Article Type: Research Article Article Citation: Ozgun Teksoy, Varol Sahinturk,
Mustafa Cengiz, Behcet İnal,
and Adnan Ayhancı. (2020). THE POSSIBLE EFFECTS
OF SILYMARIN ON CEREBRUM WITH EXPERIMENTAL HEPATIC ENCEPHALOPATHY IN RATS. International
Journal of Research -GRANTHAALAYAH, 8(8), 140-146. https://doi.org/10.29121/granthaalayah.v8.i8.2020.946 Received Date: 02 August 2020 Accepted Date: 26 August 2020 Keywords: Hepatic Encephalopathy Rat DLPFC Silymarin Immunohistochemistry Background: The relationship between liver diseases and neurological defects is well established. Hepatic encephalopathy (HE) has been seen both in people with acute liver failure (ALF) and chronic liver disease (CLF). HE is a complex neuropsychiatric syndrome that is seen in patients suffering from liver dysfunction. Silymarin (Sm) has antioxidant, anti-inflammatory, and anti-carcinogenic features. In this study, the possible protective effects of silymarin were investigated against dorsolateral prefrontal cortex (DLPFC) damage induced by thioacetamide (TAA). Method: To achieve this, male Sprague Dawley rats (200-250 g) were randomly divided into four groups, with 7 animals comprising each group: the control group, 50 mg/kg TAA group, 50 mg/kg Sm + TAA group, and 100 mg / kg Sm + TAA group. Results: Differences between the groups were determined by performing immunohistochemical analysis of the PFC. Bax, TNF-α, and TUNEL expression increased in the brain tissue of the experimental group where only TAA was administered. Conclusions: It was observed that in high doses in particular (100 mg/kg Sm + TAA group), Sm was effective in preventing PFC damage caused by TAA. It was determined that 100 mg/kg Sm significantly reduces TAA-induced inflammation (TNF-α and H&E) and apoptosis (Bax, TUNEL) in brain tissue.
1. INTRODUCTIONAbnormal behavior and cognition impairment have been observed in the
brains of patients suffering from acute and chronic liver failure, which is, in
turn, followed by the detection of edema in the brain
cells caused by increasing ammonia levels in the blood. This syndrome is called
hepatic encephalopathy (HE) and is the result of several factors, defined as intrinsic
(genetic) or extrinsic (viral, alcohol) [1]. According to the International Association of
Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN), thioacetamide (TAA),
D-galactosamine, and carbon tetrachloride have been commonly used to create an
experimental animal model of HE [2]. TAA, used in the present study,
metabolized in the liver [3]. The metabolizing process occurs in two stages:
firstly, the TAA-S-oxide (TASO) appears followed by S, S-dioxide (reactive
form, TASO2). This metabolizing process is executed through hepatic cytochrome
P450 enzymes and the FAD-containing monooxygenase (FMO) [4]. The Several drugs are commonly used in HE
treatment but, unfortunately, a host of side effects has also been reported
alongside their use. For this reason, researchers are turning to herbal
ingredients in their quest for a suitable treatment for HE. Silymarin (Sm), obtained from the seeds and fruits of Silybum marianum, is
one of them. Sm is a mixture of isomeric flavonoids
(silybin, is silybin, and silychristin) [9]. It can be used in high doses without any resulting side
effects in either humans or animals [13]. It has a hepatoprotective feature, while at the same time
demonstrating free radical scavenging and cell membrane balancing activity [13], [14], [15], [16], [17]. Several studies have shown that Sm
also has a neuroprotective effect [18]. This effect is largely related to the inhibition of
oxidative stress molecules in the brain [19] but is also a result of the induction of pathways involved in
neuroprotection, such as the inflammatory pathways [20], [21]. Moreover, a previous study demonstrated which Sm attenuates 6-OHDA-induced motor in-coordination in rats [22]. This study aims to show brain damage
caused by TAA-induced liver damage as well as to throw some light on the
protective effect of Sm on the brain in animal models
with HE. To better understand this effect, the DLPFCs of rats were examined
immunohistochemically via apoptotic markers. 2. MATERİAL-METHOD2.1. CHEMICALS
TAA (Cat
No: 62-55-5), DMSO (Cat No: 67-68-5), and Sm (Cat No:65666-07-1)were purchased from Sigma Chemical
Co. (St. Louis, MO, USA) and immunohistochemistry (IHC) antibodies were
obtained from Santa Cruz Biotech (Santa Cruz, USA). 2.2. ANIMALS
8 weeks-old
male Sprague-Dawley rats (200-250g) were housed at 25oC and exposed
to a12 h light-dark cycle. Food and tap water were provided ad libitum. Animal experiments
were performed according to the National Institutes of Health (NIH) Guide for the Care
and Use of Laboratory Animals (Reg. No.
