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European Journal of Applied Sciences – Vol. 12, No. 1
Publication Date: February 25, 2024
DOI:10.14738/aivp.121.14674
Omolara, O. O., Tosin, O. B., Faith, A., & Ayegbusi, E. O. (2024). Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of
Melatonin Against Melphalan-Induced Reproductive Toxicity. European Journal of Applied Sciences, Vol - 12(1). 487-501.
Services for Science and Education – United Kingdom
Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of
Melatonin Against Melphalan-Induced Reproductive Toxicity
Ojo Olajumoke Omolara
Department of Biochemistry, Ekiti State University Ado-Ekiti, Nigeria
Ogunbiyi Babafemi. Tosin
Department of Biochemistry, Benjamin S. Carson (Snr) School of Medicine,
Babcock University Illisan
Adeosun Faith
Department of Biochemistry, Ekiti State University Ado-Ekiti, Nigeria
Ayegbusi Emmanuel. O.
Department of Biochemistry, Ekiti State University Ado-Ekiti, Nigeria
ABSTRACT
Oxidative stress occurs when there is an imbalance between the production and
accumulation of reactive oxygen species (ROS) in cells and tissues and the ability of
a biological system to detoxify these reactive products. The effect of Nrf2/HO-1
signaling improvement by melatonin on melphalan induced testicular dysfunction
in mice was investigated. Thirty-six male Swiss albino mice were used for this study.
Male mice were divided into six groups of six animals each and treated by
intraperitoneal injection as follows; Group I: Vehicle treated control; Group II:
Melatonin alone; Group III: 1mg/kg/bwt MEP; Group IV: 3mg/kg/bwt MEP; Group
V: 5mg/kg/bwt MEP; Group VI (5mg/kg/bwt MEP + 10mg/kg/bwt Melatonin) for
twenty-eight days. Hormonal assessments, expression of Nrf2 and HO-1 genes and
evaluation of other sperm parameters are the various investigation depicted in this
study. The result shows an increase in DNA damage and Teratozoospermia index
(TZI). Raised level of malondialdehyde (MDA) and decreased level of Total
antioxidant capacity (TAC) were measured. The result demonstrated ROS
accumulation in the melphalan-treated groups, the result of this study also showed
a decrease in testosterone levels, LH and FSH levels, decreased sperm
concentrations and increased ROS upon administration of melphalan.
Administration of melatonin to mice in Group VI showed a significant increase in
the level of these hormones and antioxidant when compared to mice treated with
melphalan alone. Therefore, it can be hypothesized that melphalan effectuated
oxidative stress in the testes by disrupting Nrf2/HO-1 signaling pathway. This study
found that melatonin is a potent regulator of Nrf2 and HO-1 in melphalan induced
testicular dysfunction.
Keywords: Melphalan, Oxidative stress, Melatonin, Teratozoospermia index, Nrf2/HO-1
Signaling.
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INTRODUCTION
Chemotherapy leads to increase production of reactive oxygen species (ROS), which cause
oxidative stress resulting in the impairment of testis function. High doses of these class of drugs
persistently decline the function of normal Leydig cells, reducing androgen production, and
irreversibly impair fertility. (Dent et al., 2000). The adverse effect of Melphalan (MEP) was
reported, the results showed significant decrease in sperm parameters, antioxidant enzymes
levels, with a concomitant increase level of reactive oxidative species. The results confirmed
that MEP- treatment leads to testicular damage due to the formation of oxidative stress. (Ojo,
2020). However, both in vitro and in vivo studies have shown that melatonin or n-acetyl-5-
methoxytryptamine is a potent scavenger of the highly toxic hydroxyl radical and other oxygen
centered radicals, suggesting that it has actions not mediated by receptors. In one study,
melatonin seemed to be more effective than other known antioxidants (e.g., mannitol,
glutathione, and vitamin E) in protecting against oxidative damage. Therefore, melatonin may
provide protection against diseases that cause degenerative or proliferative changes by
shielding macromolecules, particularly DNA, from such injuries. (Reiter, 1993). Melatonin was
also reported to protect male reproductive health, which readily crosses the blood-testis
barrier and has a very low toxicity. Studies have investigated the use of melatonin to relieve the
side effects of chemotherapy drugs and environmental toxins during spermatogenesis.
