https://doi.org/10.52973/rcfcv-e34388
Received: 05/02/2024 Accepted: 02/04/2024 Published: 08/08/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34388
ABSTRACT
Gentamicin (GM) is an aminoglycoside antibiotic the most common
used in the treatment of infectious diseases in humans and animals.
However, GM causes damage to many tissues and organs in the
body, especially the kidneys. Helichrysum plicatum (Hp), native to
the Balkans and Anatolia, is a plant used in various diseases such as
diabetes, liver and kidney damage. In this study, Male Spraque Dawley
rats (n=36 and 200–250 g) were randomly divided into 6 experimental
groups: Group1: Control; received normal saline (intraperitoneally
–i.p.–), Group 2: Hp (100 mg·kg
–1
day i.p.), Group 3: Hp (200 mg·kg
–1
day i.p.), Group 4: GM (80 mg·kg
–1
day i.p.), Group 5: GM 80 + Hp 100
(mg·kg
–1
day i.p.), and Group 6: GM 80 + Hp 200 (mg·kg
–1
day i.p.).
Then kidney tissue samples were collected for evaluations. All of
our results showed that Hp (100 mg·kg
–1
day) reduced the levels of
pro–inflammatory cytokines such as IL–8, IL–6, and TNF– while
increasing the level of anti–inammatory cytokine IL–10. It was also
observed that Hp reduced the expressions of the caspase3, NOS
and Heat shock proteins such as Hsp27 and Hsp70. With this study,
we have shown that Hp probably due to its chemical properties has
a protective effect against GM induced nephrototoxicity by reducing
the values stated above to normal values.
Key words: Caspase; gentamicin; Helichrysum plicatum; HSP;
kidney; NOS
RESUMEN
La gentamicina (GM) es un antibiótico aminoglucósido de los más
utilizado en el tratamiento de enfermedades infecciosas tanto en
humanos como en animales. Sin embargo, la GM causa daños en
muchos tejidos y órganos del cuerpo, especialmente en los riñones.
Helichrysum plicatum (Hp), originaria de los Balcanes y Anatolia, es
una planta utilizada en diversas enfermedades como la diabetes
y los daños hepáticos y renales. En este estudio, se utilizaron
ratas machos Spraque Dawley (n=36 y 200–250 g) las cuales se
dividieron aleatoriamente en 6 grupos experimentales: Grupo 1:
Control; recibieron solución salina normal (vía intraperitoneal –i.p.–),
Grupo 2: Hp (100 mg·kg
–1
día i.p.), Grupo 3: Hp (200 mg·kg
–1
día i.p.),
Grupo 4: GM (80 mg·kg
–1
día i.p.), Grupo 5: GM 80 + Hp 100 (mg·kg
–1
día i.p.), y Grupo 6: GM 80 + Hp 200 (mg·kg
–1
día i.p.). A continuación,
se recogieron muestras de tejido renal para su evaluación. Todos
nuestros resultados mostraron que Hp (100 mg·kg
–1
día) redujo los
niveles de citoquinas proinamatorias como IL–8, IL–6, y TNF–α,
mientras que, aumentó el nivel de citoquina antiinamatoria IL–10.
También se observó que la Hp reducía las expresiones de la caspasa3,
la NOS y las proteínas de choque térmico como la Hsp27 y la Hsp70.
Con este estudio, hemos demostrado que la Hp probablemente
debido a sus propiedades químicas tiene un efecto protector contra
la nefrototoxicidad inducida por GM reduciendo los valores arriba
indicados a valores normales.
Palabras clave: Caspasa; gentamicina; Helichrysum plicatum; HSP;
riñón; NOS
Protective effect of Helichrysum plicatum on head shock protein
inammation and apoptosis in Gentamicin induced nephrotoxicity
Efecto protector de Helichrysum plicatum sobre la inamación de la proteína de
choque termico y la apoptosis en la nefrotoxicidad inducida por gentamicina
Ismail Bolat
1
, Kubra Asena Terim–Kapakin
1
* , Betul Apaydin Yildirim
2
, Esra Manavoğlu Kirman
1
1
Atatürk University, Faculty of Veterinary Medicine, Departments of Pathology. Erzurum, Türkiye.
