Keywords
curcumin - malondialdehyde - cochlear fibroblast - reactive oxygen species - ototoxicity
- gentamicin
Introduction
Various kinds of medicines and topical agents can cause ototoxicity.[1] Toxic reactions induced by medicines often trigger the development of hearing disorders
in some people. They can occur in one or both ears with different levels of damage,
and their effects can be identified in the short or long terms.[2] The effect of this ototoxicity can last for a day or a few weeks after the medicine
enters body circulation.[3]
Numerous medicines can be categorized as ototoxic because of their side effects on
the inner ears. Ototoxicity can be divided into cochleotoxicity (when it affects hearing)
or vestibulotoxicity (when it affects balance).[4] Cochleotoxicity occurs in 2% to 25% of patients, while vestibulotoxicity occurs
in 15% of patients who consume aminoglycosides.[3] Some medicines which contain ototoxic effects are diuretic loop, cytostatic, tuberculostatic,
quinine, and aminoglycoside drugs. Ototoxic agents damage the structure of the inner
ear and change the mechanoelectrical transduction, which causes functional problems.[5]
As wide-spectrum antibiotics, aminoglycosides have been reported to have strong bactericidal
activity against Gram-negative bacteria. Their potency to kill bacteria is preferred
over that of other medicines which only possess bacteriostatic capabilities.[6] The popularity and universal use of aminoglycosides have made its ototoxicity side
effect as the main cause of hearing disorder. Even though aminoglycoside ototoxicity
is usually associated with accumulated dosage and the administration process, cochlear
dysfunction could still occur in some patients within or below the indicated therapeutic
dosages.[7]
The molecular mechanism of ototoxicity can cause an elevated production of reactive
oxygen species (ROS), depletion of the natural antioxidant glutathione and its enzymes,
and the average increase in fat oxidation, oxidative modification of proteins, nucleic
acid damage through caspase activation, and modification of important cochlear proteins.[8] Reactive oxygem species can also cause cell dysfunction, necrosis or apoptosis in
the tissues, as well as induce posttranslational changes that can affect the function
of protein cells and signal pathways.[9] Increasing ROS can damage deoxyribonucleic acid (DNA), disintegrate endothelial
and generate apoptosis.[10] Increasing the levels of ROS in the testicles, which are linked to membrane lipids,
proteins and DNA, can affect sperm production and quality. This peroxidation damage
to the tissues indicates a biochemical basis for reperfusion injury.[11]
Oxidative stress markers have been the focus of various studies as important tools
to assess the biological redox status, the condition and course of the disease, as
well as the effects of antioxidants on human health[12] Soltani et al.[13] (2018) conducted a study on oxidative stress and malondialdehyde (MDA), superoxide
dismutase (SOD) and glutathione peroxidase (GPx) levels. It is known that the last
product of lipid oxidation is MDA, which has been widely regarded as a reliable biomarker
of oxidative stress.[14]
Curcumin, a well-known bioactive phytochemical extracted from turmeric (Curcuma longa), has been used for centuries in Chinese and Indian traditional medicines.[15] Curcumin is also used throughout Asia as a spice and seasoning to give food a certain
taste and yellowish color.[16] Numerous studies have reported that curcumin has many healing properties, such as
an antioxidant, anti-inflammatory agent, it acts in the prevention and treatment of
cancer,[17]
[18] and is an anti-angiogenic,[17] anti-thrombotic, and hepatoprotective agent.[18] Curcumin has long been known as a safe ingredient. However, there are some side
effects that have been reported in several studies, which are diarrhea, headache,
rash, yellow stool, nausea, and an increase in the serum levels of alkaline phosphatase
and lactate dehydrogenase.[19]
The present research aims to assess whether curcumin can work as an effective and
safe antioxidant to prevent and slow the development of damage to the cochlea fibroblasts
based on the decrease in MDA expression in an ototoxic model of rats.
Methods
The present experimental study employed a randomized post-test only control group
design. All procedures were approved by the Health Research Ethical Committee, Faculty
of Medicine of Universitas Sumatera Utara/H. Adam Malik General Hospital, under number
777/TGL/KEPK FK USU-RSUP HAM/2016, in accordance with the Code of Practice for the
Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes. In total,
32 adult male white rats (Rattus norvegicus of the Wistar lineage) were obtained from the Animal Experimental Unit of the Biochemistry
Laboratory, Faculty of Medicine, Universitas Airlangga, Indonesia. Before observation,
the rats were subjected to a 12-day clinical evaluation in a preconditioned environment
(20°C to 25°C, 12 hours of light) and ad libitum feeding to control body weight between
150 g and 250 g.