However, few systematic studies have investigated whether melatonin exerts a protective role
in the psychological stress-induced impairment of spermatogenesis as well as the mechanisms
by which melatonin mitigates the damage in testes (Dong, et al. 2020). Nrf2, a transcription
factor family which contains six highly conserved domains which is a key factor in the
regulation of the oxidative stress. Under normal physiological conditions, Nrf2 binds to Kelch- like epichlorohydrin-related proteins (Keap1) to form complexes. Several reports have shown
significant upregulation of HO-1 and Nrf2 along with raised activities of GSH-Px and GSH with
simultaneous suppression of ROS content in the cells. This study is therefore designed to
investigate the mechanism involved in melatonin suppression of oxidative stress following
Melphalan treatment by exploring the role of Nrf2/HO-1.
MATERIALS AND METHODS
Animals Grouping and Treatments
The protocol was approved by the Institutional Animal Ethics Committee of Ekiti State
University, Ado Ekiti. This study was carried out in strict accordance with the recommendations
in the Guide for the Care and Use of Laboratory Animals of the Institute. Male and female Swiss
albino mice weighing about 25 g were obtained from the Laboratory Animal Division of the
University. The animals were maintained under standard conditions of humidity (50 ± 5%),
temperature (25 ± 2oC) and dark and light cycles (12hr each) with free access to food and
water. Male mice were divided into six groups of six animals each and treated intraperitoneally
as follows; Group I: Vehicle-treated control; Group II: Melatonin alone, Group III: 1mg/kg/bwt
MEP; Group IV: 3mg/kg/bwt MEP; Group V: 5mg/kg/bwt MEP; Group VI (5mg/kg/bwt MEP +
10mg/kg/bwt Melatonin) for 7, 14 and 28 days via intraperitoneal injection.
Testicular Cells Preparation
Testicular cells were prepared for apoptosis analysis following a protocol adapted from Ojo
(2020). Briefly, testes were removed and decapsulated by making a small incision in the testis.
The contents of the testes were collected through the incision into a 15 ml tube containing 5 ml
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Omolara, O. O., Tosin, O. B., Faith, A., & Ayegbusi, E. O. (2024). Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of Melatonin Against
Melphalan-Induced Reproductive Toxicity. European Journal of Applied Sciences, Vol - 12(1). 487-501.
URL: http://dx.doi.org/10.14738/aivp.121.14674
ice-cold 1X PBS buffer (pH -7.4) and the contents were incubated for 40 min at 37°C with
vigorous shaking. Then, the tubes were placed on ice and incubated to allow the seminiferous
tubules to settle. The supernatants were discarded and the seminiferous tubules were washed
twice in 10 ml of PBS twice.
Hormonal Assessments
Testicular concentration of Follicle Stimulating Hormone, testosterone and Luteinizing
hormone were measured by enzyme-linked immunosorbent assay (ELISA) as described in the
instructions of the manufacturer’s kit (Demeditec Diagnostics GmbH, Germany). Briefly,
testicular proteins were extracted with phosphate buffer (50mM, pH 7.4) and centrifuged at
10,000rpm for 20mins. The supernatant was used to estimate FSH, T and LH levels were
expressed in mg/ml.
Biochemical Estimations of Testes Tissue
Testicular tissue from each mouse were stored at -20oC for different biochemical assays Tissue
homogenates (10%) (w/v) were prepared in chilled 100mM Tris-HCl buffer (pH 7.4) using
tissue homogenizer and protein quantity was estimated according to Lowry’s method (Lowry
,1951). The values were expressed mg/ml of protein.
Estimation of Total Antioxidant Capacity (TAC)
The TAC of the serum was estimated by ferric reduction antioxidant power (FRAP) assay
(Benzie and Strain, 1999). Here, 100 μl of cellular supernatant was added to 1 ml of fresh ferric
reducing antioxidant power reagent (FRAP; Tripiridyltriazine; Merck) and incubated at 37°C
for 10 min in the dark. Reading of the blue-coloured reagent was done at 595 nm every 20 sec
for 10 min. An aqueous solution of Fe II (FeSO4.7H2O) and appropriate concentrations of freshly
prepared ascorbic acid were used as blank and standard solutions, respectively. The results
were expressed as μmol/L.
