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

 

Received: 19 April 2015/Accepted: 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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*Corresponding author; email: ckienhui@hotmail.com

 

 

 

 

 

 

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