https://doi.org/10.52973/rcfcv-e34501
Received: 08/08/2024 Accepted: 23/09/2024 Published: 26/12/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34501
ABSTRACT
Lipopolysaccharide (LPS), known as a stimulant of inammation,
causes acute liver injury by inducing the production of inammatory
mediators and oxidative stress. The purpose of this study is to
determine whether of a nonsteroidal anti–inammatory drug (NSAID)
Flunixin meglumine (FM) and herbal an medicine Ginkgo biloba L.
extract (GBE) show antioxidative, anti–inammatory or antiapoptotic
effects in liver tissue in LPS–induced hepatotoxicity. Animals were
separated to 6 groups as control, sepsis (1 mg·kg
-1
, 7
th
day single dose,
intraperitoneal (ip)), sepsis + FM (1 mg·kg
-1
, 7
th
day single dose, ip +
2.2 mg·kg
-1
day, ip), sepsis + GBE (1 mg·kg
-1
, 7
th
day single dose, ip + 50
mg·kg
-1
day, gavage), FM and GBE and the study continued for 7 days.
Liver tissues taken from rats sacriced were analyzed biochemically,
histologically and immunohistochemically. Accordingly, LPS caused
liver function markers alteration, inammation, oxidative stress,
and apoptosis, as well as histopathological changes in liver tissue.
However, it was observed that LPS–induced changes were regulated
by FM and GBE application. FM and GBE was demonstrated to have
antioxidant, antiinammatory and anti–apoptotic properties in LPS–
induced hepatotoxicity.
Key words: Flunixin meglumine; Ginkgo biloba L. extract; liver
damage; sepsis
RESUMEN
El lipopolisacárido (LPS), conocido como un estimulante de la
inamación, causa daño hepático agudo al inducir la producción de
mediadores inamatorios y estrés oxidativo. El propósito de este
estudio es determinar si un fármaco antiinamatorio no esteroide
(AINE) Flunixin meglumine (FM) y un agente natural extracto de Ginkgo
biloba L. (GBE) muestran efectos antioxidantes, antiinamatorios o
antiapoptóticos en el tejido hepático en la hepatotoxicidad inducida
por LPS. Los animales se separaron en 6 grupos como control,
sepsis (1 mg·kg
-1
, dosis única el séptimo día, intraperitoneal (ip)),
sepsis + FM (1 mg·kg
-1
, dosis única el séptimo día, ip + 2,2 mg·kg
-1
día, ip), sepsis + GBE (1 mg·kg
-1
, dosis única el séptimo día, ip + 50
mg·kg
-1
día, sonda), FM y GBE y el estudio continuó durante 7 días.
Los tejidos hepáticos extraídos de ratas sacricadas se analizaron
bioquímicamente, histológicamente e inmunohistoquímicamente.
En consecuencia, el LPS provocó alteración de los marcadores de
la función hepática, inamación, estrés oxidativo y apoptosis, así
como cambios histopatológicos en el tejido hepático. Sin embargo,
se observó que los cambios inducidos por LPS fueron regulados
por la aplicación de FM y GBE. Se demostró que FM y GBE tienen
propiedades antioxidantes, antiinamatorias y antiapoptóticas en
la hepatotoxicidad inducida por LPS.
Palabras clave: Flunixina meglumina; extracto de Ginkgo biloba L.;
daño hepático; septicemia
Ginkgo biloba L. extract and unixin meglumine attenuate sepsis–
associated liver injury, oxidative stress, inammation and apoptosis in rats
El extracto de Ginkgo biloba L. y la unixina meglumina atenúan la lesión hepática, el
estrés oxidativo, la inamación y la apoptosis asociados a la sepsis en ratas
Tuba Parlak Ak
1
* , Burcu Gul
2
, Mine Yaman
3
, Ismail Seven
4
, Gurdal Dagoglu
5
, Huseyin Fatih Gul
6
1
University of Munzur, Faculty of Health Sciences, Department of Nutrition and Dietetics.Tunceli, Türkiye.