2
Atatürk University, Faculty of Veterinary Medicine, Departments of Biochemistry. Erzurum, Türkiye.
*Corresponding Author: kubra.terim@atauni.edu.tr
Protective Effect of Helichrysum plicatum in Gentamicin induced nephrotoxicity / Bolat et al. ______________________________________
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INTRODUCTION
Aminoglycosides are a group of antibiotics which are usually used
for treatment of critical and dangerous infections particularly caused
by gram‐negative and some gram-positive bacteria. Gentamicin
(GM) is an aminoglycoside antibiotic the most common used in
the treatment of infectious diseases in humans and animals [1,
2, 3]. However, they have serious side effects such as ototoxicity
[1], nephrotoxicity [2, 3, 4, 5, 6, 7], hepatotoxicity [4, 8, 9, 10, 11],
neuromuscular blockade, and allergic skin reactions [1] depending
upon the dosage. The mechanisms by which gentamicin (GM) induces
tissue damage are not fully explained, but links to the formation of
reactive oxygen species (ROS), inammation, and activation of the
apoptotic pathways [2, 3, 6, 9, 11, 12, 13]. Despite the introduction
of the new generations of antibiotics recently, aminoglycosides
are still used in various clinical situations due to their inexpensive
and high ecacy [1, 2, 3]. Heat shock proteins (Hsps) are known as
a group of proteins that can always be produced in the body and act
as a protector in the organism in various inammation and oxidative
stress situations [2, 3]. These proteins have an important place in the
intracellular and extracellular defense mechanism in kidney tissue
[14, 15, 16]. In various toxicity studies, protective effects of HSPs in
kidney tissue against toxicity have been demonstrated [17, 18].
Many natural products have been used to preserve the tissues
from toxicities caused by GM [3, 6, 8, 12, 19, 20] through neutralizing
the reactive species and enhancing the antioxidant defense. Sandy
everlasting or immortelle ower (Helichrysum plicatum) (Hp) is an
herbaceous plant, which is natural to the Balkans, Anatolia. It has been
used in folk medicine for various diseases such as diabetes, liver and
kidney damages. Its urolitic, nephroprotective, antihyperglycemic,
antioxidant, antimicrobial, hepatoprotective, and cytotoxic effect [4,
21, 22, 23] are related to presence of avones, avanones, avonols
and kalones [21, 23, 24].
In this study, we targeted to demonstrate the relationship
between Hsp27, Hsp70, caspase–3, NOS, and cytokines expressions
in protective effects of Helichrysum plicatum (Hp) on gentamicin
induced nephrotoxicity using biochemical, histopatological,
immunohistochemical and immunoresence methods.
MATERIALS AND METHODS
Experimental Animals and Design
Male Spraque Dawley rats (Rattus norvegicus) (200–250 g) were
obtained from Experimental Application and Research Institute.
These rats were housed at normal temperature of (22 ± 2°C), and 12/12
hours (h) light/dark cycle. The rats were fed ad libitum for 7 days (d) and
their adaptations were achieved. Then, experiments were applied to
rats for 8 d [4]. Gentamicin sulphate was purchased from Eczacibasi
(Istanbul, Turkey). All other chemicals of analytical grade were bought
from the Sigma Chemical Co. (St. Louis, MO). Helichrysum plicatum
specimens were collected in Bayburt, and identied at Department
of Botany. A voucher specimen was kept at the Herbarium of Atatürk
University (Voucher Number ATA9562/2013), Erzurum (Turkey) [4].
The experiment was conducted according to the ethical conditions
conrmed by the Ethical Rules of Experimental Animal Teaching
and Researcher Center, 25240 Erzurum, Turkey. [Ethical Approval
Number: 36643897–932140].
Preparation of the Test Samples
The dried Hp samples were dusted in a crusher machine (SINOMTP,
Mobile impact crusher, Shandong, China) and then 100 g of Hp was
extracted one by one within 500 mL ethanol for 2 days at 21 ± 2°C.