The curcumin used in the research was extracted directly from C. longa (The Testing Service Unit, Faculty of Pharmacy, Universitas Airlangga, Surabaya,
number 0632/SA/V/2016) with levels of 16.62 ± 0.14% w/w compared with Standard using
Thin Layer Chromatography and Densitometry, provided by professor doctor Suprapto
Ma'at, MS (Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia). Curcumin
was administered to the rats at dosages of 100 mg/kg and 200 mg/kg, and it was suspended
in 0.5% carboxymethyl cellulose (CMC) and delivered through a nasogastric tube (NGT).
The 32 rats were randomly divided into /8 groups (with 4 rats in each group), as seen
on [Table 1].
Table 1
Setup of the ototoxic model group
|
Group 1
|
Group 2
|
Group 3
|
Group 4
|
Group 5
|
Group 6
|
Group 7
|
Group 8
|
|
Control
|
gentamycin 40 mg/ml
|
gentamycin 40 mg/ml
↓
curcumin
100 mg/kg after 18 hours
|
gentamycin 40 mg/ml
↓
curcumin 200 mg/kg after 18 hours
|
gentamycin 40 mg/ml
↓
curcumin
100 mg/kg for 7 days
|
gentamycin 40 mg/ml
↓
curcumin 200 mg/kg for 7 days
|
Days 1–3: curcumin 100 mg/kg per day
↓
gentamycin 40 mg/ml
|
Days 1–3: curcumin 200 mg/kg per day
↓
gentamycin 40 mg/ml
|
|
↓
|
↓
|
↓
|
↓
|
↓
|
↓
|
↓
|
↓
|
|
Termination in 18 hours
|
Termination in 18 hours
|
Termination in day 9
|
Termination in 18 hours
|
To induce ototoxicity on the test subjects, 0.1 ml of gentamicin (40 mg/ml) was injected
(intratympanic) at the anterosuperior part within 60 to 80 minutes of the anesthesia
(7.5 mg/kg of xylazine and 100 mg/kg of ketamine intraperitonially).[20] The functional and morphological changes in the cochlea were observed in terminated
test subjects 18 hours after the administration of gentamicin.[21]
It is known that the administration of 100 mg/kg of curcumin once a day for 7 days
to posthepatectomy rats could decrease MDA significantly.[22] Another research[23] showed that curcumin was able to induce the proliferation of Langerhans cells and
protect against oxidative stress in damaged cell tissues related to diabetic cognitive
disorder. In the present research, we also tested a double dose to see if we could
achieve a better result.
We used the MDA NB 100–62737 antibody (Novusbio, Novus Biologicals, Centennial, CO,
US), the biotinylated secondary antibody (anti-rabbit), and peroxidase-labeled streptavidin.
After the administration, the test subjects were euthanized by cervical dislocation,
and a necropsy of temporal bone tissues was conducted to obtain tissue samples, which
were fixed with 10% formalin buffer and subjected to ethylenediaminetetraacetic acid
(EDTA) decalcification for 4 weeks. The laboratory examination was performed through
fixation by deparaffinized tissue blocks dyed with haematoxylin eosin (HE). The MDA
expression was assessed using a immunohistochemical (IHC) grading technique,[24] by multiplying 0% of the cells labeled in the IHC (it was said 0 when it was 0%,
1 when it was < 30%, 2 when it was between 30% and 60%, and 3 when it was > 60%) with
the intensity of the IHC reaction (it was called 0 when there was no reaction, 1 when
the reaction was week, 2 when it was moderate, and 3 when it was strong), so that
the final value was between 0 and 9.
The results were submitted to analysis of variance (ANOVA; p ≤ 0.05) using the Statistical Package for the Social Sciences (IBM Corp., Armonk,
NY, US) software, version 22.0. Data comparison was performed using the Mann-Whitney
test followed by the Kruskal-Wallis test to determine the statistical differences
between each group.
Results
MDA as the Marker of Oxidative Stress
After the desired cochlea areas were identified through HE pigmentation, we performed
the IHC grading to measure MDA expression in the fibroblast of the lateral walls of
the cochlea ([Fig. 1]).
Fig. 1 Supporting tissues and cochlea lateral walls with immunohistochemical (IHC) pigmentation
(magnification: 10x). The directional arrow indicates the lateral walls.