2
University of Firat, Faculty of Health Sciences, Department of Nursing. Elazig, Türkiye.
3
University of Firat, Faculty of Veterinary Medicine, Department of Histology and Embryology. Elazig, Türkiye.
4
University of Firat, Vocational School of Sivrice, Department of Plant and Animal Production. Elazig, Türkiye.
5
University of Firat, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology. Elazig, Türkiye.
6
University of Kafkas, Faculty of Medicine, Department of Biochemistry. Kars, Türkiye.
*Corresponding author: tubaparlakak@munzur.edu.tr
Ginkgo biloba L. in apoptosis in rats / Parlak Ak et al. _______________________________________________________________________________
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INTRODUCTION
Lipopolysaccharide (LPS), known as a stimulant of inammation,
is the main constituent of Gram–negative bacteria and induces the
manufacture of uncontrolled inammatory mediators and oxidative
stress concluded acute hepatic injury [1]. LPS indicates a pro–
oxidative effect on this damage by activating liver macrophages that
produce inammatory cytokines inclusive tumor necrosis factor alpha
(TNF–ɑ), interleukin–1ß (IL–1ß) and interleukin–6 (IL–6) [2] and inducing
excessive production of reactive oxygen species (ROS) [3]. Therefore,
it is important to develop the diversity and currency of preferred
anti–inammatory agents for the inhibition of these cytokines.
Nonsteroidal anti–inammatory drugs (NSAIDs) have the capability
to scavenge free radicals and play as robust antioxidants. Flunixin
meglumine (FM), an NSAID, is proven to have antitoxemic mechanisms
and is frequently used in the treatment of endotoxemia [4]. FM strongly
obstructs cyclooxygenase (COX) and the synthesis of eicosanoids and
attunes acute hemodynamic alterations throughout endotoxemia [5].
However, such drugs have diverse side effects with long–term use [6].
For this reason, it is noteworthy that natural produce are discovered
in the improve of new treatments that are more attractive and have
minimal toxicity [7] and they are the focus of recent research in the
phytochemical–based management of diseases [6].
Ginkgo biloba L. extract (GBE), which is very widely used universally,
has diverse pharmacological properties [8]. The benecial inuences
of Ginkgo biloba extract 761 (EGb 761) obtained from the leaves based
on these pharmacological properties are due to its active ingredients
consisting of avonoids (24%) and terpene lactones (6%) [9]. It has
been reported that GBE, which has antioxidant, anti–inammatory,
anti–apoptotic and antigenotoxic effects, is effective in various toxicities
[10], and especially prevents oxidative stress and decrease in antioxidant
defense in hepatotoxicity [11]. It is known that the antioxidant capacity
of Ginkgo biloba, which causes this hepatic renovation, is related
to increasing glutathione content, reducing lipid peroxidation and
hydroperoxide levels, and restoration of antioxidant enzyme activity [12].
EGb 761 has been reported to decrease LPS–induced oxidative stress,
especially through ROS and nitric oxide (NO) [13]. Also, the robust anti–
inammatuar characteristics of GBE components occur through their
inhibitive effects on TNF–α, IL–6, prostaglandin E2 (PGE2), inducible nitric
oxide synthase (iNOS) mRNA and COX–2 mRNA values in LPS–induced
macrophages [14]. This work designed to specied the ameliorative
activities of FM and GBE in LPS–induced liver injury by reducing some
inammatory mediators, preventing the formation of oxidative stress
and inhibiting cell apoptosis.
MATERIALS AND METHODS
Chemicals and animals
Lipopolysaccharide (Escherichia coli, O55:B5, Sigma, USA), FM
(Vilsan Pharmaceuticals, TR) and EGb 761 (Abdi İbrahim İlaç, TR)
were acquired from the indicated companies. The rest agents were
provided by Sigma–Aldrich Company (USA). Thirty–six male Spraque–
Dawley rats (Rattus norvegicus) (male, 6–8 weeks old, 280–300 g
weight) were acquired from Firat University Experimental Research
Center (Elazig, Turkey). The animals were provided with optimal
situation (50–60% humidity, 22–24°C temperature, feed and optional
water, 12h light/12–h cycle) throughout the experimental applications.