Glutathione (GSH) Concentration
Glutathione (GSH) concentration was estimated by centrifuging an aliquot of 10% homogenates
of the tissues in 100 mM Tris-HCL buffer (pH 7.4) containing 0.16 M KCL at 1000 g for 5 min.
The supernatant was used to measure the rate of reduction of 5’ 5’- dithiobis-(2 nitrobenzoate)
to 2-nitro-5 thiobenzoate. The absorbance was read at 412 nm. Glutathione content was
expressed in μM/mg protein. For determination of GSSG content 0.1M NaOH was used instead
of Tris-HCL buffer.
Glutathione Peroxidase (GPX) Level
Glutathione peroxide activity was determined according to the method of Wendel, 1980. The
reaction mixture containing 48 mM sodium phosphate, 0.38 mM EDTA, 0.12 mM NADPH, 0.95
mM sodium azide, 3.2 units glutathione reductase, 1 mM glutathione, 0.02 mM DTT and 0.0007
% (v/v) H2O2 were used to monitor the enzyme activity. Enzyme activity was determined by
measuring the change in absorbance at 340 nm for 3 min at 30 sec interval and expressed in
units/mg protein.
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Measurement of Reactive Oxygen Species (ROS) Level
The ROS assay was performed. Briefly, 50 μl of testicular tissue homogenate and 1400 μl
sodium acetate buffer was transferred to a cuvette. After then, 1000 ul of reagent mixture (N,
N-diethyl paraphenylenediamine 6 mg/ml with 4.37 μM of ferrous sulfate dissolved in 0.1M
sodium acetate buffer pH- 4.8) was added at 37°C for 5 minutes. The absorbance was measured
at 505 nm using spectrophotometer (Molecular Devices.) ROS levels from the tissue were
calculated from a calibration of H2O2 and expressed as U/mg of protein (1 unit = 1.0 mg H2O2/L).
Malondialdehyde Measurement
Testicular tissues were homogenized in 10 ml TCA (trichloroacetic acid) which is at the rate of
10%, and then centrifuged at 4°C for 15 minutes. 750 μl of the supernatant which was obtained
was mixed with 0.67% TBA (thiobarbituric acid) in a ratio of 1:1. Afterwards, the solution was
left in the water bath for 15 minutes. Finally, the absorbance was measured
spectrophotometrically at 535 nm. The results were presented as μmol/L.
Teratozoospermia Index (TZI)
The TZI or multiple anomalies index is the number of defects per abnormal spermatozoon. Each
abnormal spermatozoon may have one to four abnormalities including head, neck/mid piece
and tail defects or presence of cytoplasmic residues. TZI values have been read between 1.00
(each abnormal spermatozoon has only one defect) and 3.00 (each abnormal spermatozoon
has a head, mid-piece, and tail defects) as described by Krassas et al. (2008).
Sperm DNA Damage Evaluation
The assessment of DNA damage in epididymal sperm will be performed using the orange
acridine dye. The result of test will be expressed as percentage of DNA fragmentation.
RNA Extraction and RT- PCR
Total RNA will be extracted from testis samples using the Trizol reagent (Invitrogen, Carlsbad,
CA, USA) and reverse transcribed into cDNA will be done using standardization of cDNA
amplification condition and optimization of annealing temperature for primer use. Primers to
be used for quantitative RT-PCR for amplifications of genes involved in anti-oxidation, Tm and
amplicon size.
Statistical Analysis
All statistical comparisons between the groups were made using analysis of variance (ANOVA)
by Prism statistics software. Results were presented as mean ± SEM (Standard Error Mean).
Values of p < 0.05 were considered as statistically significant.
RESULTS
Effect on Testosterone
The concentration of testosterone was reduced when treated with Melphalan in comparison
with the control group. Meanwhile, there was a significant increase (p<0.05) in the
concentration of testosterone of mice in Group VI (5mg/kg/bwt MEP + 10mg/kg/bwt
Melatonin) compared to the mice treated with melphalan only (Group III, Group IV and Group
V).
