Sains Malaysiana 45(3)(2016): 451–458
Hydrogen Peroxide Induces Acute
Injury and Up-regulates Inflammatory Gene Expression in Hepatocytes:
An in vitro Model
(Hidrogen Peroksida Mengaruh Kecederaan
Akut dan Mengatur Naik Ekspresi Gen Inflamasi dalam Hepatosit:
Suatu Model in vitro)
LING LING
LIAU1,
MAKPOL
SUZANA2,
ABDUL
GHANI
NUR
AZURAH3
& KIEN HUI CHUA1*
1Department
of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia,
Jalan Yaacob Latif, 56000 Kuala Lumpur, Malaysia
2Department of Biochemistry,
Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob
Latif, 56000 Kuala Lumpur, Malaysia
3Department of Obstetrics
and Gynaecology, Faculty of Medicine, Universiti Kebangsaan Malaysia
Jalan
Yaacob Latif, 56000 Kuala Lumpur, Malaysia
Diserahkan: 19
April 2015/Diterima: 28 September 2015
ABSTRACT
In the past, many in
vitro hepatocyte injury models developed for liver regeneration
used carbon tetrachloride as irritant chemical. Recently, carbon
tetrachloride usage was prohibited due to serious deleterious
effects to human and environment. There is an urgent need to develop
a new acute chemical-induced hepatocyte injury model using other
chemical compound to replace carbon tetrachloride. In this study,
we used hydrogen peroxide (H2O2)
to induced hepatocyte injury with HepG2 as the liver
cell model. HepG2 injury was established by exposing
the cells to CC50 of
H2O2 at
the concentration of 2.4 mM, predetermined via MTT assay
for 2 h exposure. Aspartate aminotransferase (AST)
activity was measured to determine the extent of cellular injury
and quantitative PCR was carried out to determine the expression of inflammatory
genes of the cells 24 h after H2O2 exposure.
The results showed that AST activity increased with time and
peak at 24 h after H2O2 exposure.
Quantitative PCR analysis demonstrated that expression of inflammatory
genes (TGF-β1, MMP-3, NF-κβ,
IL-8
and IL-6) increased significantly. In addition, the gene expression
of GPX, an anti-oxidant enzyme was also increased significantly
in response to oxidative stress. In summary, H2O2 demonstrated
excellent capability in inducing oxidative injury to HepG2
and together they represent an ideal acute chemical-induced injury
model that can be used for liver regeneration study. Our results
also provide input for inflammatory gene expression in the hepatocyte
injury model.
Keywords: Hepatocytes; H2O2; inflammatory
genes; in vitro; liver injury model
ABSTRAK
Banyak model kecederaan hepatosit
in vitro yang telah dibangunkan untuk kajian regenerasi hepar
pada masa lalu adalah berdasarkan kepada iritasi karbon tetraklorida.
Kebelakangan ini, karbon tetraklorida telah dilarang penggunaaannya
disebabkan ia boleh membawa kemudaratan kepada manusia dan persekitaran.
Maka, satu model kecederaan hepatosit akut aruhan bahan kimia
yang baru untuk menggantikan karbon tetraklorida kepada bahan
kimia lain perlu dibangunkan dengan kadar segera. Dalam kajian
ini, kami menggunakan hidrogen peroksida (H2O2)
untuk mengaruh kecederaan hepatosit dengan menggunakan HepG2
sebagai model sel hepar. Kecederaan HepG2 diaruh dengan
mendedahkan sel kepada CC50 H2O2 pada
kepekatan 2.4 mM yang telah dipratentukan melalui asai MTT selama
2 jam pendedahan. Aktiviti aspartat transaminase (AST)
diukur untuk menentukan tahap kecederaan sel dan PCR kuantitatif
dijalankan untuk menentukan ekspresi gen-gen inflamasi pada masa
24 jam selepas pendedahan kepada H2O2.
Hasil kajian mendapati aktiviti AST meningkat dengan masa dan
mencapai puncak kepekatan pada masa 24 jam selepas pendedahan
kepada H2O2.
PCR
kuantitatif menunjukkan peningkatan ekspresi gen-gen
inflammasi (TGF-β1,
MMP-3,
NF-κβ,
IL-8
and IL-6) secara signifikan. Selain itu, ekspresi
gen GPX, sejenis enzim antioksidan juga meningkat secara signifikan
sebagai respons kepada tekanan oksidatif. Secara kesimpulan, H2O2 menunjukkan
keupayaan yang baik untuk mengaruh kecederaan oksidatif pada HepG2
dan kombinasi H2O2 dengan
HepG2 menghasilkan model kecederaan hepatosit akut
aruhan bahan kimia yang ideal untuk kajian regenerasi hepar. Di
samping itu, hasil kajian ini juga memberi input terhadap ekspresi
gen inflamasi dalam model kecederaan hepatosit.
Kata kunci: Gen inflamasi; hepatosit; H2O2; in vitro; model kecederaan
hepatosit
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*Pengarang untuk surat-menyurat; email:
ckienhui@hotmail.com