Malate, 1mM ADP and 1mg/
ml of  BSA. The process of  translation was initiated by 
adding 50 μM of  each dNTP to the medium followed 
by an incubation for 5 hours in gentle shaker. At the 

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end of  incubation process, mitochondria were pelleted 
by a high-speed centrifugation at 12000g for 10mins at 
4°C. The pelleted mitochondria were then suspended 
in Lysis buffer (150mM NaCl, 20mM Tris-HCl, 20mM 
EDTA, 1%SDS) at pH-8.75 in the presence of  10 μM 
Proteinase K and 10 μM RNase A followed by Agarose 
gel electrophoresis (Garrido et al., 2008).

Mitochondrial DNA Isolation and DNA Quantification
Intact mitochondrial DNA was isolated by following the 
exact experimentation procedure mentioned by Martia, 
et al. Phenol, Cholorform and Isoamyl alcohol was used 
for biphase separation followed by precipitation of  
DNA by 100% ethanol. The precipitated was washed 
thrice using 75% ethanol and the dried DNA pellet was 
dissolved in elution buffer AE (5mM Tris-Hcl) at pH 8.5. 
The DNA sample were then quantified using a nanodrop 
spectrophotometer by Thermo Fisher Scientific 
(NanoDrop 2000 Spectrophotometer) (Enriquez et al., 
1996). 

Statistical Analysis
All the statistical analysis involved through the study was 
carried out using Prism version 8 (Graph Pad Software 
Inc., San Diego, CA, USA). The data analysis was carried 
out using Two-way analysis of  variance (ANOVA), 
followed by Dunnet’s post-test. The experimental results 
were expressed as mean ± SD, and a P < 0.05 was 
considered statistically significant.

RESULTS AND DISCUSSION
5‐Azacytidine preserves the mitochondrial function 
during IR in an isolated mitochondrial system
Mitochondrial dysfunction is one of  the key events to 
occur during ischemia reperfusion injury. This has an 
irreversible impact over the mitochondria even after 

various therapeutic intervention. So, an ideal therapeutic 
should overcome permanent mitochondrial dysfunction. 
Mitochondrial integrity with 5-Azacytidine pre-treatment 
was evaluated. From the evaluation we were able to 
identify that 0.5 μM 5-Azacytidine was protective 
against IR. So, an optimal dose 0.5 μM was fixed as 
standard throughout all the experimentation procedures 
involving isolated mitochondria. We further evaluated 
the electron transport chain enzyme activity to asses the 
mitochondrial function. The IR groups pre-treated with 
5-Azacytidine has improved levels of  electron transport 
chain activity. 5-Azacytidine pre-treatment in IR preserved 
mitochondrial electron transport chain complex activity 
of  complex I, II, III and IV by %, %, % & % in the 
mitochondria isolated from heart, when compared to 
mitochondrial groups subjected to IR. 5-Azacytidine 
pre-treatment in IR preserved mitochondrial electron 
transport chain complex activity of  complex I, II, III 
and IV by %, %, % & % in the mitochondria isolated 
from kidney, when compared to mitochondrial groups 
subjected to IR.

Figure 1: Dosage Determination: Optimal concentration 
of  5-Azacytidine drug was estimated to be 0.5 µm 
using NQR assay for an isolated mitochondrial system 
subjected to IR

Figure 2: Effect of  5-Azacytidine on mitochondrial electron transport chain complex activity in isolated rat heart 
mitochondria

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Effect of  5-Azacytidine on mitochondrial electron 
transport chain complex activity in isolated rat heart 
mitochondria. 
(A) Complex I activity (NQR- NADH dehydrogenase) 
was measured as μmol NADH oxidized/min/mg protein, 
(B) Complex II activity (SQR- Succinate dehydrogenase) 
was measured in nmol DCPIP reduced/min/mg protein, 

(C) Complex III activity (QCR- Cytochrome bc1) was 
measured in nmol Cytochrome C reduced/min/mg 
protein, and (D) Complex IV activity (COX- Cytochrome 
c oxidase) was measured in nmol Cytochrome C oxidized/
min/mg protein.(5-Aza,5-Azacytidine; DMSO, Dimethyl 
sulfoxide; IR, Ischemia-reperfusion.) Data were represented 
as mean ± SD. ( n=6 per group). *p < 0.05 versus IR. 

Figure 3: Effect of  5-Azacytidine on mitochondrial electron transport chain complex activity in isolated rat kidney 
mitochondria

Effect of  5-Azacytidine on mitochondrial electron 
transport chain complex activity in isolated rat kidney 
mitochondria. 
(A) Complex I activity (NQR- NADH dehydrogenase) was 
measured as μmol NADH oxidized/min/mg protein, (B) 
Complex II activity (SQR- Succinate dehydrogenase) was 
measured in nmol DCPIP reduced/min/mg protein, (C) 
Complex III activity (QCR- Cytochrome bc1) was measured 
in nmol Cytochrome C reduced/min/mg protein, and 
(D) Complex IV activity (COX- Cytochrome c oxidase) 
was measured in nmol Cytochrome C oxidized/min/mg 
protein.(5-Aza,5-Azacytidine; DMSO, Dimethyl sulfoxide; 
IR, Ischemia-reperfusion.) Data were represented as mean 
± SD. ( n=6 per group). *p < 0.05 versus IR.