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Omolara, O. O., Tosin, O. B., Faith, A., & Ayegbusi, E. O. (2024). Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of Melatonin Against
Melphalan-Induced Reproductive Toxicity. European Journal of Applied Sciences, Vol - 12(1). 487-501.
URL: http://dx.doi.org/10.14738/aivp.121.14674
Figure 1: Bar chart showing the effect on Testosterone. Note: All the values are expressed as
mean ± SEM (n=5). “**P” and “**q” indicates significantly different as compared to control at
(p< 0.05), and to melatonin alone at (p< 0.05) respectively.
Effect on Luteinizing Hormone (LH)
There was a significant increase (p<0.05) in the concentration of luteinizing hormone of mice
in Group VI (5mg/kg/bwt MEP + 10mg/kg/bwt Melatonin) compared to the mice treated with
Group V (5mg/kg/bwt MEP) after six hours (6hrs). Also, there was a significant increase
(p<0.05) in the concentration of luteinizing hormone of mice in Group VI (5mg/kg/bwt MEP +
10mg/kg/bwt Melatonin) compared to the mice treated with 5mg/kg/bwt of melphalan, after
fourteen days (14 days). After twenty-eight days (28 days), the group of mice treated
3mg/kg/bwt and 5mg/kg/bwt of melphalan respectively, had significant decrease (p<0.05) in
their Luteinizing hormone levels compared to that of the group of mice in Group VI
(5mg/kg/bwt MEP + 10mg/kg/bwt Melatonin).
Figure 2: Bar chart showing effect on LH. Note: All the values are expressed as mean ± SEM
(n=5). “**P” and “**q” indicates significantly different as compared to control at (P< 0.05), and
to melatonin alone at (P< 0.05) respectively.
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Effect on Follicle Stimulating Hormone (FSH) (mlU/ml)
There was a significant decrease (p<0.05) in the concentration of Follicle Stimulating Hormone
of groups of mice treated with melphalan compared to the control group of mice. Also, there
was a significant increase (p<0.05) in the concentration of Follicle Stimulating Hormone of mice
in Group VI (5mg/kg/bwt MEP + 10mg/kg/bwt Melatonin) compared to the mice in Group IV
and Group V across the groups. After twenty-eight days, the group of mice treated 3mg/kg/bwt
and 5mg/kg/bwt of melphalan respectively, had significant decrease (p<0.05) in their
concentration of Follicle Stimulating Hormone, compared to that of the group of mice in Group
IV, V and VI.
Figure 3: Bar chart showing the effect on FSH. Note: All the values are expressed as mean ± SEM
(n=5). “**P” and “**q” indicates significantly different as compared to control at (P< 0.05), and
to melatonin alone at (P< 0.05) respectively.
Effect on levels of Reactive Oxygen Species (ROS)
There was a significant decrease (p<0.05) in the level of ROS of mice in Group VI (5mg/kg/bwt
MEP + 10mg/kg/bwt Melatonin) compared to the mice treated with melphalan only (Group V).
Also, the levels of Malondialdehyde (MDA) were increased when treated with Melphalan in
comparison with the vehicle treated control.
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Omolara, O. O., Tosin, O. B., Faith, A., & Ayegbusi, E. O. (2024). Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of Melatonin Against
Melphalan-Induced Reproductive Toxicity. European Journal of Applied Sciences, Vol - 12(1). 487-501.
URL: http://dx.doi.org/10.14738/aivp.121.14674
Figure 4: Bar chart showing the effect on ROS. Note: All the values are expressed as mean ± SEM
(n=5). “**P” and “**q” indicates significantly different as compared to control at (P< 0.05), and
to melatonin alone at (p< 0.05) respectively.
Effect on Levels of Malondialdehyde (MDA) (U/mg)
There was a significant decrease (p<0.05) in the level of malondialdehyde of mice in Group VI
(5mg/kg/bwt MEP + 10mg/kg/bwt Melatonin) compared to the mice treated with melphalan
only (Group IV and Group V). Also, the levels of Malondialdehyde (MDA) were increased when
treated with Melphalan in comparison with the vehicle treated control.
Figure 5: Bar chart showing effect on MDA. Note: All the values are expressed as mean ± SEM
(n=5). “**P” and “**q” indicates significantly different as compared to control at (P< 0.05), and
to melatonin alone at (p< 0.05) respectively.
