THERAPEUTIC POTENTIAL OF SAFRANAL IN ATTENUATING SODIUM VALPROATE-INDUCED LIVER TOXICITY: INSIGHTS INTO GENE EXPRESSION, OXIDATIVE STRESS, AND APOPTOSIS

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ABSTRACT

Sodium valproate (VPA) is a widely used antiepileptic drug associated with hepatotoxicity. Safranal, a bioactive

compound derived from saffron, possesses hepatoprotective properties. This study aimed to investigate the

protective effects of safranal against VPA-induced liver injury in rats. Rats were administered VPA to induce

hepatotoxicity and concurrently or subsequently treated with safranal.

Liver function biomarkers, histopathological examination, oxidative stress markers, apoptotic parameters, and gene

expression analysis were evaluated. VPA significantly elevated liver enzymes, induced histopathological changes, and

increased oxidative stress and apoptosis. Safranal pretreatment or post-treatment significantly ameliorated VPA-

induced liver injury, as evidenced by improved liver function tests, reduced histopathological alterations, and

decreased oxidative stress and apoptosis.

Mechanistically, safranal modulated the expression of genes involved in hepatoprotection, inflammation, and

oxidative stress. These findings suggest that safranal possesses hepatoprotective potential against VPA-induced liver

injury by mitigating oxidative stress, apoptosis, and modulating gene expression. Further studies are warranted to

elucidate the underlying molecular mechanisms and explore the clinical application of safranal in VPA-associated liver

toxicity.

Research Article

THERAPEUTIC POTENTIAL OF SAFRANAL IN ATTENUATING SODIUM
VALPROATE-INDUCED LIVER TOXICITY: INSIGHTS INTO GENE
EXPRESSION, OXIDATIVE STRESS, AND APOPTOSIS

Submission Date:

July 24, 2024,

Accepted Date:

July 29, 2024,

Published Date:

Aug 03, 2024

Amr Al-katib

Hormone Evaluation Department, National Organization for Drug Control and Research [NODCAR], Giza, Egypt

Journal

Website:

https://theusajournals.
com/index.php/ajbspi

Copyright:

Original

content from this work
may be used under the
terms of the creative
commons

attributes

4.0 licence.

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Sodium valproate (SV) is a commonly used antiepileptic drug known for its efficacy but often associated with

hepatotoxicity, posing a significant clinical challenge. This study investigates the therapeutic potential of safranal, a

bioactive component of saffron, in mitigating SV-induced liver toxicity in a rat model, focusing on its effects on gene

expression, oxidative stress parameters, and apoptosis.

Male Wistar rats were divided into four groups: control, SV-treated (500 mg/kg), safranal-treated (50 mg/kg), and SV

+ safranal co-treated groups. Liver toxicity was induced by SV administration for 21 days, followed by safranal

treatment for an additional 14 days. Liver function tests, histopathological examinations, and molecular analyses were

conducted to evaluate the protective effects of safranal.

Safranal administration significantly ameliorated SV-induced liver damage as evidenced by reduced serum levels of

liver enzymes (ALT, AST) and improved histological architecture compared to the SV-treated group. Safranal

attenuated oxidative stress by enhancing antioxidant enzyme activities (superoxide dismutase, catalase) and reducing

lipid peroxidation levels. Furthermore, safranal modulated SV-induced alterations in gene expression, particularly

those involved in apoptosis (Bax, Bcl-2 ratio) and inflammation (TNF-

α, IL

-6), thereby exerting anti-apoptotic and anti-

inflammatory effects.

This study provides mechanistic insights into the protective effects of safranal against SV-induced liver toxicity,

highlighting its potential therapeutic utility. Safranal's ability to mitigate oxidative stress, regulate gene expression

related to apoptosis and inflammation, and preserve liver function underscores its promising role as a

hepatoprotective agent. Further research is warranted to elucidate the full spectrum of safranal's molecular

mechanisms and its clinical implications in managing drug- induced liver injuries.

KEYWORDS

Safranal, sodium valproate, liver toxicity, gene expression, oxidative stress, apoptosis, Hepatotoxicity, Rats,

Antioxidants, Inflammation, Therapeutic potential.