The administration of gentamicin triggered the incidence of the oxidative process
indicated by the increase in MDA expression in the cochlear fibroblast, marked by
the browning of cytoplasm. This was also clearly observed with the increase in the
brownish intensity of the cells in the fibroblast area within the group without curcumin
administration (group 2). Curcumin could decrease MDA expression in the cochlear fibroblast
of ototoxic model of rats (groups 3 to 8) compared with those in group 2. Group 8
had the lowest MDA expression. The change in MDA expression could be seen in the IHC
description, as it was shown in [Fig. 2].
Fig. 2 Malondialdehyde (MDA) expression in each group (magnification: 40x): (A) group 1; (B) group 2; (C) group 3; (D) group 4; (E) group 5; (F) group 6; (G) group 7; and (H) group 8. The directional arrow indicates the brownish color that characterizes MDA
expression in the cochlear fibroblast.
Analysis of the Data
Based on the statistical assessment of the measurement result, group 2 had the highest
mean value of MDA expression, while the lowest mean value was found in group 8, as
shown in [Fig. 3].
Fig. 3 The mean value of MDA expression in the fibroblast of cochlear lateral walls in an
ototoxic rat model during the IHC examination.
In [Table 2], a significant difference (p ≤ 0.05) in fibroblast MDA expression is observed between the induced-ototoxicity
group (group 2) and the control group (group 1), but there was no significant difference
between the induced-ototoxicity group and the groups who only received one dose of
curcumin (groups 3 and 4). There was a significant difference (p ≤ 0.05) in the ototoxic model groups who received only 1 dose of curcumin (groups
3 and 4) compared with the ototoxic model groups who underwent 7 days of curcumin
treatment (groups 5 and 6). No significant differences were observed between groups
7 and 8; but, overall, there was a significant difference between the groups who underwent
the curcumin treatment and those who did not.
Table 2
Analysis of variance for malondialdehyde expression
|
Group
|
Group
|
Standard deviation
|
p-value
|
|
Group 1
|
2
|
5.000* ± 1.070
|
.000*
|
|
3
|
4.250* ± 1.070
|
.001*
|
|
4
|
3.500* ± 1.070
|
.003*
|
|
5
|
1.750 ± 1.070
|
.115
|
|
6
|
.250 ± 1.070
|
.817
|
|
7
|
.000 ± 1.070
|
1.000
|
|
8
|
.25 ± 1.070
|
.817
|
|
Group 2
|
3
|
.750 ± 1.070
|
.490
|
|
4
|
1.500 ± 1.070
|
.174
|
|
5
|
3.250* ± 1.070
|
.006*
|
|
6
|
4.750* ± 1.070
|
.000*
|
|
7
|
5.000* ± 1.070
|
.000*
|
|
8
|
5.250* ± 1.070
|
.000*
|
|
Group 3
|
4
|
.750 ± 1.070
|
.490
|
|
5
|
1.750 ± 1.070
|
.028*
|
|
6
|
3.250* ± 1.070
|
.001*
|
|
7
|
4.250* ± 1.070
|
.001*
|
|
8
|
4.500* ± 1.070
|
.000*
|
|
Group 4
|
5
|
1.750 ± 1.070
|
.115
|
|
6
|
3.250* ± 1.070
|
.006*
|
|
7
|
3.500* ± 1.070
|
.003*
|
|
8
|
5.750* ± 1.070
|
.002*
|
|
Group 5
|
6
|
1.500 ± 1.070
|
.174
|
|
7
|
1.750 ± 1.070
|
.115
|
|
8
|
2.000 ± 1.070
|
.074
|
|
Group 6
|
7
|
.250 ± 1.070
|
.817
|
|
8
|
.500 ± 1.070
|
.645
|
|
Group 7
|
8
|
.250 ± 1.070
|
.817
|
Note: * p ≤ 0.05: statistically significant difference.
Discussion
The present research assessed MDA expression in the cochlear fibroblast of white rats,
R. norvegicus of the Wistar lineage. Rodents (especially Mus musculus and R. norvegicus) have been widely used as guinea pigs in biomedical research for years.[25] Fluorescent imaging shows that gentamicin attaches to the stria vascularis, especially
to its marginal cells. Huth et al.[3] point out that, after aminoglycoside enters the systemic current, it enters cochlea
in only a few minutes through capillary blood vessels. Aminoglycosides can also be
seen in the basal and fibrotic cells. Schacht et al.[4] point out that the outer hairs of corti cells are easily damaged, which gradually
affects the lower apex regions. The damage usually occurs from the first up to the
third layers, and inner hair cells gradually disappear. Aminoglycoside is also found
in the stria vascularis, where degradation of tissue and marginal cells occur. Damage
to the ganglion spiral cells usually follows the loss of hair cells, which can persist
long after the therapy has finished. It is also pointed out that anomalies in the
stria vascularis can occur without damage to the hair cells.