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Omolara, O. O., Tosin, O. B., Faith, A., & Ayegbusi, E. O. (2024). Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of Melatonin Against
Melphalan-Induced Reproductive Toxicity. European Journal of Applied Sciences, Vol - 12(1). 487-501.
URL: http://dx.doi.org/10.14738/aivp.121.14674
Figure 8: Bar chart showing the effect on Reduced Glutathione. Note: All the values are
expressed as mean ± SEM (n=5). “**P” and “**q” indicates significantly different as compared to
control at (p< 0.05), and to melatonin alone at (p< 0.05) respectively.
Effect on Protein Level
Protein level was reduced across the groups in comparison with the control. Meanwhile, there
was a significant increase (p<0.05) in the level of protein of testes in Group VI (5mg/kg/bwt
MEP + 10mg/kg/bwt Melatonin) compared to the mice treated with melphalan only (Group III,
Group IV and Group V).
Figure 9: Bar chart showing the effect on the Protein level. Note: All the values are expressed as
mean ± SEM (n=5). “**P” and “**q” indicates significantly different as compared to control at
(p< 0.05), and to melatonin alone at (p< 0.05) respectively.
Effect on Teratozoospermia Index (TZI)
The concentration of Teratozoospermia index (TZI) was increased when treated with
Melphalan in comparison with the control group. Meanwhile, there was a significant decrease
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
7days 14days 28days
**p **p **p **p **p **p **p **P **p
**q **q
**q
GSH U/L
Dose Groups
Group I (Control) Group II Group III Group IV Group V Group VI
**p
**p
**p
**q
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Group I
(Control)
Group II (
Melatonin
alone)
Group III
(1mg/kg/bwt
MEP)
Group IV
(3mg/kg/bwt
MEP)
Group V
(5mg/kg/bwt
MEP)
Group VI
(5mg/kg/bwt
MEP +
10mg/kg/bwt
Melatonin)
Protein mg/ml
Dose groups 7days 14days 28days
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Omolara, O. O., Tosin, O. B., Faith, A., & Ayegbusi, E. O. (2024). Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of Melatonin Against
Melphalan-Induced Reproductive Toxicity. European Journal of Applied Sciences, Vol - 12(1). 487-501.
URL: http://dx.doi.org/10.14738/aivp.121.14674
The mRNA expression of Nuclear Factor Erythroid 2–related Factor 2 and Heme Oxygenase-1
genes were upregulated when treated with Melphalan in comparison with the vehicle treated
control. Downregulation of Nrf2 and HO-1 mRNA expression were observed in the group of
mice treated with 5mg/kg/bwt MEP + 10mg/kg/bwt Melatonin, compared to the mRNA
expression of Nrf2 and HO-1 genes of group of mice treated with melphalan only (Group IV and
Group V).
Figure 12: Bar chart showing the effect of melatonin on expression of Nrf2 and HO-1 genes in
testis. Note: All the values are expressed as mean ± SEM (n=5). “**P” and “**q” indicates
significantly different as compared to control at (p< 0.05), and to melatonin alone at (p< 0.05)
respectively.
DISCUSSION
The anticancer activity of melphalan was accidentally discovered many years ago. People have
a deep understanding of the chemical and biochemical changes leading to its medicinal
properties. Melphalan is a chemotherapeutic drug which is capable of causing excessive ROS
production. In the current study, it was found that chemotherapy-induced testicular tissue
changes, including Leydig cell disruption, may be caused by oxidant/antioxidant imbalances.
Moreover, chemotherapy has a long-lasting effect on Leydig cells in cancer patients (Al-Bader
and Kilarkaje, 2015). Hypothalamic neurons secrete GnRH, which triggers the release of LH and
FSH from the pituitary into the peripheral circulation. The main function of LH is to stimulate
Leydig cells to secrete testosterone. Therefore, Leydig cell damage may lead to the abnormal
secretion of testosterone. More and more studies have reported that chemotherapeutic drugs
can cause oxidative stress and abnormal gonadal hormone secretion. A study found that
chemotherapeutic drug melphalan and cisplatin decreased the levels of testosterone, LH, and
FSH, decreased antioxidant enzyme activity, and increased the levels of oxidative stress (Afsar
et al. 2017).