Effect of  5-Azacytidine in alleviating damage induced 
to mitochondria due to oxidative stress during IRI
A robust release of  free radicals is one of  the most critical 
events involved in IR injury pathology. This is also one of  
the key contributors to mitochondrial dysfunction. The 
therapeutic effect of  5-Azcytidine in overcoming and 
altering the damages due oxidative stress induced by IR 
is vital.
Mitochondrial oxidative stress parameters were assessed 
with 5-Azacytidine pre-treatment. From the evaluation, 
the IR groups pre-treated with 5-Azacytidine has 

improved levels of  catalase and superoxide dismutase 
activity by % and % in the mitochondria when compared 
to mitochondrial groups subjected to IR, isolated from 
heart. 5-Azacytidine showed improved levels of  catalase 
and superoxide dismutase activity by % and % in the 
mitochondria when compared to mitochondrial groups 
subjected to IR, isolated from kidney.
Further experimental evaluation revealed that the IR 
groups pre-treated with 5-Azacytidine has improved 
levels of  GSH:GSSG ratio by % in the mitochondria 
when compared to mitochondrial groups subjected to 
IR, isolated from heart. 5-Azacytidine showed improved 
levels of  GSH:GSSG ratio by % in the mitochondria 
when compared to mitochondrial groups subjected to IR, 
isolated from kidney.

Assessment of  oxidative stress damage in cardiac 
mitochondria subjected to 5-Azacytidine pre-
treatment.
(A) Catalase enzyme activity, (B) SuperOxide Dismutase 
(SOD) enzyme activity, and (C) GSH:GSSG activity; all 
measured in isolated mitochondrial lysates of  rat kidneys. 
Values are represented as mean ± SD of  six individual 
animals per group. *p < 0.05 versus IR. Abbreviation:5-
Aza,5-Azacytidine; DMSO, Dimethyl sulfoxide; IR, 
Ischemia-reperfusion. 

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Assessment of  oxidative stress damage in renal 
mitochondria subjected to 5-Azacytidine pre-treatment.
(A) Catalase enzyme activity, (B) SuperOxide Dismutase 
(SOD) enzyme activity, and (C) GSH:GSSG activity; all 
measured in isolated mitochondrial lysates of  rat kidneys..
Values are represented as mean ± SD of  six individual 
animals per group. *p < 0.05 versus IR. Abbreviation:5-
Aza,5-Azacytidine; DMSO, Dimethyl sulfoxide; IR, 
Ischemia-reperfusion

5-Azacytidine pre-treatment overcomes impaired 
protein translational ability in an isolated mitochondrial 
system
In vitro protein translation ability of  mitochondria in an 
isolated mitochondria system is assessed and visualised in 
the experiment. Epigenetic modification during IRI is one 
of  the most reasons for translational inaccuracy. Hampered 
protein synthesis is one of  the main reasons behind 
irreversible recovery from IRI. The ameliorative effect 
of  5-Azacytidine in overcoming translational inaccuracy 
when subjected is explored in our study. The significant 
improvement in IR groups treated with 5-Azacytidine 
compared to IR groups can be clearly visualised through 
the electrograms of  the SDS PAGE gels.
Mitochondrial translational ability was assessed after 
5-Azacytidine pre-treatment. Before subjecting the 
mitochondria from heart to in vitro protein translation, 
the mitochondrial protein was normalised to mg/ml 
across all groups. After the process of  in vitro translation, 
the IR groups pre-treated with 5-Azacytidine showed an 
increase in mitochondrial protein by % when compared 

to mitochondrial groups subjected to IR in heart.
Before subjecting the mitochondria from kidney to 
in vitro protein translation, the mitochondrial protein 
was normalised to mg/ml across all groups. After the 
process of  in vitro translation, the IR groups pre-treated 
with 5-Azacytidine showed an increase in mitochondrial 
protein by % when compared to mitochondrial groups 
subjected to IR in kidney.

Figure 4: Assessment of  oxidative stress damage in cardiac mitochondria subjected to 5-Azacytidine pre-treatmen

Figure 5: Assessment of  oxidative stress damage in renal mitochondria subjected to 5-Azacytidine pre-treatment

Table 1: Quantitative Mitochondrial Protein Estimation 
by Bradfords Method in Heart Tissue Before In Vitro 
Protein Translation  
S.No Group Name Protein Unit
1. Normal 0.67333333 mg/ml
2. Normal + 5-Aza 0.67333333 mg/ml
3. Normal + DMSO 0.67333333 mg/ml
4. IR 0.67333333 mg/ml
5. IR + 5–Aza 0.67333333 mg/ml
6. IR+DMSO 0.67333333 mg/ml

Table 2: Quantitative Mitochondrial Protein Estimation 
by Bradfords Method in Heart Tissue after in Vitro 
Protein Translation  
S.No Group Name Protein Unit
1. Normal 0.909 mg/ml
2. Normal + 5-Aza 0.97 mg/ml
3. Normal + DMSO 0.612 mg/ml
4. IR 0.42 mg/ml
5. IR + 5–Aza 0.873 mg/ml
6. IR+DMSO 0.359 mg/ml

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In-vitro cardiac mitochondrial translated proteins 
analysis using SDS-PAGE gel electrophoresis. 
(A) SDS PAGE gel electrogram, (B) Protein estimation, 
(C) Total Band Intensity, and (D) Representations for gel 

electrogram lanes. Values are represented as mean ± SD 
of  six individual animals per group. *p < 0.05 versus IR. 
(5-Aza,5-Azacytidine; DMSO, Dimethyl sulfoxide; IR, 
Ischemia-reperfusion.) 