KU/IAEC/PhD/100 dated 26.07.2012). This
study was carried out with the permission of the Eskişehir
Osmangazi University Local Animal Ethics Committee
(No: 513-2 / 2016). 2.3. THE EXPERIMENTAL DESIGN
The groups
were determined in accordance with previous studies [23]. Untreated group: 0.5 mL of water with 0.2% DMSO was administered orally
to each animal for the first 14 days, followed by an intraperitoneal injection
of 0.5 mL serum physiologic for the remaining 14 days. TAA group: 50 mg/kg TAA (1 mL/kg b.w.) was
intraperitoneally injected to each animal for the second period of 14 days. Low dose treatment group: 50 mg/kg Sm (1 mL/kg
b.w.) was administered orally to each animal for the
first 14 days, followed by 50 mg/kg TAA (1 mL/kg b.w.) intraperitoneally injected into each animal for the remaining
second 14 days. High dose treatment group: 100 mg/kg Sm (1
mL/kg b.w.) was administered orally to each animal
for the first 14 days, followed by 50 mg/kg TAA (1 mL/kg b.w.) intraperitoneally injected to each animal for the remaining
second 14 days. 5% dextrose
containing 0.9% NaCl and potassium (20 mEq/L) was
injected into all animals daily to prevent weight loss, hypoglycemia,
and renal failure [24]. 1 day after
the last TAA injection, a laparotomy was drastically performed under sterile
conditions and all rats were anesthetized with ketamine/xylazine (5/1 ratio); blood
samples were then collected from the left ventricle of the heart and
centrifuged at 3000 rpm for 10 mins after which serum was obtained. 2.4. IMMUNOSTAINING
Immunostaining was performed based on the standard
procedure with some modifications. Zivic Rat Brain
Slicers were used to cut the brain into slices. After fixing the cerebrum
tissues (DLPFC) (56oC, 1 night), they were deparaffinized with
xylene and then rehydrated in ethanol of various percentages. They were then immersed
in fresh and ice-cold methanol containing 1% H2O2forabout
20 mins to eliminate the effect of endogenous peroxidase. Samples rinsed with
PBS were incubated in blocking buffer (PBS containing 3% BSA, 0,1% Tween-20) at
25oC [25]. Samples
were incubated with anti-Bax (Abcam, cat: ab53154)
and anti-TNF-α (Abcam, cat: ab6671) antibodies (Millipore, cat: S7101) in a
blocking solution for 1/2 day at 4°C and washed with PBS (25oC).
This was then followed by incubation with a horseradish peroxidase (HRP)
antibody (Abcam) (1:2500) in blocking solution for 120 minutes at 25oC.
After washing with PBS, the samples were incubated with 0,2 3, 3′%-diaminobenzidine
(DAB), and then later rinsed with distilled water. All samples were
counterstained with hematoxylin. Immunoreactivity
between groups was examined under a fluorescent microscope with a camera
(Olympus-DP70 camera). Apoptotic cells were detected according to the apoptotic detection kit guide, (Millipore, Cat No: S7101). After the sections had been sliced at 5 µm thicknesses, they were treated with 20 µg/ml proteinase K in 0.1 mol/l Tris–HCl buffer (pH 7.4) for 15 minutes. They were then incubated with 100 µL equilibration buffer at 25oC for 15 min and later with rTdT incubation buffer (45 µL equilibration buffer, 5 µL nucleotide mix, 1 µL rTdT enzyme) at 37oC-60 minutes in a humidified chamber. The reaction was completed by treating the samples with 2 x SSC for 15 minutes at 25oC. The samples were stained with 1 µg/ml propidium iodide for analyzing in a fluorescence microscope (620nm). 500 healthy cells were examined in a fluorescence microscope and those that had a red appearance were considered TUNEL positive [26]. 3. RESULTS AND DISCUSSIONSThis study
has described how brain damage due to TAA-induced liver depredation was treated
by100mg/kg Sm. It was observed that apoptosis (Bax,
TUNEL) and inflammation (TNF-α) reduced in the TAA group depending on the
administration of Sm treatment. However, Bax, TUNEL, and TNF-α expression levels did not reduce
in the 50 mg/kg Sm+TAA group (Figures 1-3). The Bcl-2
family (Bcl-2, Bax, Bim,
Bid, Bak, and Bcl-xL) plays
an important role in initiating an intrinsic apoptotic pathway [26], [27], [28], [29], [30]. Bax, a
pro-apoptotic member of the family, promotes cell death via permeabilization of
the mitochondrial outer membrane. In contrast, Bcl-2 (an anti-apoptotic member)
prevents apoptosis by blocking Bax activity [28], [29]. The balance between Bax
and Bcl-2 can determine cellular fate. Mutagenic chemicals such as TAA trigger
apoptosis, with some studies, successfully demonstrating increased expression
of Bax and decreased Bcl-2 expression following
TAA-induced hepatotoxicity [24]. Brain damage caused by TAA has also been demonstrated
[30], [31], [32], [33], [34], [35]. Another study led by El-Ghazaly
established that when TAA-induced Wistar HE rats are administrated local or
whole-body low dose γ radiation (0.5 Gy),
increased caspase-3 expression level occurs in the brain immunochemically [32]. Khanna and Trigun
(2016) found that 100mg/kg TAA in Wistar rats significantly increased the level
of cerebrum Bax expression [33]. Tumour
necrosis factor-alpha (TNF-α), a cytokine, plays a role in inflammation
and is responsible for the process which results in cell death. As a result of
excessive oxidative stress, neuronal apoptosis begins depending on
mitochondrial dysfunction or TNF family receptors activation [34]. El-Marasy et al.