Melphalan causes testicular toxicity, leading to oxidative stress, sperm toxicity, DNA damage
and increased tetratozoospermia index, resulting in infertility. Numerous studies indicated that
melphalan significantly decreased the number of spermatogonia, Leydig cells, and Sertoli cells,
the testicular volume, the height of the germinal epithelium, glutathione, and Glutathione
peroxidase enzyme activities, and serum testosterone levels in the testes of rats. In contrast,
*
*
** ** **
** ** **
**
**
0
0.5
1
1.5
2
2.5
3
3.5
Nrf2 HO-1
Nrf2 and HO-1 Genes
Dose Groups
Group I (Control)
Group II ( Melatonin
alone)
Group III
(1mg/kg/bwt MEP)
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the numbers of malondialdehyde (MDA) levels were increased in the testes of melphalan- treated rats. Interestingly, this current study also demonstrated that the Total antioxidant
capacity (TAC) level decreased and the content of MDA increased in the melphalan-treated
mice. The Total antioxidant capacity (TAC) levels were still low, and the content of MDA further
increased after the mice were treated with melphalan and recovered for 7 days, 14 days and 28
days (Cui et al., 2017).
Stressful conditions lead to excessive production of free radicals that cause an imbalance in the
oxidant/anti- oxidant system, and oxidative stress is one of the major factors that induce
testicular cells apoptosis in testes. For instance, reactive oxygen species induces enhanced
oxidative stress, oxidative damage, and reduction in the total antioxidant levels in the testes of
rats (Zhang and Zhang, 2014). A previous study found that scrotal heat induced severe
oxidative stress in mouse testes, which consequently caused testicular cells death. On the other
hand, the antioxidants are well-known for protecting the testicular cells against oxidative
damage. Melatonin is a well-established antioxidant exerting a protective role against oxidative
stress and apoptosis in somatic and germ cells (Maneesh et al., 2005).
However, whether melatonin can protect against restraint stress-mediated oxidative damage
of testes is yet unknown. Therefore, we assessed the level of ROS in testes and explored the
effects of melatonin. Our results demonstrated that ROS accumulation occurred in the stress
group, which was diminished by melatonin treatment. In addition, antioxidants, such as GSH,
were significantly decreased in the stressed mice that recovered almost to baseline level in the
melatonin-treated stress group. SOD and GSH constitute the major antioxidant system. GSH is
sensitive to intracellular ROS, which could be neutralized by ROS as one the major endogenous
antioxidants (Schafer and Buettner, 2001). In the present study, it was shown that oxidative
stress induced mice had a reduced level of glutathione peroxidase (GPx) and glutathione (GSH)
in the testes, thereby suggesting that GPx and GSH were likely attenuated by increased ROS in
melphalan induced testes. Considering the antioxidant functions of melatonin, it was further
shown that the redox balance was improved by melatonin as evident from the normalized
levels of GSH and GPx in the melatonin-treated group (Kanatsu-Shinohara et al., 2016). Thus,
melatonin may alleviate restraint stress-induced testicular cells apoptosis by relieving the
oxidative stress. Melatonin and its derivatives are potent antioxidants and directly scavenge
free radicals. Melatonin possesses anti-aging, anti-inflammatory, and anti-apoptotic
capabilities, as well as immunity-enhancing and cancer-fighting characteristics (Xiang et al.,
2019). Melatonin also has a significant function in male reproduction by regulating the release
of steroid hormones. Melatonin protects the testes against high temperatures, environmental
chemicals, and medicines. Notably, the Nrf2 pathway plays an important role in preventing
oxidative stress. Oxidative stress stimulates the translocation of Nrf2 from the cytoplasm to the
nucleus, where it binds to antioxidant-response elements (AREs) and promotes the expression
of many genes encoding antioxidant enzymes, such as HO-1 (Hayes and Dinkova-Kostova,
2014). Nrf2 regulates the transcriptional activity of endogenous antioxidant proteins, including
GSH and GPX. The Nrf2 antioxidant response element pathway is an important system for
preventing oxidative stress. Several studies indicated that H2O2 caused oxidative stress and
lowered the protein levels of Nrf2 and HO-1 in mice Leydig cells (Dong et al., 2020).