The extract was ltered and evaporated to dryness in a vacuum
(Biomec, ECO370, Turkiye) under reduced pressure at 40°C with
a evaporator (IKA, RV 05, Türkiye). These extracts obtained were
stored (Arçelik, 4252 EY, Türkiye) at 4°C. It was prepared according
to method described in our previous study [4].
Experimental Protocol
Male Spraque Dawley rats (n=36) were randomly divided into 6
experimental groups: Group 1: Control. Received normal saline
(intraperitoneally –i.p.–); Group 2: Hp (100 mg·kg
–1
day i.p.); Group3: Hp
(200 mg·kg
–1
day i.p.); Group 4: GM (80 mg·kg
–1
day i.p.); Group5: GM +
Hp 100 (mg·kg
–1
day i.p.); and Group 6: GM + Hp 200 (mg·kg
–1
day i.p.).
Administration was done for 8 days and then rats were sacriced with
pentobarbital sodium (60 mg·kg
–1
i.p.) under anesthesia. The kidney tissue
samples were taken for evaluations biochemically, histopathologically
immunohistochemically and immunooresence. Then, the kidney tissue
samples were stored at -80°C for biochemical analysis.
Kidney tissues homogenization for ELISA and cytokines levels
determination by
The kidney tissues were homogenized in a Qiagen Tissue Lyser II
homogenizer using a buffer of 0.1 M phosphate buffer (pH 7.4) to obtain
a 1:10 (w/v) homogenate. The tissues homogenate preparation method
required for all ELISA (enzyme–linked immunosorbent assay) kits
have been given in our previous study [4]. The supernatant obtained
was used for Interleukin 6 (IL–6), Interleukin 8 (IL–8), Interleukin 10
(IL–10), and Tumor necrosis factor alpha (TNF–α) analyzes. The IL–10,
Il–6, IL–8 and TNF– levels were analyzed in the kidney using an ELISA
according to the manufacturer’ instructions (Epoch, BioTeK; Winooski,
VT, USA) commercially.
Histopathological examination
For histopathological and immunohistochemical examinations,
the tissue samples were xed with 10% neutral buffered formalin
for 48 h. Then, the samples were dehydrated through alcohol series,
cleared with xylene and embedded in paran blocks. After routine
procedures, sections of 4–5 μm thickness were cut (LEICA RM2255,
Germany) and mounted onto slides. At last, the sections were stained
with Hematoxylin and Eosin (H&E) and Periodic Acid Schiff (PAS), and
examined under a light microscope (OLYMPUS BX51, Japan) [25].
Immunohistochemical examination
The sections washed with phosphate buffered saline (PBS) were
incubated (BINDER ED 56, Germany) with polyclonal rabbit Hsp27
antibody (clone: ab78806, dilution 1/100; Abcam, UK), Hsp70 (clone:
sc66036, Santa Cruz Biotecnology, dilution 1/200), and caspase–3
(clone: ND 600–1235, Novus Biological, USA, diluted 1:200) at 37
o
C
for 30 min. Immunohistochemical analysis was carried out as
recommended by the manufacturer [Abcam, UK, HRP Detection
IHC Kit (Catalog No: ab80436)] and 3,3–diaminobenzidine (DAB, Dako
Cytomation) was used as the chromogen. Ground staining was achieve
with Mayer’s hematoxylin [26, 27].