(2018) showed that the TNF-α content in brain tissue was enhanced 1.46-fold
compared to normal rats in TAA (100 mg/kg) induced-Wistar HE rats. They also
noted that plasma TNF-α levels were significantly increased in Wistar rats
given 150 mg/kg TAA when compared to the control group [36]. Terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method used for apoptosis detection
and it detects DNA fragmentation by labeling the
3′- hydroxyl terminal in dsDNA [37]. Bustamante and colleagues found
that the apoptosis rate in a rat MHE (minimal hepatic encephalopathy) model performed
by surgery increased by 2.3 times compared to the control group [37]. Several
studies have been conducted on the neuroprotective effect of Sm. Haddadi et al.
treated neurotoxicity induced by 6-hydroxydopamine (6-OHDA) in Wistar rats with
Sm daily, 100, 200, and 300 mg/kg injected into SNc (substantia nigra pars
compacta) [26]. As a consequence, the Bcl-2 level increased
while the Bax and TUNEL levels decreased. However, the caspase-3 level did not change significantly
in any group. Our experimental results show that Silymarin may have
neuroprotective effects on neurotoxicity caused by hepatic encephalopathy. Figure 1: Bax
immunoreactivity in neurons in brain cortex sections of rats belonging to
experimental groups. Bax immunoreactivity is
indicated by a neuron that reacts positively (arrow). A: Weak reaction in
neurons in the control group.B:
Strong positive reaction in the TAA group.C: Weak
reaction in the TAA + low dose Sm group and D: Weak
reaction in the TAA + high dose Sm group. All bars
are 100 μm. Figure 2: TUNEL
immunoreactivity in neurons in the brain cortex sections of rats belonging to
experimental groups. Some of the neurons that reacted positively are indicated
by (arrows). A: Weak reaction in neurons in the control group.B: Strong positive reaction in the TAA treated
group. C: Moderate reaction in the TAA + low dose Sm
group and D: Weak reaction in the TAA + high dose Sm
group. All bars are 100 μm. Figure 3: TNF-α immunoreactivity in neurons in brain cortex sections of rats
belonging to experimental groups. Some of the neurons that reacted positively
are indicated by (arrows). A: Weak reaction in neurons in the control group. B:
Strong positive reaction in the TAA treated group. C: Moderate reaction in the
TAA + low dose Sm group and D: Weak reaction in the
TAA + high dose Sm group. All bars are 100 μm. 4. CONCLUSIONSilymarin
is an extremely good hepatoprotective agent. For this reason, it has the
potential to be used as a protective agent in hepatic encephalopathy, which
causes both liver and brain damage. Our experimental findings support this
hypothesis. However, since the bioavailability of Sm
is rather low, we propose two different strategies to increase its
effectiveness. First, the active ingredients of Sm
should be investigated more comprehensively, considering synergistic and
antagonistic effects. Secondly, new materials containing Sm
should be prepared to increase the bioavailability of Sm,
such as sugar-coated tablets, its own micro emulsifying drug delivery system
(SMEDDS), or beta-cyclodextrin inclusion. SOURCES OF FUNDINGThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. CONFLICT OF INTERESTThe author have declared that no competing interests exist. ACKNOWLEDGMENTThis study (coded
2017-1621) from Özgün Teksoy's
doctoral dissertation was supported by Eskişehir
Osmangazi University. REFERENCES
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