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Omolara, O. O., Tosin, O. B., Faith, A., & Ayegbusi, E. O. (2024). Upregulation of Nrf2/Ho-1 Signaling and Protective Effect of Melatonin Against
Melphalan-Induced Reproductive Toxicity. European Journal of Applied Sciences, Vol - 12(1). 487-501.
URL: http://dx.doi.org/10.14738/aivp.121.14674
Melatonin stimulated the production of Nrf2 and HO-1 in H2O2. Nrf2 activation is critical for the
protective effect of melatonin. Melatonin reduces oxidative stress and apoptosis by promoting
the Nrf2/HO-1 signaling pathway in mouse testicular cells. Melatonin can also inhibit stress- induced spermatogenic injury. Melatonin inhibits hyperhomocysteinemia-induced oxidative
stress and apoptosis in rat smooth muscle cells via the Nrf2/HO-1 pathway. (Tang et al., 2019)
found that psychological stress can induce oxidative stress and apoptosis in testicular cells.
Melatonin ameliorates oxidative stress and apoptosis by upregulating the Nrf2/HO-1 signaling
pathways. This current study showed that the oxidative damage was elevated in the testes of
melphalan-treated mice. Melatonin has a protective effect on melphalan-induced oxidative
stress. A recent study found that melatonin protected human sperm by activating Nrf2 and its
downstream gene HO-1 (Tang et al., 2019).
Given the vital role of ROS in stress condition, it is imperative to address how to limit ROS- induced spermatogenic impairment. Melatonin is known to activate the antioxidant enzymes.
Nrf2 plays a significant role in preventing the development of oxidative stress in
spermatogenesis. HO-1 has also been reported to be involved in the testicular response to
stress. In the current study, it was shown that mRNA expression of Nrf2 and HO-1 genes of the
groups of mice induced with melphalan only (Group III, Group IV and Group V) had significant
upregulations compared to the control group of mice. Therefore, it can be hypothesized that
melphalan effectuated oxidative stress in the testes by disrupting Nrf2/HO-1 signaling
pathway. Several studies found that melatonin is a potent regulator of Nrf2 and HO-1 (Zhang
and Zhang, 2014).
Melatonin has been shown to modulate neuroinflammation and oxidative stress in
experimental diabetic neuropathy via the regulation of Nrf2 pathways. A recent study reported
that melatonin activates the Nrf2/HO-1 pathway when it exhibits a protective effect against
early brain injury in a subarachnoid hemorrhage model. Another study indicated that
melatonin modulates the expression of Nrf2 to protect against cyclophosphamide-induced
urotoxicity (Zaninotto et al., 2006). On the other hand, melatonin is demonstrated as a regulator
of HO-1, especially, it alleviates the cadmium-induced cellular stress in association with the
effect of HO-1. In addition, the ability of melatonin to regulate the Nrf2 pathway is associated
with the regulation of HO-1 expression (Nakamura, 2010). Testicular cells apoptosis was
accompanied by increased ROS in this current study, which could induce the damage of
testicular cells indeed. Therefore, it is possible that ROS overproduction can induce testicular
cells apoptosis by activating apoptotic signaling pathways, which could be ameliorated by
melatonin.
CONCLUSION
This study mainly investigated the suppression of oxidative stress via Nrf2/HO-1 signaling by
melatonin in testicular tissue of Melphalan-treated mice. Additionally, the expression and
localization of melatonin receptors were studied in induced testicular dysfunction mice with
oxidative stress caused by melphalan chemotherapeutic drugs. Whether melatonin can protect
mice testes and Leydig cells from melphalan-induced injury by activating the Nrf2/HO-1
signaling pathway; the results show that melphalan chemotherapeutic drugs can lead to
testicular injury, decreased testosterone levels, decreased LH and FSH levels, decreased sperm
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concentrations, and increased ROS and malondialdehyde. Melatonin pre-treatment can
alleviate melphalan-induced testicular damage in mice.
ACKNOWLEDGMENT
We are appreciating all P8 Research Team for their hard work.
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