TABLE I
The levels of IL–10, IL–6, IL–8, and Tnf– in the kidney
tissues of all the experimental groups
Groups
IL–10
(pg·mg
-1
)
IL–6
(pg·mg
-1
)
IL–8
(pg·mg
-1
)
Tnf–α
(pg·mg
-1
)
1: Control 30.54 ± 0.21
a
131.24 ± 0.32
c
86.67 ± 0.50
d
1.33 ± 0.01
cd
2: Hp100 30.42 ± 0.20
a
130.86 ± 0.40
c
86.70 ± 0.32
d
1.31 ± 0.00
d
3: Hp200 29.60 ± 0.25
a
131.00 ± 0.28
c
87.45 ± 0.33
d
1.34 ± 0.01
c
4: GM 14.86 ± 0.25
c
371.69 ± 1.81
a
187.07 ± 0.31
a
2.33 ± 0.01
a
5: GM+Hp100 29.31 ± 0.18
a
132.66 ± 0.35
bc
94.70 ± 0.94
c
1.31 ± 0.00
d
6: GM+Hp200 23.18 ± 0.85
b
135.86 ± 0.75
b
98.62 ± 0.20
b
1.37 ± 0.00
b
P *** *** *** ***
The results were expressed as mean ± standard error means (SEM) for 6 rats in each
group. Dierent lowercase letters (
a–d
) in the same row indicate signicant dierence
among each group (
P<0.05). The lowest value is marked with “a” and the sign letter is
changed gradually when the statistical dierence occurs IL–8, IL–6, Tnf–α, and IL–10
FIGURE 1. Histopathological changes in the kidney tissues of all the experimental
groups. Degeneration (**, P=0.0022); Necrosis (**, P=0.0022); Nephritis (**, P=0.0065).
(n=6) (nsd=no standard deviation)
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Immunouorescence examination
The sections washed with phosphate buffered saline (PBS) were
incubated (BINDER ED 56, Germany) with NOS antibody (clone:
sc–7271, dilution 1/100, US), at 37
o
C for 30 min. Then, secondary
uorescent antibody (FITC, clone: ab6717 Dilution Ratio: 1/500, UK)
was dropped and incubated for 45 min. The washed sections were
covered with a coverslip with a mixture of glycerine and distilled water
(1/9) and examined under a uorescent microscope (ZEISS AXIO,
Germany). Hsp70, Hsp27, Caspase–3, NOS positivity were scored as
follows: none (−), mild (+), moderate (++) , severe (+++), more severe
(++++), and images were taken [26, 27].
Statistical analysis
The data obtained were evaluated statistically using the GraphPad
Prisim Version 8.0.2 statistical package program. The mean, standard
error, and median values of the data were determined using the
Descriptive Statistics–Explore test. The nonparametric Kruskal–
Wallis test was used to determine whether there was a statistical
difference between the parameters and the data obtained from the
groups. P values less than 0.05 were evaluated signicant.
RESULTS AND DISCUSSION
Biochemical ndings
Levels of renal cytokines TNF–α, IL–6, and IL–8 were detected an
increase in the GM group while IL–10 level was found a decrease as
compared to the control group (P<0.05). It was observed that TNF–α,
IL–6, and IL–8 levels were signicantly a decrease whereas IL–10 level
was an increase in all experimental groups treated with Hp. However, in
the group treated with GM + Hp100 (mg·kg
–1
day i.p.) TNF–α, IL–6, IL–8,
and IL–10 levels were observed to reach levels similar to the control
group (P<0.05). TABLE I summarized renal cytokines levels results.
Histopathological ndings
Histopathological changes in the kidney tissues of all the
experimental groups are demonsrated in FIG. 1. Histopathological
changes were not observed in the kidney tissues in the control group. In
the GM group there were very severe tubular epithelial degeneration and
necrosis, and severe lymphocyte and macrophage cells in intertubular
regions. In addition, intratubular hyaline cast and enlargement of the
bowman capsule were seen in the kidney tissues. In the GM+Hp200
group there were severe tubular epithelial degeneration and necrosis,
and severe lymphocyte and macrophage cells in intertubular regions,
and intratubular hyaline cast. In the GM+Hp100 group there were mild
tubular epithelial degeneration and necrosis. In the Hp200 group there
were moderate tubular epithelial degeneration and necrosis, and mild
lymphocyte and macrophage cells in intertubular regions. In the Hp100
group there were mild tubular epithelial degeneration (FIG. 2).