This study was approved by Firat University Animal Experiments Local
Ethics Committee (20.11.2013/09–127).
Experimental design
Animals were randomly separated to six groups (n=6) and the
study continued for 7 days (d). The average solution volume applied
to all groups was determined as 0.5 mL. For the control group,
physiological saline solution was received through intraperitoneally
(ip) injection for 7 d. For the sepsis group, 1 mg·kg
-1
LPS solved in
saline was received through ip injection only on the 7
th
day [15]. For
the FM group, 2.2 mg·kg
-1
d FM was received through ip injection
for 7 d [4]. For the GBE group, 50 mg·kg
-1
day EGb 761 was received
through gavage for 7 d [16]. For the sepsis + FM group, 1 mg·kg
-1
LPS
was received only on the 7
th
d following 2.2 mg·kg
-1
d FM application
for 7 d. For the sepsis + GBE group, 1 mg·kg
-1
LPS was received only
on the 7
th
d following 50 mg·kg
-1
d EGb 761 application for 7 d. The rats
were sacriced under anesthesia then blood specimens and liver
tissues were taken and evaluated for biochemical, histological and
immunohistochemical examinations.
Serum analysis
Serum TNF–ɑ (E–EL–R0019, Elabscience, USA; pg·mL
-1
), PGE2
(E–EL–0034, Elabscience, USA; pg·mL
-1
) and IL–1ß (E–EL–R0012,
Elabscience, USA; pg·mL
-1
) levels were specied by commercial
enzyme linked immunosorbent assay (ELISA) kit and studied in
accordance with the specied procedures. All process were applied
according to Ilhan et al. [13]. Serum glucose, albumin, globulin, total
protein, aspartate aminotransferase (AST), alanine transaminase
(ALT), total cholesterol, triglyceride, high–density lipoprotein (HDL),
low–density lipoprotein (LDL), and very low–density lipoprotein (VLDL)
analyzes were determined with Olympus AU 600 (Optical Co., Ogaki,
JAPAN) autoanalyzer.
Biochemical analysis
Malondialdehyde (MDA) and glutathione (GSH) levels and superoxide
dismutase (SOD) and catalase (CAT) activities were determined
spectrophotometrically in homogenate of liver tissues. MDA levels
were expressed as nmol·ml
-1
[17], GSH levels as nmol·mg
-1
protein
[18], SOD levels percent inhibition·mg
-1
protein [19], CAT levels as
k·g
-1
protein [20].
Histological evaluation
The tissues were paranized after 10% formalin xation and blocks
were prepared. For histological examination, hematoxylin–eosin (H&E)
applied sections were photographed with Olympus BX–51 microscope.
(Olympus Optical Co., Ltd., Tokyo, JAPAN). Different histological
elds were appreciated semi–quantitatively at 20x magnication to
determine the extent of histopathological alterations in the tissues
of all groups. The analysis scores of histopathological changes was
calculated according to the criteria of none (0), slight (1), medium (2)
and severe (3) [21].
Immunohistochemical examination
Caspase–3 (Casp 3) (#RB–1197–B, Thermo Fisher Scientic Co.,
USA) immunohistochemistry reactivity in the tissues was dened
by the Avidin–Biotin–Peroxidase Complex method [22]. Background
staining was performed with haematoxylin. Immunoreactivity was
computed by the formula area x intensity (intensity; none (0), very
little (0.5), little (1), moderate (2), severe (3) x area; 0.1 (<25%), 0.4
(26–50%), 0.6 (51–75%), 0.9 (76–100%) [22].