Figure 6: In-vitro cardiac mitochondrial translated proteins analysis using SDS-PAGE gel electrophoresis

Table 3: Quantitative mitochondrial protein estimation 
by Bradfords method in kidney tissue before in vitro 
protein translation
S.No Group Name Protein Unit
1. Normal 0.94833333 mg/ml
2. Normal + 5-Aza 0.94833333 mg/ml
3. Normal + DMSO 0.94833333 mg/ml
4. IR 0.94833333 mg/ml
5. IR + 5–Aza 0.94833333 mg/ml
6. IR+DMSO 0.94833333 mg/ml

Table 4: Quantitative mitochondrial protein estimation 
by Bradfords method in kidney tissue after in vitro 
protein translation  
S.No Group Name Protein Unit
1. Normal 1.1505 mg/ml
2. Normal + 5-Aza 1.2545 mg/ml
3. Normal + DMSO 0.9008 mg/ml
4. IR 0.69 mg/ml
5. IR + 5–Aza 1.1896 mg/ml
6. IR+DMSO 0.67087 mg/ml

In-vitro renal mitochondrial translated proteins 
analysis using SDS-PAGE gel electrophoresis. 
(A) SDS PAGE gel electrogram, (B) Protein estimation, 
(C) Total Band Intensity, and (D) Representations for gel 

electrogram lanes. Values are represented as mean ± SD 
of  six individual animals per group. *p < 0.05 versus IR. 
(5-Aza,5-Azacytidine; DMSO, Dimethyl sulfoxide; IR, 
Ischemia-reperfusion.). 

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Figure 7: In-vitro renal mitochondrial translated proteins analysis using SDS-PAGE gel electrophoresis

5-Azacytidine enhances DNA replication machinery 
in an isolated mitochondrial system
In vitro DNA replication ability of  mitochondria in an 
isolated mitochondria system is assessed and visualised in 
the experiment. DNA hypermethylation during IRI is one 
of  the most reasons for transcriptional impairment and 
translational inaccuracy. Upregulation of  DNMT1 during 
IR is one of  the major reasons behind extensive and far-
fetched damage. The therapeutic effect of  5-Azacytidine 
in overcoming impaired DNA replication machinery 
when subjected is explored in our study. The significant 
enhancement in mitochondrial DNA copy number in 
IR groups treated with 5-Azacytidine compared to IR 
groups can be clearly visualised through the electrograms 
of  the Agarose gels.
Mitochondrial DNA replication ability was assessed 
after 5-Azacytidine pre-treatment. Before subjecting the 
mitochondria from heart to in vitro DNA replication, the 
mitochondrial protein was normalised to mg/ml across 
all groups. After the process of  in vitro DNA replication, 
the IR groups pre-treated with 5-Azacytidine showed an 

increase in mitochondrial DNA by % when compared to 
mitochondrial groups subjected to IR in heart.
Before subjecting the mitochondria from kidney to in 
vitro DNA replication, the mitochondrial protein was 
normalised to mg/ml across all groups. After the process 
of  in vitro DNA replication, the IR groups pre-treated 
with 5-Azacytidine showed an increase in mitochondrial 
DNA by % when compared to mitochondrial groups 
subjected to IR in kidney.

Table 5: Quantitative mitochondrial protein estimation 
by Bradfords method in heart tissue before in vitro 
DNA replication
S.No Group Name Protein Unit
1. Normal 0.763 mg/ml
2. Normal + 5-Aza 0.763 mg/ml
3. Normal + DMSO 0.763 mg/ml
4. IR 0.763 mg/ml
5. IR + 5–Aza 0.763 mg/ml
6. IR+DMSO 0.763 mg/ml

Table 6: Mitochondrial DNA quantification in Heart using NanoDrop Spectrophotometer after in vitro 
DNA replication
S.No Group Name Nucleic acid Unit 260/280 260/230 Sample Type
1. Normal 578.25 ng/µl 1.81 2.12 DNA
2. Normal + 5-Aza 583.14 ng/µl 1.82 2.14 DNA
3. Normal + DMSO 462.69 ng/µl 1.9 2.11 DNA
4. IR 258.18 ng/µl 1.9 2.12 DNA
5. IR + 5–Aza 565.82 ng/µl 1.89 2.17 DNA
6. IR+DMSO 264.28 ng/µl 1.83 2.0 DNA

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Figure 8: Agarose gel electrophoresis analysis of  In-vitro cardiac mitochondrial DNA replication

Agarose gel electrophoresis analysis of  In-vitro 
cardiac mitochondrial DNA replication. 
(A) Agarose gel electrogram, (B) Total Band Intensity, 
and (C) Representations for gel electrogram lanes. Data 
were represented as mean ± SD. ( n=6 per group). *p 
< 0.05 versus IR. Abbreviation:5-Aza,5-Azacytidine; 
DMSO, Dimethyl sulfoxide; IR, Ischemia-reperfusion.