FIGURE 2. Histopathological view of the kidney tissues H&E, Bar: 50 μm, A: Control
group, B: GM group, C: GM+Hp200 group, D: GM+Hp100 group, E: Hp200 group and
F: Hp100 group. Degeneration in tubular epithelium (arrow head), lymphocyte
and macrophage cells inltration in intertubular regions (arrow), and hyaline
casts (star) and PAS staining (Above plate B, Bar: 20 μm)
FIGURE 3. Hsp 27, HSP 70, Caspase 3 and NOS level in the kidney tissues of all
the experimental groups. HSP 27 (*, P=0.0130); HSP 70 (**, P=0.0022); Caspase 3
(*, P=0.0130); NOS (**, P=0.0065) (n=6), (nsd=no standard deviation)
Protective Effect of Helichrysum plicatum in Gentamicin induced nephrotoxicity / Bolat et al. ______________________________________
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Immunohistochemical ndings
Hsp27 and Hsp70 levels in the kidney tissues of all the experimental
groups are demonstrated in FIG. 3. The Hsp27 (FIG. 4) and Hsp70
(FIG. 5) expressions were seen in the kidney tubular epithelial cells,
renal mesangial cells, and endothelial cells of blood vessels in all
experimental groups. However, high expression levels were observed
in the groups GM and GM+Hp200 whilst low secretion levels in the
control group were observed. The mildest expressions Hsp27 and
Hsp70 were detected in the GM+Hp100 group. Caspase–3 levels in
the kidney tissues of all the experimental groups are demonstrated in
FIG. 3. The expression of caspase–3 (FIG. 6) were showed in the kidney
mesangial cells, epithelial cells of kidney tubules and, endothelial cells
of blood vessels, as well as inammatory cells. A stronger expressions
were observed in the GM and GM+Hp200 groups than the other groups,
the weak expression was in the control group (P<0.05). The mildest
caspase–3 expression was detected in the GM+Hp100 group.
Immunouorescence ndings
NOS levels in the kidney tissues of all the experimental groups are
demonstrated in FIG 7. The NOS expressions (FIG. 3) were seen in the
kidney tubular epithelial cells, renal mesangial cells, and endothelial
cells of blood vessels in all experimental groups. However, high
expression levels were observed in the groups GM and GM+Hp200
whilst low secretion levels in the control group were observed. The
mildest expressions NOS were detected in the GM+Hp100 group.
The NOS expression were decreased in Group GM+Hp100 when
FIGURE 4. Hsp27 expressions in tubular epithelial cells (arrow heads), in the
kidney tissues, Bar: 50
μm. A: Control group, B: GM group, C: GM+Hp200 group,
D: GM+Hp100 group, E: Hp200 group and F: Hp100 group
FIGURE 5. Hsp70 expressions in tubular epithelial cells (arrow heads) in the
kidney tissues, Bar: 50 μm. A: Control group, B: GM group, C: GM+Hp200 group,
D: GM+Hp100 group, E: Hp200 group and F: Hp100 group
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TNF–α levels were increased after GM administration in kidney
tissue samples.
IL–10, one of the anti–inflammatory cytokines, suppresses
the inammatory response by inhibiting the production of pro–
inflammatory cytokines like TNF–α [11]. In previous studies, a
signicant decrease in IL–10 level was reported in treated with GM
at different doses [11, 32]. In our study, it was observed that the level
of IL–10 in kidney tissue decreased in GM group.
Previous histopathological studies reported that GM administration
were caused nephrotoxicity [2, 4, 5, 10, 33]. The obtained kidney
histopathologic results in our current study are in agreement
with previous studies. In this study showed that histopathological
examinations supported the biochemical effects of GM on kidney tissues.
Although many natural products such as, phytochemicals, different
plants extracts and plants formulas have been used in order to stop
or cure tissues damage caused by adverse effect of GS, there is still
not available specic treatment. Some studies have reported that
various plants extracts such as virgin coconut oil [6], Hesperidin [30],
berberine [12], curcumin [8], Malva sylvestris extract [20], Panax
ginseng [19], Moringa oleifera seed oil [3] are used for preventing
or treatment the tissue damage related to GM treatment. In these
compared with groups GM and GM+Hp200, and there was a statistically
signicant difference when compared with the GM group (P<0.05).