TABLE I
Eect of FM and GBE on LPS–induced liver function biomarkers
Parameter Control Sepsis FM GBE Sepsis+FM Sepsis+GBE P
Glucose (mg·dl
-1
) 78.83 ± 2.24
a
113.80 ± 3.12
c
79.03 ± 2.03
a
80.01 ± 2.60
a
88.62 ± 2.30
b
89.38 ± 3.01
b
*
Albumin (g·dl
-1
) 3.45 ± 0.06
a
2.98 ± 0.08
b
3.43 ± 0.06
a
3.41 ± 0.07
a
3.22 ± 0.08
a
3.23 ± 0.06
a
*
Globulin (g·dl
-1
) 2.36 ± 0.03 2.30 ± 0.02 2.35 ± 0.03 2.35 ± 0.04 2.41 ± 0.03 2.47 ± 0.01 NS
T.protein (g·dl
-1
) 5.81 ± 0.03
a
5.28 ± 0.05
b
5.78 ± 0.04
a
5.76 ± 0.05
a
5.63 ± 0.05
a
5.70 ± 0.03
a
*
ALT (U·L
-1
) 77.30 ± 2.30
a
107.50 ± 2.70
c
77.10 ± 2.10
a
76.40 ± 3.20
a
90.10 ± 2.80
b
89.20 ± 2.60
b
**
AST (U·L
-1
) 192.10 ± 8.70
a
289.00 ± 7.20
c
189.70 ± 5.10
a
190.00 ± 6.30
a
234.10 ± 3.10
b
232.90 ± 4.60
b
**
T. cholesterol (mg·dl
-1
) 43.00 ± 1.13
a
74.80 ± 2.07
c
43.80 ± 2.12
a
44.30 ± 1.62
a
58.50 ± 1.80
b
57.80 ± 1.37
b
*
Triglyceride (mg·dl
-1
) 47.30 ± 3.5
a
86.40 ± 4.71
b
47.60 ± 5.01
a
48.20 ± 3.67
a
59.30 ± 4.02
a
57.10 ± 3.80
a
*
HDL (mg·dl
-1
) 12.90 ± 0.45
a
29.46 ± 0.60
b
10.81 ± 0.91
a
10.50 ± 0.74
a
16.90 ± 0.86
a
17.20 ± 0.67
a
*
VLDL (mg·dl
-1
) 9.46 ± 0.97
a
15.23 ± 1.25
c
10.40 ± 0.82
a
10.80 ± 0.93
a
11.60 ± 0.79
b
11.80 ± 0.87
b
*
LDL (mg·dl
-1
) 5.50 ± 0.73
a
9.66 ± 0.90
c
7.21 ± 0.18
b
7.30 ± 0.74
b
6.71 ± 0.52
b
6.72 ± 0.91
b
*
a,b,c
: Dierent superscripts in the same row indicate the signicant dierence, NS: Non–signicant, * P<0.05: statistically signicant, ** P<0.01: statistically signicant,
AST: aspartate aminotransferase, ALT: alanine transaminase, HDL: high–density lipoprotein, LDL: low–density lipoprotein, VLDL: very low–density lipoprotein
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34501
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Statistical analysis
SPSS 21.0 (SPSS Inc., Chicago, IL, USA) software was preferred.
Differences between groups were carried out using one–way analysis
of variance subsequently the posthoc Tukey test. The data were
presented as mean ± standard deviation format. The values with
differences of P<0.05 were deemed statistically signicant.
RESULTS AND DISCUSSIONS
Sepsis is a complicated pathophysiological process involving an
excessive inammatory and immune response that cause multiorgan
failure. It is a global health problem that continues to cause large
numbers of deaths today. LPS is considered a extremely pathogenic
endotoxin responsible for these dysfunctions in sepsis. Therefore,
studies are still ongoing to identify new therapeutic drugs for
effective sepsis treatment [23]. Many studies have shown that natural
and medicine produces such as curcumin [24], metformin [25],
aminoguanidine [26] have a protective and therapeutic effects on
LPS induced hepatotoxicity. However, there is a lack of data regarding
the effectiveness of FM and GBE.