Agarose gel electrophoresis analysis of  In-vitro renal 
mitochondrial DNA replication. 
(A) Agarose gel electrogram, (B) Total Band Intensity, 

Table 7: Quantitative mitochondrial protein estimation 
by Bradfords method in kidney tissue before in vitro 
DNA replication
S.No Group Name Protein Unit
1. Normal 0.984 mg/ml
2. Normal + 5-Aza 0.984 mg/ml
3. Normal + DMSO 0.984 mg/ml
4. IR 0.984 mg/ml
5. IR + 5–Aza 0.984 mg/ml
6. IR+DMSO 0.984 mg/ml

Table 8: Mitochondrial DNA quantification in Kidney using NanoDrop Spectrophotometer after in vitro 
DNA replication 
S.No Group Name Nucleic acid Unit 260/280 260/230 Sample Type
1. Normal 578.25 ng/µl 1.89 2.0 DNA
2. Normal + 5-Aza 583.14 ng/µl 1.82 2.1 DNA
3. Normal + DMSO 462.69 ng/µl 1.91 2.1 DNA
4. IR 258.18 ng/µl 1.8 2.12 DNA
5. IR + 5–Aza 565.82 ng/µl 1.83 2.17 DNA
6. IR+DMSO 264.28 ng/µl 1.9 2.0 DNA

Figure 9: Agarose gel electrophoresis analysis of  In-vitro renal mitochondrial DNA replication

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and (C) Representations for gel electrogram lanes. Data 
were represented as mean ± SD. ( n=6 per group). *p 
< 0.05 versus IR. Abbreviation:5-Aza,5-Azacytidine; 
DMSO, Dimethyl sulfoxide; IR, Ischemia-reperfusion

DISCUSSION
Ischemia reperfusion injury is one of  the most common 
and unavoidable forms of  injury to occur during a 
revascularisation procedure or because of  therapeutic 
interventions to an atherosclerotic plaque or an 
ischemic stroke (Piper et al., 1998). The injury might 
seem inevitable because of  huge number of  variable 
complications arising after interventional therapy. Till 
date, a lot of  different therapeutics have been tested, 
repurposed and trailed for the treatment of  IRI but a lot 
of  them fails in one or many concern. In previous study 
by Rahavi, et al., has reported the ameliorative effect of  
5-Azacytidine against IRI preliminarily due to its inherent 
ability to combat the DNMT1 by inhibiting this action 
(A.A.Mangoni et al. 2018). The role of  5-Azacytinde in 
combating cancer is also well explored. But, the role of  
5-Azacytidine in alleviating mitochondrial damage due 
to IRI is not explored. As mitochondria is one of  the 
key players in regulation and resuscitating cell survival 
during IRI, it is of  immense importance to have a 
deep understanding of  the mechanism through which 
5-Azacytidine helps alleviating IRI. The mechanistic 
action on drug on mitochondria is not well documented 
and is unexplored. So, having a better understanding 
of  the way through which mitochondria mediates IR 
protection would help enable a complete overlay (D.Jain 
et al. 2017). Even though there are multiple drugs to 
treat IRI, such multifaceted view on the action of  drug 
is absent thereby most drugs fail to transition even into 
clinical trials. Therefore, phrenological manipulative 
ability without enough substantial evidence is not fruitful 
yield (I.Andreadou et al. 2020).
Many drugs like methotrexate, hydroxychloroquine which 
are used for the treatment of  various diseases like cancer, 
malaria have been repurposed to treat cardiovascular 
diseases as well (A.Daiver et al. 2021). 5-Azacytidine 
mediated epigenetic reprogramming is used extensively in 
cancer research. The mechanism of  action and epigenetic 
modulation mediated by 5-Azacytidine is clearly 
established and well documented (E.Hervout et al. 2013). 
Also, many studies have reported the cardioprotective, 
nephroprotective and vasculo-protective nature of  the 
compound. But the effect of  this compound on cardiac 
IRI and renal IRI are not well established (S.Sou et al, 
2016). Therefore, a proper evidence-based study would 
help us repurpose the drug effectively to manage many 
cardiovascular and renal complications in a clinical 
scenario as a potent pharmacological agent (L.Badimon 
et al. 2019). From our study, 5-Azacytidne has been to 
shown to provide an ameliorative effect against IRI in 
an Isolated Mitochondrial system thereby, providing a 
promising role as a pharmacological intervention.
The organs heart and kidney have been shown to exhibit 

an elevated levels of  mitochondrial dysfunction because 
of  the free radical stack and extensive calcium overload 
(H.Ma et al.  2011). This immediately results in the change 
in functionality of  the mitochondria leading to loss of  
mitochondrial bioenergetics (K.Yang et al. 2017). Based 
on these evidences, we identified a significantly decreased 
mitochondrial electron transport chain activity due to 
deterioration in function of  mitochondrial complexes I, 
II, III, IV. There was also decreased tendency to scavenge 
the free radical resulting increased oxidative stress. Our 
studies have shown that pre-treatment with 5-Azacytidine 
resulted in improved mitochondrial electron transport 
chain activity. The results have also indicated that the 
scavenging effect of  5-Azacytidine but it was not that 
considerable when compared to its other therapeutic 
effect. However, the compound enhanced the survival key 
for mitochondria when subjected to IRI by considerably 
enhancing the mitochondrial functionality and improved 
resistance to oxidative stress induced mitochondrial 
damage. 
Translational inaccuracy and reduced mitochondrial copy 
number are another key deteriorative feature of  IRI. So, 
a pharmacological agent that has the ability to modify the 
epigenetics of  a cellular system is vital (P.E.Nikolaou et al. 
2019). Often times we fail to realise that mitochondria is a 
system of  its own and it has a trivial role to play in cellular 
epigenetics as well (B.A.Hemmings et al. 2015). When 
subjected to IRI, the levels of  mitochondrial protein 
translation were significantly reduced and it also resulted 
in decreased mitochondrial copy number. However, once 
the groups were pre-treated with 5-Azacytidine and when 
subjected to IRI hey showed improved to resistance to 
translational inaccuracy by enhanced protein synthesis. 
The epigenetic modification that 5-Azacytidine imparts 
on the DNA replication machinery also resulted improved 
mitochondrial copy number even when to IRI. Based on 
the results obtained from these independent experiments 
throughout the long run we could demonstrate the 
therapeutic effect of  5-Azacytidine to Attenuate the 
Ramifying Repercussions of  Ischemia-Reperfusion 
Injury on Mitochondrial Molecular Machinery.

