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

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.