In this study, the liver function marker levels of all groups are
presented in TABLE I. It was determined that the glucose, total
cholesterol, triglyceride, HDL, LDL, VLDL levels (P<0.05), AST and
ALT levels (P<0.01) signicantly increased in the sepsis group in
comparison to the control. On the contrary, it was defined that
signicant for albumin and total protein levels (P<0.05) decreased
in sepsis group than control. Additionally, it was observed that the
AST and ALT levels (P<0.01) and other liver function marker levels
(P<0.05) a signicantly decreased in the sepsis + FM and sepsis +
GBE groups in comparison to the sepsis. In particular, triglyceride
and HDL levels were found to reach the control group levels, while
albumin, globulin and total protein levels were higher than in the sepsis
group. Some researchers have stated that this markers increase
with LPS administration [6, 25, 26, 27]. It has been stated that FM
[4] and GBE [28, 29] treatments signicantly decreased the LPS–
induced increase in AST and ALT levels similar to this study. There
are also studies in the literature in which this increases caused by
LPS is decreased with different agents [24, 25, 26]. It is reported
that LPS–induced sepsis causes tissue damage due to mitochondrial
dysfunction and cell rupture [30], and the decrease in increased AST
and ALT levels indicating this tissue damage may be attributed to
the physiological defense mechanism of FM and GBE through tissue
regenerative effects.
LPS severely stimulates immune cells and triggers extreme
inflammation by increasing the synthesis and release of
proinammatory components including [2]. In an experimental with
LPS, it was reported that the levels of proinammatory cytokines
such as TNF–ɑ, IL–1ß and IL–6 levels increased signicantly [31]. Our
study results showed that TNF–α, PGE2 and IL–1β levels increased
signicantly in the sepsis group (P<0.001) compared to the control
group with LPS administration. It was also determined that these
values were decreased in the sepsis + FM and sepsis + GBE groups
(P<0.001) compared to the sepsis group. These values of all groups
are presented FIG. 1. In some LPS–induced studies, increased
proinammatory cytokine levels have been shown to be regulated
using different agents [6, 25, 26]. In our study, LPS–induced increased
TNF–α and IL–1β levels were recorded to be decreased by FM and GBE
administration, similar to hepatotoxicity studies [28, 32]. It has also
been recorded that GBE decreases the values of these cytokines in
liver ischemia/reperfusion [8]. Therefore, it can be said that FM and
GBE exhibit anti–inammatory effects in sepsis by regulating the
production and release of various inammatory mediators.
Oxidative stress activates a number of transcription factors
and induces the expression of many genes, containing various
proinammatory cytokines [21]. It has been determined that TNF–ɑ and
IL–1ß, which are proinammatory cytokines, induce the production
of ROS and this damage further increases the production of these
cytokines [33]. LPS induced sepsis models, it has been shown by
many studies that ROS levels increases, especially in the liver [24,
34]. In our study, MDA and GSH levels and SOD and CAT activities of
all groups are presented in FIG. 2. MDA levels showed a signicant
increase in the sepsis group compared to the control group (P<0.01).
Also, a decrease in GSH levels, SOD (P<0.01) and CAT activities (P<0.05)
was detected. These data are consistent with previous studies
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0
10
20
30
40
50
60
TNF-α (pg·mL
-1
)
c
c
c
b
b
a
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0
1
2
3
4
5
6
MDA (nmol·mL
-1
homogenate)
a
b
b
bc
bc
c
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0.0
0.2
0.4
0.6
0.8
1.0
CAT (k/g protein)
b
b
a
a
a
c
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0
5
10
15
20
25
30
GSH (nmol·mg
-1
protein)
b
b
c
a
a
a
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0
10
20
30
40
50
60
70
SOD (Inhibition %)
a
a
a
b
b
c
Control Seps is FM GBE Sepsis+FM Sepsis+GBE
0
20
40
60
80
100
120
140
PGE2 (pg·mL
-1
)
b
b
c
c
c
a
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0
20
40
60
80
100
120
140
160
IL-1β (pg·mL
-1
)
c
c
c
b
b
a
FIGURE 1. Serum levels of TNF–ɑ (A), PGE2 (B) and IL–1ß (C) analyzed by ELISA method. Data are stated as mean ± standard deviation, P<0.001. TNF–ɑ:tumor necrosis
factor alpha, PGE2: prostaglandin E2, IL–1ß: interleukin–1ß, ELISA: enzyme–linked immunosorbent assay
FIGURE 2. Liver levels of MDA (A) and GSH (B) and activities of SOD (C) and CAT (D) analyzed by spectrophotometrical
method. Data are stated as mean ± standard deviation, P<0.01 for all analyzes apart from CAT activitiy (P<0.05).
