Fas mRNA expression and calcium influx change in H2O2-induced apoptotic hepatocytes in vitro.
OBJECTIVE
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.


CONCLUSIONS
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.
Lu
Qi-Ping
QP
Department of General Surgery, Wuhan General Hospital of Guangzhou Militray Command, Wuhan 430070, Hubei Province, China. luyiping@medmail.com.cn
Tian
Lei
L
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D022781:Hepatocytes / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008854:Microscopy, Electron; D016877:Oxidants / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
D017304:Annexin A5; D016877:Oxidants; D012333:RNA, Messenger; D019014:fas Receptor; D006861:Hydrogen Peroxide; D002118:Calcium