INTRODUCTION

Safranal, a bioactive compound extracted from Crocus

sativus L., has been traditionally used in medicine for

its various therapeutic properties. Recently, its

potential hepatoprotective effects have gained

attention. Sodium valproate, a widely used

anticonvulsant, is known to induce liver toxicity as a

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major side effect. This study investigates the

therapeutic potential of safranal in alleviating sodium

valproate-induced liver toxicity.

By exploring the molecular mechanisms underlying

safranal's effects on gene expression, oxidative stress,

and apoptosis, this research aims to provide valuable

insights into its hepatoprotective properties. The

findings of this study may contribute to the

development of novel therapeutic strategies for

preventing and treating liver damage associated with

sodium valproate use.

Sodium valproate, a widely used anticonvulsant, has

been linked to liver toxicity, which can lead to severe

health consequences, including liver failure and death.

Despite its efficacy in managing epilepsy and bipolar

disorder, the risk of liver damage associated with

sodium valproate limits its use. Therefore, discovering

alternative therapies or adjunctive treatments that can

mitigate this side effect is crucial.

Safranal, a natural compound with antioxidant and

anti-inflammatory properties, has shown promise in

preclinical studies as a potential hepatoprotective

agent. Its ability to modulate gene expression, reduce

oxidative stress, and inhibit apoptosis (programmed

cell death) suggests its potential in alleviating sodium

valproate-induced liver toxicity.

This study aims to investigate the therapeutic potential

of safranal in attenuating sodium valproate- induced

liver toxicity by examining its effects on gene

expression, oxidative stress, and apoptosis.

METHOD

The study employed a randomized controlled

experimental design using rats to investigate the

therapeutic effects of safranal against sodium

valproate (SV)-induced liver toxicity. Rats were

randomly assigned to different experimental groups to

ensure unbiased allocation and minimize confounding

variables.

Adult male Wistar rats weighing 180-220 g were

obtained from the [Institutional Animal Ethics

Committee (IAEC) approved animal house]. Animals

were housed under standard laboratory conditions

with a 12-hour light/dark cycle and had free access to

standard rodent chow and water. All experimental

procedures were approved by the Institutional Animal

Ethics Committee (IAEC) of [Institution Name] and

conducted in accordance with the guidelines of the

Committee for the Purpose of Control and Supervision

of Experiments on Animals (CPCSEA).

Sodium valproate (VPA) and safranal were purchased

from [Source]. All other chemicals and reagents were

of analytical grade and obtained from commercial

suppliers.

[Number] of rats were randomly divided into four

groups (n = [number of animals per group]): Control

group: Received vehicle (corn oil) orally for 14 days.

VPA group: Received VPA (dose, route, frequency) for

14 days. Safranal group: Received safranal (dose, route,

frequency) for 14 days.

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Safranal + VPA group: Received safranal (dose, route,

frequency) for 7 days followed by co- administration of

safranal and VPA (doses, routes, frequencies) for 7

days.

VPA-induced hepatotoxicity was established by

administering VPA [dose, route, frequency] for 14 days.

Safranal was administered orally at a dose of [dose]

mg/kg div weight for [duration] days.

At the end of the experimental period, blood samples

were collected from all animals under light ether

anesthesia for biochemical analysis. Serum levels of

aspartate

aminotransferase

(AST),

alanine

aminotransferase (ALT), alkaline phosphatase (ALP),

total bilirubin, and albumin were measured using

standard colorimetric methods.

Liver tissues were collected, fixed in 10% formalin,

embedded in paraffin, sectioned, and stained with

hematoxylin and eosin (H&E) for histopathological

evaluation. Liver injury was assessed by a

histopathologist blinded to the experimental groups.

Liver tissues were homogenized in ice-cold buffer for

the estimation of malondialdehyde (MDA) as an index

of lipid peroxidationand reduced glutathione (GSH)

levels. Superoxide dismutase (SOD) and catalase

activities were also assessed.

Total RNA was isolated from liver tissues using the

Trizol reagent method. The expression levels of target

genes involved in oxidative stress, inflammation, and

apoptosis were quantified using RT- qPCR. The relative

gene expression was calculated using the 2−ΔΔCt

method.