MDA: malondialdehyde, GSH: glutathione, SOD: superoxide dismutase, CAT: catalase
A
A
B
B
C
C D
Ginkgo biloba L. in apoptosis in rats / Parlak Ak et al. _______________________________________________________________________________
4 of 8
reporting that oxidative stress markers such as MDA increase and
GSH, SOD and CAT decrease in LPS–induced hepatotoxicity. Some
researchers have associated the irregularity in these levels with
LPS–induced liver damage [24, 31, 34]. This study is consistent with
other studies in that the imbalance in LPS–induced oxidative stress
markers can potentially be regulated by antioxidant agents such as
FM and GBE. In the sepsis + FM and sepsis + GBE groups, MDA levels
were reduced compared to sepsis group (P<0.01), while GSH levels,
SOD (P<0.01) and CAT activities (P<0.05) were signicantly increased.
Consistent with this study data, it was recorded that FM regulates
impaired oxidant and antioxidant systems in LPS–induced sepsis
[4]. Moreover, Tatli Seven et al. [32] suggested that FM normalizes
oxidative stress markers in liver toxicity. Parimoo et al. [35] stated that
the imbalance in oxidative stress markers was regulated by GBE, an
antioxidant agent. In addition, it is stated that GBE attenuates hepatic
oxidative stress through modulation of redox imbalance [36]. It has
been also shown that the impaired oxidant and antioxidant systems
LPS induced are regulated in studies by using different antioxidant
agents [26, 37]. It can be predicted that antioxidant agents used
against excessive ROS production in LPS–induced sepsis may be a
good choice to reverse liver damage.
Control FM GBE Histological Score
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Histological score
a
b
b
Sepsis Sepsis Sepsis + FM Sepsis + GBE
FIGURE 3. Photomicrographs of histopathological changes on the liver tissues of all groups. Black thick arrow: hyperemia in Vena centralis, white thick arrow: congestion
in sinusoid, yellow thick arrow: Kuper cell activation, black thin arrow: karyomegaly in hepatocytes, white thin arrow: necrosis in hepatocytes, yellow thin arrow:
inammatory cell inltration. Scale bar=100 µm for the groups apart from high magnication (Scale bar=50 µm) of sepsis group. H&E
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34501
5 of 8
Endotoxic shock, one of the most critical symptoms of LPS–induced
sepsis, causes various pathological changes. It is known that these
changes are tissue damage caused by the rising in cytokine release,
oxidative stress and mitochondria dysfunction [30]. Some research
showed various histopathological alterations such as serious necrosis,
hemorrhage, congestion, edema, inammatory cell inltration, and
signicant destruction of hepatolobular structure with LPS application
[6, 25]. Similarly, a study revealed that LPS impaired the hepatic
architecture along with vacuolization, degeneration, and inammatory
cell inltration in the liver [24]. In our study, lobuler structure, central
vein and portal areas were in normal histological architecture and
hepatocytes were radially located in control, FM and GBE groups. In
contrast, it was noted signicant alterations as disorganization in
hepatic cord areas, hyperemia in vena centralis and congestion in
sinusoids in the sepsis group. In addition to Kupffer cell activation,
karyomegaly and necrosis in hepatocytes were noted. Inammatory
cell inltration, mainly neutrophil granulocytes, was also detected.