CONCLUSION
Ischemia reperfusion injury mediated mitochondrial 
damage is inevitable and without proper pharmacological 
intervention it can be irreversible. This study shows 
the therapeutic effect of  5-Azacytidine in attenuating 
IRI mediated mitochondrial dysfunction. 5-Azacytidine 
pre-treated groups showed improved mitochondrial 
translational accuracy and increased mitochondrial copy 
number. These evidences indicated the rooted ability of  
5-Azacytidine to act on mitochondria to ameliorate IRI 
induced mitochondrial damage and thereby contributing 
towards cellular homeostasis.

FUTURE PROSPECTS
From our findings pre-treatment with 5- Azacytidine 
has enhanced the ability of  protein translation, increased 

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mitochondrial copy number and have restored the damage 
caused to mitochondrial function when subjected to 
ischemia reperfusion injury in an isolated mitochondrial 
system. An isolated mitochondrial system acts as great 
tool in understanding the specificity, targeted action 
and therapeutic potential of  5-Azacytidine. However, 
this system doesn’t exactly replicate environment and 
interactions of  an organ system, whereas it replicates the 
environment of  the cell to sustain a healthy mitochondria 
population. So, extensive animal studies consisting 
animals pre-treated with 5-Azacytidine are required 
to further evaluate the effect of  5-Azacytidine on 
mitochondria wherein a lot of  other factors can interplay 
to contribute, impair or enhance the therapeutic effect. 
Also, the effect of  5-Azacytinde on mitochondrial gene 
expression associated with ischemia reperfusion and need 
to be studied. Furthermore, studies can also be extended 
to analyse the efficacy of  5-Azacytidine in attenuating 
any repercussions on distant organ due to ischemia 
reperfusion injury.
Acknowledgements
Funding: We would like to acknowledge The Department 
of  Science and Technology (DST), Government of  
India, New Delhi, for funding the instrumentation 
facility required for research through grand‐in‐aid 
(EMR/2017/000669). 

REFERENCES
Andreadou, I., Schulz, R., Papapetropoulos, A., Turan, 

B., Ytrehus, K., Ferdinandy, P., Daiber, A., & Di Lisa, 
F. (2020). The role of  mitochondrial reactive oxygen 
species, NO and H2 S in ischaemia/reperfusion 
injury and cardioprotection. Journal of  cellular and 
molecular medicine, 24(12), 6510–6522. https://doi.
org/10.1111/jcmm.15279

Boovarahan, S., & Kurian, G. (2021). Preconditioning 
the rat heart with 5‐azacytidine attenuates myocardial 
ischemia/reperfusion injury via PI3K/GSK3β and 
mitochondrial K ATP signaling axis. Journal Of  
Biochemical And Molecular Toxicology, 35(12). https://
doi.org/10.1002/jbt.22911

Braunwald, E., & Kloner, R. A. (1985). Myocardial 
reperfusion: a double-edged sword?. The Journal of  
clinical investigation, 76(5), 1713–1719. https://doi.
org/10.1172/JCI112160

Badimon, L., Casaní, L., Camino-Lopez, S., Juan-Babot, 
O., & Borrell-Pages, M. (2019). GSK3β inhibition and 
canonical Wnt signaling in mice hearts after myocardial 
ischemic damage. PloS one, 14(6), e0218098. https://
doi.org/10.1371/journal.pone.0218098

Barrientos, A., Fontanesi, F., & Díaz, F. (2009). Evaluation 
of  the mitochondrial respiratory chain and oxidative 
phosphorylation system using polarography 
and spectrophotometric enzyme assays. Current 
protocols in human genetics, 63(1), 19-3. https://doi.
org/10.1002/0471142905.hg1903s63

Bechtel, W., McGoohan, S., Zeisberg, E. M., Müller, 
G. A., Kalbacher, H., Salant, D. J., Müller, C. A., 

Kalluri, R., & Zeisberg, M. (2010). Methylation 
determines fibroblast activation and fibrogenesis in 
the kidney. Nature medicine, 16(5), 544–550. https://
doi.org/10.1038/nm.2135

Christman, J. (2002). 5-Azacytidine and 5-aza-2′-
deoxycytidine as inhibitors of  DNA methylation: 
mechanistic studies and their implications for cancer 
therapy. Oncogene, 21(35), 5483-5495. https://doi.
org/10.1038/sj.onc.1205699 

Caccioppo, A., Franchin, L., Grosso, A., Angelini, F., 
D’Ascenzo, F., & Brizzi, M. F. (2019). Ischemia 
Reperfusion Injury: Mechanisms of  Damage/
Protection and Novel Strategies for Cardiac Recovery/
Regeneration. International journal of  molecular sciences, 
20(20), 5024. https://doi.org/10.3390/ijms20205024