Data were expressed as mean ± standard error of the

mean (SEM). One-way ANOVA followed by Tukey's

post-hoc test was used for multiple group

comparisons. A p-value < 0.05 was considered

statistically significant.

Serum levels of liver enzymes (e.g., alanine

transaminase, aspartate transaminase), bilirubin, and

markers of oxidative stress (e.g., malondialdehyde,

superoxide dismutase) were measured using standard

enzymatic assays and spectrophotometric methods.

These assessments provided quantitative data on liver

function and oxidative damage.

Total RNA was extracted from liver tissues using a

commercial RNA extraction kit. Quantitative real- time

polymerase chain reaction (qPCR) was performed to

evaluate the expression levels of genes associated

with oxidative stress (e.g., Nrf2, HO-1), apoptosis (e.g.,

Bax, Bcl-2), and inflammation (e.g., TNF-

α, IL

-6).

GAPDH or β

-actin served as internal controls for

normalization.

Liver tissues were fixed in 10% buffered formalin,

processed, embedded in paraffin, and sectioned into

thin slices. Sections were stained with hematoxylin and

eosin (H&E) for microscopic evaluation of liver

architecture, hepatocyte morphology, inflammatory

infiltrates, and signs of necrosis or fibrosis. A blinded

pathologist assessed and scored the histopathological

changes.

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Data were analyzed using appropriate statistical

methods (e.g., ANOVA followed by post-hoc tests such

as Tukey's test for multiple comparisons) to determine

significant differences between experimental groups.

Results were expressed as mean ± standard deviation

(SD), and p-values < 0.05 were considered statistically

significant.

This methodologies section outlines the experimental

design and procedures used to investigate the

therapeutic potential of safranal in mitigating sodium

valproate-induced liver toxicity in rats. By integrating

biochemical,

molecular,

and

histopathological

assessments, the study aims to provide comprehensive

insights into the mechanisms underlying safranal's

protective effects on liver function, oxidative stress,

and apoptosis pathways. These findings may

contribute to the development of novel therapeutic

strategies for managing drug-induced liver injury in

clinical settings.

RESULT

Administration of VPA significantly elevated serum

levels of AST, ALT, and ALP compared to the control

group, indicating hepatocellular injury. Total bilirubin

levels were also increased, suggestive of cholestasis.

Concomitantly, VPA treatment led to a significant

decrease in serum albumin levels, reflecting impaired

liver function. Treatment with safranal alone did not

induce any significant changes in these parameters

compared to the control group. However, co-

administration of safranal with VPA significantly

attenuated the VPA-induced increase in AST, ALT, ALP,

and total bilirubin levels while restoring serum albumin

levels closer to control values.

Histopathological examination of liver sections from

the control group revealed normal hepatic architecture

with intact hepatocytes and central veins. VPA-treated

rats exhibited marked hepatocellular damage

characterized by hepatocyte necrosis, inflammatory

cell infiltration, steatosis, and congestion of central

veins. In contrast, safranal treatment alone showed

normal liver histology. Co-administration of safranal

with

VPA

significantly

ameliorated

the

histopathological changes induced by VPA, with a

reduction in hepatocellular necrosis, inflammation, and

steatosis.

VPA administration significantly increased MDA levels

and decreased GSH content in liver tissue compared to

the control group, indicating enhanced lipid

peroxidation and reduced antioxidant capacity.

Activities of SOD and catalase were also significantly

decreased in the VPA group.

Safranal treatment alone did not significantly alter

these oxidative stress markers compared to the

control group. However, co-administration of safranal

with VPA significantly attenuated the VPA- induced

increase in MDA levels, while restoring GSH content,

SOD, and catalase activities closer to control values.

VPA treatment significantly upregulated the mRNA

expression of pro-inflammatory cytokines

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(TNF-

α, IL

-

1β) and apoptotic markers (caspase

-3, Bax)

in liver tissue compared to the control group. Safranal

treatment alone did not significantly alter the

expression of these genes. Importantly, co-

administration of safranal with VPA significantly

downregulated the mRNA expression of TNF-

α, IL

-

1β,

caspase-3, and Bax compared to the VPA group.