The significant MDA expression among the gentamycin-induced ototoxic model groups
compared with the control group supports the statement of Ayala et al.,[14] which point out that MDA constitutes a reliable marker to pinpoint oxidative stress
in clinical studies; therefore, MDA reactivity and toxicity are often correlated in
biomedical research. Dierckx et al.[26] found high levels of MDA in the lipid autoxidation pathway in diabetic patients
compared with healthy groups.
This is also in accordance with the theory that states that aminoglycoside induces
the establishment of ROS, which are central in the molecular path to ototoxicity.
Aminoglycosides are known to directly modulate the enzymatic activity related to ROS
metabolism by interfering with other antioxidants.[27] Reactive oxygen species can be produced in several sources, including the mitochondria,
xanthine oxidase, and unoccupied nitric oxide synthase.[9] The excessive production of ROS promotes oxidative stress that causes many molecular
changes such as nucleic acid mutations and protein misfolding that leads to diseases.[15] An increase in ROS levels can produce oxidative stress, activated apoptosis, and
cause damage to DNA in the tissues.[28] A study reported that excess ROS decrease sperm motility and morphology, resulting
in DNA damage and apoptosis.[29] These factors play their role in initiating cell death as the result of the oxidative
response.[30] Numerous pathways are believed to trigger the apoptosis of cochlear cells through
ROS activity, such as the caspase-dependent pathway, caspase-independent pathway,
lipid autoxidation, and induction of Ca2+ influx.[31] Lipid peroxidation (autoxidation) is a chain reaction which continuously provides
the supply of peroxide-free radicals and initiates the next peroxidation in the lipoprotein
membrane by ROS; among which MDA belongs.[32]
Oxidative damage can occur as the result of overproduction of ROS and/or the lack
of antioxidant scavenging ability to neutralize free radicals. There are at least
three strategies to avoid the development of oxidative stress within the inner ears:
ROS detoxification with antioxidants, ROS inhibition by oxidant scavengers, or cutting
off the associated downstream ROS signal pathway.[27] Curcumin shows a strong scavenging activity regarding various ROS, including anionic
superoxide, hydroxyl radicals, and nitrogen dioxide radicals.[33]
A significant difference in MDA expression in both ototoxic models was observed between
the groups in whom the duration of the curcumin treatment varied (18 hours and 7 days).
The results could mean that the longer curcumin treatment with either dose (100 mg/kg
or 200 mg/kg) might yield better results.
The decrease in MDA expression caused by curcumin activity within the cochlea fibroblast
could be inferred from previous medical observations. Correa et al.[34] pointed out that oxidative stress constituted the basis of the risk factor for mortality
in senior hemodialysis patients. It was also found that curcumin could decrease cardiac
complications in some patients.
A significant difference was observed between model groups with curcumin administration
as therapy (curcumin was administered after gentamycin) and the groups with curcumin
supplement as prevention measures (curcumin was administered before gentamycin). Kuhad
et al.[35] point out that curcumin has protective effects in cases of cisplatin-induced nephrotoxicity,
hinting its function as a strong anti-inflammatory and antioxidant. It is believed
that curcumin would also be suitable to prevent the hearing impairment associated
with oxidative stress.
Considering that aminoglycosides cannot be metabolized and persist in hair cells for
several months, it is necessary to find a potential compound which can suppress the
development of ototoxicity during the long-term therapy.[3] Alrawaiq and Abdullah[36] noted that curcumin was considered a strong phytochemical to prevent and alleviate
various diseases, and could be used as potential therapeutic agent. The importance
of MDA as an important marker of oxidative stress and its role in causing health disorders
should be further explored, including the development of better and more reliable
testing methods to be applied in nutritional and medical studies.[37]
From the results obtained, it is expected that curcumin can become an effective and
safe plant-based medication to prevent and treat hearing loss due to ototoxicity in
a biomolecular level. Despite this, animals are not representive of human beings.
Therefore, it is necessary to do further research on human beings.The present research
is expected to be the basis for further research to assess cochlear function using
tools such as otoacoustic emissions (OAEs).
Conclusion
The results show that curcumin could effectively prevent and decrease gentamycin-induced
oxidative stress by limiting MDA expression in the cochlea fibroblast. It is implied
that the antioxidative properties of curcumin could slow and prevent the development
of ototoxicity. We encourage further exploration of this research and turning it into
proper clinical research.