Consolini, A. E., Ragone, M. I., Bonazzola, P., & 
Colareda, G. A. (2017). Mitochondrial Bioenergetics 
During Ischemia and Reperfusion. Advances in 
experimental medicine and biology, 982, 141–167. https://
doi.org/10.1007/978-3-319-55330-6_8

Czibere, A., Bruns, I., Kröger, N., Platzbecker, U., Lind, 
J., Zohren, F., Fenk, R., Germing, U., Schröder, T., 
Gräf, T., Haas, R., & Kobbe, G. (2010). 5-Azacytidine 
for the treatment of  patients with acute myeloid 
leukemia or myelodysplastic syndrome who 
relapse after allo-SCT: a retrospective analysis. Bone 
marrow transplantation, 45(5), 872–876. https://doi.
org/10.1038/bmt.2009.266

Cao, D., Li, D., Huang, Y., Ma, Y., Zhang, B., Zhao, C., 
Deng, S., Luo, M., Yin, T., Wei, Y. Q., & Wang, W. (2017). 
5-Azacytidine promotes invadopodia formation and 
tumor metastasis through the upregulation of  PI3K 
in ovarian cancer cells. Oncotarget, 8(36), 60173–60187. 
https://doi.org/10.18632/oncotarget.18580 

Cao, L., Zhu, T., Lang, X., Jia, S., Yang, Y., Zhu, C., Wang, 
Y., Feng, S., Wang, C., Zhang, P., Chen, J., & Jiang, 
H. (2020). Inhibiting DNA Methylation Improves 
Survival in Severe Sepsis by Regulating NF-κB 
Pathway. Frontiers in immunology, 11, 1360. https://doi.
org/10.3389/fimmu.2020.01360

Daiber, A., Andreadou, I., Oelze, M., Davidson, S. M., & 
Hausenloy, D. J. (2021). Discovery of  new therapeutic 
redox targets for cardioprotection against ischemia/
reperfusion injury and heart failure. Free radical biology 
& medicine, 163, 325–343. https://doi.org/10.1016/j.
freeradbiomed.2020.12.026

Frank, A., Bonney, M., Bonney, S., Weitzel, L., Koeppen, 
M., & Eckle, T. (2012). Myocardial ischemia reperfusion 
injury: from basic science to clinical bedside. Seminars 
in cardiothoracic and vascular anesthesia, 16(3), 123–132. 
https://doi.org/10.1177/1089253211436350.

Fenaux, P., Mufti, G. J., Hellstrom-Lindberg, E., Santini, V., 
Finelli, C., Giagounidis, A., Schoch, R., Gattermann, 
N., Sanz, G., List, A., Gore, S. D., Seymour, J. F., 
Bennett, J. M., Byrd, J., Backstrom, J., Zimmerman, 
L., McKenzie, D., Beach, C., Silverman, L. R., & 
International Vidaza High-Risk MDS Survival Study 
Group (2009). Efficacy of  azacitidine compared with 

https://journals.e-palli.com/home/index.php/ajcp


Pa
ge

 
19

https://journals.e-palli.com/home/index.php/ajcp

Am. J. Chem. Pharm. 2(1) 8-20, 2023

that of  conventional care regimens in the treatment of  
higher-risk myelodysplastic syndromes: a randomised, 
open-label, phase III study. The Lancet. Oncology, 
10(3), 223–232. https://doi.org/10.1016/S1470-
2045(09)70003-8

Ferrera, R., Benhabbouche, S., Bopassa, J. C., Li, B., & 
Ovize, M. (2009). One hour reperfusion is enough to 
assess function and infarct size with TTC staining in 
Langendorff  rat model. Cardiovascular drugs and therapy, 
23(4), 327–331. https://doi.org/10.1007/s10557-
009-6176-5

Gerczuk, P. Z., & Kloner, R. A. (2012). An update on 
cardioprotection: a review of  the latest adjunctive 
therapies to limit myocardial infarction size in clinical 
trials. Journal of  the American College of  Cardiology, 59(11), 
969–978. https://doi.org/10.1016/j.jacc.2011.07.054

Gilbert, K., Godbout, R., & Rousseau, G. (2016). 
Caspase-3 Activity in the Rat Amygdala Measured 
by Spectrofluorometry After Myocardial Infarction. 
Journal of  visualized experiments, JoVE, 107, e53207. 
https://doi.org/10.3791/53207

Hemmings, B. A., & Restuccia, D. F. (2015). The PI3K-
PKB/Akt pathway. Cold Spring Harbor perspectives 
in biology, 7(4), a026609. https://doi.org/10.1101/
cshperspect.a026609

Hausenloy, D. J., & Yellon, D. M. (2013). Myocardial 
ischemia-reperfusion injury: a neglected therapeutic 
target. The Journal of  clinical investigation, 123(1), 92–
100. https://doi.org/10.1172/JCI62874

Hausenloy, D. J., & Yellon, D. M. (2013). Myocardial 
ischemia-reperfusion injury: a neglected therapeutic 
target. The Journal of  clinical investigation, 123(1), 92–
100. https://doi.org/10.1172/JCI62874. 

