Basic Research
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 28, 2005; 11(4): 534-537
Published online Jan 28, 2005. doi: 10.3748/wjg.v11.i4.534
Fas mRNA expression and calcium influx change in H2O2-induced apoptotic hepatocytes in vitro
Qi-Ping Lu, Lei Tian
Qi-Ping Lu, Lei Tian, Department of General Surgery, Wuhan General Hospital of Guangzhou Military Command, Wuhan 430070, Hubei Province, China
Author contributions: All authors contributed equally to the work.
Supported by National Natural Science Fundation of China, No.39770938; and Whole Army Medical Scientific Research Task during the Fifteen the Five-year Plan, No. 01MA040
Correspondence to: Dr. Qi-Ping Lu, Department of General Surgery, Wuhan General Hospital of Guangzhou Militray Command, No 627 Wuluo Street, Wuhan 430070, Hubei Province, China. luyiping@medmail.com.cn
Telephone: +86-27-68878501 Fax: +86-27-68878108
Received: February 11, 2004
Revised: February 15, 2004
Accepted: March 2, 2004
Published online: January 28, 2005
Abstract

AIM: To investigate the relationship between Fas gene expression and calcium influx change in peroxide-induced apoptotic hepatocytes and the possible molecular mechanism of Rxa in protecting hepatocytes.

METHODS: Single-cell Fas mRNA expression in H2O2-exposed L02 hepatocytes with or without treatment of Rxa, an extract from an anti-peroxidant, Radix Salviae Miltiorrhizae, was determined by all-cell patch clamp and single-cell reverse transcriptase polymerase chain reaction (RT-PCR). Transient calcium influx change ([Ca2+]i) in the cells was evaluated with all-cell patch clamp micro-fluorescence single-cytosolic free Ca2+ concentration technique. Fas protein expression, early apoptotic index (annexin-V+) and cell membrane change in the cells were evaluated by immunohistochemistry, flow cytometry (FCM) and scan electron microscopy respectively.

RESULTS: In cells exposed to H2O2 for 2 h, the specific lane for Fas mRNA was vivid on electrophoresis, with increased Fas protein expression, [Ca2+]i (from 143.66±34.21 to 1115.28±227.16), annexin-V+ index (from 4.00±0.79 to 16.18±0.72) and membrane vesicle formation. However, in cells exposed to H2O2 but pre-treated with Rxa, there was no increase in Fas mRNA or protein expression and [Ca2+]i (103.56±28.92). Annexin-V+ index (8.92±1.44) was lower than the controls (P<0.01), and the cell membrane was intact.

CONCLUSION: H2O2 induces apoptosis of L02 cells by increasing cytosolic [Ca2+]i, and inducing Fas mRNA and protein expression. Rxa protects the L02 cells from apoptosis through anti-peroxidation, inhibition of calcium overloading and prevention of the activation of cytosolic Fas signal pathway.

Keywords: Hepatocytes, Apoptosis, Fas protein, Gene expression, Calcium