Interestingly, it was noted in our study that FM and GBE alleviated
these necrotic, hemorrhagic and infiltrative changes caused by
LPS. The histopathology and histopathological score of all our study
groups are presented in FIG 3. It has been reported that FM improves
histopathological damage in the liver by restoring liver function markers
that are impaired in sepsis with gluconeogenic and ureogenic activity
and by regulating the inammatory response and oxidative stress status
with its anti–inammatory and antioxidant activities. Also, it was stated
that FM application reduced leukocyte inltration and eliminated the
prevalence of necrosis in LPS–induced hepatotoxicity [4]. Similarly,
other researchs have reported that FM exhibits hepatopreventive
property by reducing degenerative and necrotic changes [32]. The
cleavage activity of GBE plays a role in supplying pharmacological
preservation of cellular functions in pathological cases [38]. In an
experimental hepatoxicity model, it was reported that GBE provided
a signicant improvement in the histopathological changes [36].
Besides, in another research, it was detected that GBE remarkably
inhibited hepatocyte denaturation and necrosis due to toxicity [29].
Therefore, it can be said that FM and GBE improve the histopathological
injury produced by LPS by regulating the inammatory response and
strengthening the antioxidant defense system.
LPS–induced ROS increase aggravates acute liver injury, leading
to apoptotic cell death [39]. Also, Killilea et al. [27] reported that
the rise in LPS–induced liver proinammatory mediators causes
increased Casp3 activity and expression, apoptosis and aggravated
hepatic damage. Another research announced that LPS application
rised the protein expression level of the pro–apoptotic gene Casp3
and the number of TUNEL positive hepatocytes [37]. The Casp3
immunoreactivity and the immunohistochemical histoscore of all
this study groups are offered in FIG. 4. Similar to the above data, in
our study, it was determined that Casp3 immunoreactivity increased
Control FM GBE
Histoscore
(prevalence × intensity)
Control Sepsis FM GBE Sepsis+FM Sepsis+GBE
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Histoscore (prevalence x intensity)
a
b
c
dd
d
Sepsis Sepsis Sepsis + FM Sepsis + GBE
FIGURE 4. Casp–3 immunoreactivity evaluated on the liver tissues of all groups. Arrows: Casp–3 immunoreactive cells. Scale bar=100 µm for the groups apart from high
magnication (Scale bar=50 µm) of sepsis group
Ginkgo biloba L. in apoptosis in rats / Parlak Ak et al. _______________________________________________________________________________
6 of 8
in the sepsis group due to LPS–induced apoptotic activity compared
to the control, FM, and GBE groups. This immunostaining intensity
increased in the nucleus and cytoplasm of hepatocytes in the
sepsis group than control. Morever, it was observed that FM and
especially GBE signicantly reduced this increased activity. Casp3
immunoreactivity was found reduced in the sepsis + FM and especially
sepsis + GBE groups than sepsis. In this case, the statistical data of
Casp3 immunoreactivity was determined to be highest in the sepsis
(2.50 ± 0.22, P<0.05) compared to the sepsis + FM and sepsis + GBE
groups (1.33 ± 0.25 and 1.19 ± 0.31, P<0.05, respectively). As expected,
the lowest data was in control (0.29 ± 0.10). Avila et al. [4] stated
that FM application reduced LPS–induced apoptotic cell increase.
It has been reported that the increased number of apoptotic cells
and staining intensity decreased in studies using FM against other
hepatotoxic agents [32]. Furthermore, GBE has been reported to
inhibit induced hepatocyte apoptosis [35, 40] and signicantly reverse
increased Casp3 immunoreactivity in liver toxicity [28]. Considering
the anti–apoptotic abilities of FM and GBE, it can be said that they
contribute to tissue regeneration by reducing Casp3 immunoreactivity
that occurs in liver damage.
CONCLUSION
This study results suggest that FM, and especially GBE, has a
ameliorative act on LPS–induced liver toxicity and dysfunction. These
effects may be attributed to their capacity to reduce LPS–induced
inammatory responses, oxidative stress and apoptotic activity.
The results suggest that GBE, specically a natural product, may
represent a new candidate for ameliorating liver damage. Further
researches are required to completely comprehend the molecular
mechanism of the potential act of this agent.
Conict of Interests
The authors declare that there is no conict of interest.
ACKNOWLEDGMENTS
The authors would like to thank Munzur University Scientific
Research Projects Coordination Unit (MUNIBAP) for supporting this
work by Grant Code: MFTUB014–04.
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