Hervouet, E., Cheray, M., Vallette, F. M., & Cartron, P. 
F. (2013). DNA methylation and apoptosis resistance 
in cancer cells. Cells, 2(3), 545–573. https://doi.
org/10.3390/cells2030545

Jain, D., Ahmad, T., Cairo, M., & Aronow, W. (2017). 
Cardiotoxicity of  cancer chemotherapy: identification, 
prevention and treatment. Annals of  translational 
medicine, 5(17), 348. https://doi.org/10.21037/
atm.2017.06.35

Kurian, G. A., Rajagopal, R., Vedantham, S., & Rajesh, M. 
(2016). The Role of  Oxidative Stress in Myocardial 
Ischemia and Reperfusion Injury and Remodeling: 
Revisited. Oxidative medicine and cellular longevity, 2016, 
1656450. https://doi.org/10.1155/2016/1656450

Kalogeris, T., Baines, C. P., Krenz, M., & Korthuis, R. J. 
(2012). Cell biology of  ischemia/reperfusion injury. 
International review of  cell and molecular biology, 298, 229–
317. https://doi.org/10.1016/B978-0-12-394309-
5.00006-7

Kim, Y. S., Kang, W. S., Kwon, J. S., Hong, M. H., 
Jeong, H. Y., Jeong, H. C., Jeong, M. H., & Ahn, 
Y. (2014). Protective role of  5-azacytidine on 
myocardial infarction is associated with modulation 
of  macrophage phenotype and inhibition of  fibrosis. 
Journal of  cellular and molecular medicine, 18(6), 1018–

1027. https://doi.org/10.1111/jcmm.12248
Kumar, P., Nagarajan, A., & Uchil, P. (2018). Analysis of  

Cell Viability by the MTT Assay. Cold Spring Harbor 
Protocols, 2018(6), pdb.prot095505. https://doi.
org/10.1101/pdb.prot095505

Kasibhatla, S., Amarante-Mendes, G. P., Finucane, 
D., Brunner, T., Bossy-Wetzel, E., & Green, D. R. 
(2006). Acridine Orange/Ethidium Bromide (AO/
EB) Staining to Detect Apoptosis. CSH protocols, 
2006(3), pdb.prot4493. https://doi.org/10.1101/
pdb.prot4493

Moore, L. D., Le, T., & Fan, G. (2012). DNA Methylation 
and Its Basic Function. Neuropsychopharmacology, 38(1), 
23–38. https://doi.org/10.1038/npp.2012.112 Yang, 
M., Linn, B. S., Zhang, Y., & Ren, J. (2019). Mitophagy 
and mitochondrial integrity in cardiac ischemia-
reperfusion injury. Biochimica et biophysica acta. 
Molecular basis of  disease, 1865(9), 2293–2302. https://
doi.org/10.1016/j.bbadis.2019.05.007

Mangoni, A. A., Tommasi, S., Zinellu, A., Sotgia, S., 
Carru, C., Piga, M., & Erre, G. L. (2018). Repurposing 
existing drugs for cardiovascular risk management: a 
focus on methotrexate. Drugs in context, 7, 212557. 
https://doi.org/10.7573/dic.212557

Ma, H., Guo, R., Yu, L., Zhang, Y., & Ren, J. (2011). 
Aldehyde dehydrogenase 2 (ALDH2) rescues 
myocardial ischaemia/reperfusion injury: role of  
autophagy paradox and toxic aldehyde. European heart 
journal, 32(8), 1025–1038. https://doi.org/10.1093/
eurheartj/ehq253

Nikolaou, P. E., Boengler, K., Efentakis, P., 
Vouvogiannopoulou, K., Zoga, A., Gaboriaud-
Kolar, N., Myrianthopoulos, V., Alexakos, P., 
Kostomitsopoulos, N., Rerras, I., Tsantili-Kakoulidou, 
A., Skaltsounis, A. L., Papapetropoulos, A., 
Iliodromitis, E. K., Schulz, R., & Andreadou, I. (2019). 
Investigating and re-evaluating the role of  glycogen 
synthase kinase 3 beta kinase as a molecular target 
for cardioprotection by using novel pharmacological 
inhibitors. Cardiovascular research, 115(7), 1228–1243. 
https://doi.org/10.1093/cvr/cvz061

Neri, M., Riezzo, I., Pascale, N., Pomara, C., & 
Turillazzi, E. (2017). Ischemia/Reperfusion Injury 
following Acute Myocardial Infarction: A Critical 
Issue for Clinicians and Forensic Pathologists. 
Mediators of  inflammation, 2017, 7018393. https://doi.
org/10.1155/2017/7018393

Okamoto, H., Kamitsuji, Y., Komori, Y., Sasaki, N., 
Tsutsumi, Y., Miyashita, A., Tsukamoto, T., Mizutani, 
S., Shimura, Y., Kobayashi, T., Uoshima, N., & Kuroda, 
J. (2021). Durable Remission of  Chemotherapy-
Refractory Myeloid Sarcoma by Azacitidine. The 
Tohoku journal of  experimental medicine, 254(2), 101–105. 
https://doi.org/10.1620/tjem.254.101

Oran, B., de Lima, M., Garcia-Manero, G., Thall, P. F., 
Lin, R., Popat, U., Alousi, A. M., Hosing, C., Giralt, 
S., Rondon, G., Woodworth, G., & Champlin, R. E. 
(2020). A phase 3 randomized study of  5-azacitidine 

https://journals.e-palli.com/home/index.php/ajcp


Pa
ge

 
20

https://journals.e-palli.com/home/index.php/ajcp

Am. J. Chem. Pharm. 2(1) 8-20, 2023

maintenance vs observation after transplant in 
high-risk AML and MDS patients. Blood advances, 
4(21), 5580–5588. https://doi.org/10.1182/
bloodadvances.2020002544

Palmer, J. W., Tandler, B., & Hoppel, C. L. (1977). Biochemical 
properties of  subsarcolemmal and interfibrillar 
mitochondria isolated from rat cardiac muscle. The 
Journal of  biological chemistry, 252(23), 8731–8739.

