Qi-Ping Lu1, Lei Tian. 1. Department of General Surgery, Wuhan General Hospital of Guangzhou Militray Command, Wuhan 430070, Hubei Province, China. luyiping@medmail.com.cn
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.
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.
Authors: C Scaffidi; S Fulda; A Srinivasan; C Friesen; F Li; K J Tomaselli; K M Debatin; P H Krammer; M E Peter Journal: EMBO J Date: 1998-03-16 Impact factor: 11.598
Authors: Andrey Belous; Clayton Knox; Ian B Nicoud; Janene Pierce; Christopher Anderson; C Wright Pinson; Ravi S Chari Journal: Transplantation Date: 2003-05-27 Impact factor: 4.939
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