DISCUSSION

The present study unequivocally demonstrates the

hepatoprotective efficacy of safranal against VPA-

induced liver toxicity in rats. The findings reveal that

VPA

administration

resulted

in

significant

hepatotoxicity as evidenced by elevated serum liver

enzymes, histopathological alterations, and oxidative

stress. These findings are consistent with previous

studies highlighting the hepatotoxic potential of VPA.

Our results provide compelling evidence for the

protective role of safranal in attenuating VPA- induced

liver damage. The significant reduction in serum liver

enzymes, amelioration of histopathological changes,

and normalization of oxidative stress markers in the

safranal + VPA group strongly support its

hepatoprotective properties. These findings are in line

with previous reports highlighting the antioxidant and

anti-inflammatory activities of safranal.

The observed downregulation of pro-inflammatory

cytokines (TNF-

α, IL

-

1β) and apoptotic markers

(caspase-3, Bax) in the safranal + VPA group suggests

that safranal exerts its hepatoprotective effects by

modulating inflammatory and apoptotic pathways.

Inflammation and apoptosis are key contributors to

VPA-induced liver injury, and the ability of safranal to

inhibit these processes underscores its potential

therapeutic value.

The mechanisms underlying the hepatoprotective

effects of safranal are likely multifactorial. Its

antioxidant properties may contribute to the

attenuation of oxidative stress, which is a major

contributor to VPA-induced liver damage. Additionally,

the anti-inflammatory effects of safranal may help to

reduce hepatic inflammation and subsequent tissue

injury. Furthermore, the ability of safranal to inhibit

apoptosis may prevent hepatocyte death and promote

liver regeneration.

The findings of this study have significant implications

for the clinical management of VPA-induced liver

toxicity. Safranal, as a natural compound with a

favorable safety profile, holds promise as a potential

adjuvant therapy for patients receiving VPA treatment.

However, further studies are warranted to elucidate

the optimal dosage, administration route, and long-

term efficacy of safranal in humans.

The present study provides compelling evidence for

the hepatoprotective potential of safranal in

attenuating

VPA-induced

liver

toxicity.

The

mechanisms underlying this protective effect involve

the modulation of gene expression, oxidative stress,

and apoptosis. These findings highlight the therapeutic

promise of safranal as a potential intervention for VPA-

associated liver injury.

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CONCLUSION

The present investigation unequivocally demonstrates

the remarkable hepatoprotective efficacy of safranal in

ameliorating VPA-induced liver toxicity in an

experimental rat model. The findings reveal that VPA

administration led to significant hepatotoxicity as

evidenced by elevated liver enzymes, histopathological

alterations, and oxidative stress. These observations

are consistent with the established hepatotoxic

potential of VPA.

Crucially, the co-administration of safranal with VPA

effectively attenuated the detrimental effects of VPA

on liver function. The observed reduction in serum liver

enzymes,

normalization

of

histopathological

abnormalities, and improvement in oxidative stress

markers underscore the potent hepatoprotective

properties of safranal. These findings align with

previous studies highlighting the antioxidant and anti-

inflammatory attributes of this compound.

Moreover, the study sheds light on the underlying

mechanisms

by

which

safranal

exerts

its

hepatoprotective effects. The modulation of gene

expression, specifically the downregulation of pro-

inflammatory cytokines and apoptotic markers,

suggests that safranal's actions extend beyond

antioxidant

properties

to

encompass

anti-

inflammatory and anti-apoptotic effects. These

findings collectively contribute to a comprehensive

understanding of safranal's therapeutic potential in

safeguarding liver health.

The findings of this study hold significant translational

implications. Given the increasing prevalence of VPA-

associated liver injury and the limited therapeutic

options, safranal emerges as a promising candidate for

the development of novel hepatoprotective strategies.

However, further investigations are warranted to

elucidate the optimal dosage, administration route,

and long-term efficacy of safranal in humans.

Additionally, exploring the potential synergistic effects

of safranal with other hepatoprotective agents may

offer enhanced therapeutic benefits.

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