Qian, Q., Qian, H., Zhang, X., Zhu, W., Yan, Y., Ye, S., 
Peng, X., Li, W., Xu, Z., Sun, L., & Xu, W. (2012). 
5-Azacytidine induces cardiac differentiation of  
human umbilical cord-derived mesenchymal stem 
cells by activating extracellular regulated kinase. 
Stem cells and development, 21(1), 67–75. https://doi.
org/10.1089/scd.2010.0519

Ravindran, S., Boovarahan, S. R., Shanmugam, K., 
Vedarathinam, R. C., & Kurian, G. A. (2017). Sodium 
Thiosulfate Preconditioning Ameliorates Ischemia/
Reperfusion Injury in Rat Hearts Via Reduction of  
Oxidative Stress and Apoptosis. Cardiovascular drugs and 
therapy, 31(5-6), 511–524. https://doi.org/10.1007/
s10557-017-6751-0

Suhaeri, M., Subbiah, R., Van, S. Y., Du, P., Kim, I. G., 
Lee, K., & Park, K. (2015). Cardiomyoblast (h9c2) 
differentiation on tunable extracellular matrix 
microenvironment. Tissue engineering. Part A, 21(11-
12), 1940–1951. https://doi.org/10.1089/ten.
TEA.2014.0591

Sánchez-Hernández, C. D., Torres-Alarcón, L. A., 
González-Cortés, A., & Peón, A. N. (2020). Ischemia/
Reperfusion Injury: Pathophysiology, Current Clinical 
Management, and Potential Preventive Approaches. 
Mediators of  inflammation, 2020, 8405370. https://doi.
org/10.1155/2020/8405370

Sedlak, J., & Lindsay, R. H. (1968). Estimation of  total, 
protein-bound, and nonprotein sulfhydryl groups in 
tissue with Ellman’s reagent. Analytical biochemistry, 
25(1), 192–205. https://doi.org/10.1016/0003-
2697(68)90092-4

Tabaei, S., & Tabaee, S. (2019). DNA methylation 
abnormalities in atherosclerosis. Artificial Cells, 

Nanomedicine, And Biotechnology, 47(1), 2031-2041. 
https://doi.org/10.1080/21691401.2019.1617724

Thygesen, K., Alpert, J. S., Jaffe, A. S., Simoons, M. L., 
Chaitman, B. R., White, H. D., Joint ESC/ACCF/
AHA/WHF Task Force for Universal Definition of  
Myocardial Infarction, Authors/Task Force Members 
Chairpersons, Thygesen, K., Alpert, J. S., White, H. 
D., Biomarker Subcommittee, Jaffe, A. S., Katus, H. 
A., Apple, F. S., Lindahl, B., Morrow, D. A., ECG 
Subcommittee, Chaitman, B. R., Clemmensen, P. M., 
… Wagner, D. R. (2012). Third universal definition 
of  myocardial infarction. Journal of  the American 
College of  Cardiology, 60(16), 1581–1598. https://doi.
org/10.1016/j.jacc.2012.08.001

Yoshimoto, G., Mori, Y., Kato, K., Odawara, J., Kuriyama, 
T., Ueno, T., Obara, T., Yurino, A., Yoshida, S., 
Ogawa, R., Ohno, Y., Iwasaki, H., Eto, T., Akashi, K., 
& Miyamoto, T. (2021). Azacitidine for the treatment 
of  patients with relapsed acute myeloid leukemia 
after allogeneic stem cell transplantation. Leukemia & 
lymphoma, 62(12), 2939–2948. https://doi.org/10.108
0/10428194.2021.1941937

Yang, C., Yi, J., Gong, X., Ge, P., Dai, J., Lin, L., Xing, 
Y., & Zhang, L. (2017). Anti-oxidative and anti-
inflammatory benefits of  the ribonucleoside analogue 
5-azacitidine in mice with acetaminophen-induced 
toxic hepatitis. International immunopharmacology, 48, 
91–95. https://doi.org/10.1016/j.intimp.2017.05.001

Yu, S. M., & Kim, S. J. (2016). 5-Azacytidine regulates 
matrix metalloproteinase-9 expression, and the 
migration and invasion of  human fibrosarcoma 
HT1080 cells via PI3-kinase and ERK1/2 pathways. 
International journal of  oncology, 49(3), 1241–1247. 
https://doi.org/10.3892/ijo.2016.3612

Yang, K., Chen, Z., Gao, J., Shi, W., Li, L., Jiang, S., Hu, 
H., Liu, Z., Xu, D., & Wu, L. (2017). The Key Roles 
of  GSK-3β in Regulating Mitochondrial Activity. 
Cellular physiology and biochemistry. International 
journal of  experimental cellular physiology, biochemistry, 
and pharmacology, 44(4), 1445–1459. https://doi.
org/10.1159/000485580

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