Gentamicin (GM) is an antibiotic the most widely used in the
treatment of infectious diseases caused by gram‐negative and some
gram–positive bacteria. Although they are preferred due to their high
eciency and cheapness [1, 2, 3], they cause serious adverse effects
such as nephrotoxicity [2, 3, 4, 5, 6, 7]. The mechanism of GM–induced
adverse effects is not fully understood, but there are studies showing
that gentamicin increases the formation of Reactive Oxygen Species
(ROS) by suppressing non–enzymatic and enzymatic antioxidants.
Free radicals cause cellular injury, inammation, degeneration, and
necrosis by some mechanisms such as peroxidation of phospholipids
membrane, protein denaturation, DNA damage and apoptosis [3, 12,
28]. IL–6, IL–8, and TNF–α are pro–inammatory cytokine that they
are primarily involved in the regulation of immune system, apoptosis
and inammatory responses [29].
Several studies have been reported that increasing levels of
pro–inammatory cytokines such as TNF–α [8, 11], IL–1β [3], IL–6
[3, 30] and IL–8 [31] were positively correlated with the inltration
of inflammatory cells in both tissues of GM administered rats
signicantly. In the present study, we observed that IL–6, IL–8 and
FIGURE 6. Caspase–3 expression in tubular epithelial cells (arrow heads) in
kidney tissues, Bar: 50 μm. A: Control group, B: GM group, C: GM+Hp200 group,
D: GM+Hp100 group, E: Hp200 group and F: Hp100 group
FIGURE 7. NOS expression in tubular epithelial cells (arrow heads) in the
kidney tissues, Bar: 50 μm. A: Control group, B: GM group, C: GM+Hp200 group,
D: GM+Hp100 group, E: Hp200 group and F: Hp100 group
Protective Effect of Helichrysum plicatum in Gentamicin induced nephrotoxicity / Bolat et al. ______________________________________
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induced GM by decreasing the levels of TNF–α, IL–6, IL–8, by increasing
IL–10. These results made us to think, that the protective effects of
Hp on kidney damage may be due to the inhibition of inammation.
Heat shock proteins (Hsps) are a highly conserved group of proteins
produced by all living organisms that protect cells and organisms
from different damage such as inammation, exposure to toxins
and oxidative stress [14, 16, 26, 38]. Hsps have been classified
into six major families on the basis of their size, construction and
assignment: the Hsp110, Hsp40, Hsp90, Hsp70, Hsp60 and small Hsp
families including Hsp27 [16, 38]. Hsps has an important place in the
intracellular and extracellular defense mechanism of kidney tissues
[14, 15, 16]. In previous studies, Hsp47, Hsp72/73 [39], Hsp72 [40],
Hsp90 [41] and Hsp70 [42] were detected in glomerular mesangial
cells, in proximal tubular epithelial cells after GM administration. In
these studies, it was reported that Hsp90 and Hsp72 levels started
to increase in 36 h after GM administration compared to the control
group, reached the highest level in 12 d, and started to decrease in 18
d [40, 41]. It was reported that Hsp47 expression reached its highest
level on the 7
th
day after GM administration and decreased to the
control group level on the 28
th
d [39]. In another study, it was reported
that Hsp70 expression was more severe in the GM group compared
to the control group after GM administration, but it was noted that
positivity was only in the interstitial cells in both groups [43].
studies, they reported that the severity of damage caused by GM
in kidney tissues was reduced by multiple targeted therapeutic
mechanisms such as prevention of oxidative stress, inammation,
and apoptosis and regulation of autophagy [8, 12, 19, 20, 30].
Helichrysum plicatum DC. (Asteraceae) is a herbaceous naturel plant
used traditional treatment method [21, 23, 24]. In recent years, that
has been used in the treatment many diseases due to its antiapoptotic,
antineurodegenerative, nephro–hepatoprotective, antihyperglycemic,
antioxidant, antimicrobial properties [4, 22, 23]. The therapeutic
medicinal feature of Helichrysum species is considered because of
the presence of avones, avanones, avonols, kalones, triterpenoids,
and steroids [21, 23, 24].
In our previous study we reported that the advers effects of
GM on the kidney were reducesed depending on the dose of Hp,
histopathologically [4]. The obtained histopathologic ndings in
our current study are in consistent with our previous studies on this
subject. In previous studies different members of the Helichrysum
species have been shown to inhibit pro–inammatory cytokines and
reduce anti–inammatory cytokines [34, 35, 36, 37]. Nevertheless,
there are no data found the role of cytokines the effect of Hp on GM
induced nephrotoxicity. In the present study, it was seen GM+Hp100
treatment (100 mg·kg
–1
day i.p. 8 d) inhibited the kidney inammation
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In our study Hsp70 and Hsp27expressions was observed
immunohistochemically in proximal, distal and collecting tubular
epithelium, glomerular mesangial cells, and vascular endothelial cells
in kidney tissue of the all experimental groups. In our study, it observed
that Hsp70 and Hsp27 levels, which increased in kidney tissues after
the GM administration, the level of decreased after the Hp (100 mg·kg
–1
day i.p. 8 d) treated. In our study, it observed that the Hsp27and Hsp70
expressions were decreased in kidney tissues Group GM+Hp100 when
compared with groups GM and GM+Hp200, and there was a statistically
signicant difference when compared with the GM group (P<0.05).
Apoptosis is a physiological cellular reaction to all external and
internal stimuli. The purpose of apoptosis is to destroy unwanted
cells like demaged cells without giving any harm to healthy cells.
The apoptotic process, can be stimulated (Bax, caspases family) or
inhibited (Bcl–2 family of genes, Hsps) by different signals or cytokines
[8, 10, 19, 30, 43, 44]. Previously researchers, have demonstrated that
caspase–3 expression were detected increased in kidney proximal
tubul epithelial and mesangial cells [6, 31, 44, 45]. The obtained kidney
results in the present study were similar to mentioned studies.
To our best knowledge, the antiapoptotic effect of Hp on
nephrotoxicity due to GM hasn’t been studied immunohistochemically.
The caspase–3 expression in kidney tissues were decreased in Group
GM+Hp100 when compared with groups GM and GM+Hp200, and there
was a statistically signicant difference when compared with the GM
group (P<0.05). In our study the antiapoptotic effect of Hp (100 mg·kg
–1
day i.p. 8 d) on GM induced nephrototoxicity was demonstrated with
caspase–3 immunohistochemically.
Nitric oxide (NO) is secreted by many cells in the body when oxidative
stress increases in the body. Nitric oxide synthase (NOS) inactivates these
free radicals and eliminates the negative effects of oxidative stress on
the body. In some gentamicin toxicity studies on kidney tissues [45,
46, 47], it has been reported that gentamicin administration increases
the NOS expression level in the body. Some studies have shown that
Hp inhibits oxidative damage by suppressing oxidative stress in the
body. In an experimental study, it has been shown that Hp suppresses
the oxidative stress that occurs after gentamicin administration and
protects the tissues against damage in tissues and organs [4, 48]. In
this study, it was observed that gentamicin administration increased
NOS expressions by increasing oxidative damage in kidney tissues,
in accordance with the literature. Again, it was determined that Hp
application suppressed oxidative stress and protected tissues against
gentamicin toxication against gentamicin applications.
All of our results showed that Hp (100 mg·kg
–1
day) reduced the levels
of pro–inammatory cytokines such as IL–8, IL–6, and TNF– while
increasing the level of anti–inammatory cytokine IL–10. It was also
observed that Hp reduced the expressions of the apoptotic marker
caspase–3, NOS and Heat shock proteins such as Hsp27 and Hsp70.
During the evaluation of the results of the study, it was observed that
high doses of Helichrysum plicatum may also have slightly adverse
effects on kidney tissues.
CONCLUSION
As a conclusion, we have shown that Hp probably due to its chemical
properties has a protective effect against GM–induced nephrotoxicity
by reducing the values stated above to normal values. However, in
the future, comprehensive studies are needed to elucidate the
mechanism of Hp’s nephroprotective effect.
Conict of interest
The Authors declare that there is no conict of interest.
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