The accumulation of
glycochenodeoxycholate (GCDC) induced hepatocyte apoptosis in
cholestasis. However, many hepatocytes still survived GCDC-induced apoptosis. The molecular mechanism for the survival of hepatocytes remains unclear. In the present study, isolated rat hepatocytes were cultured in William's E medium and treated with 50 microM GCDC.
DNA,
RNA, cell lysate, and
nuclear proteins were collected at different intervals for DNA fragmentation assay, reverse transcription PCR, Western blotting, and gel mobility shift assay, respectively. GCDC-induced active
caspases were detected as early
as 2 h by Western blotting and kinetic
caspase assay, whereas hepatocyte apoptosis was found at 4 h by DNA fragmentation and
terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling assay. When GCDC was removed, the increased
caspases as well as
NF-kappaB could be restored to control level. A1/
Bfl-1 and
inducible nitric oxide synthase (iNOS) were up-regulated in 2 h of GCDC stimulation. After GCDC was removed, hepatocytes decreased expression of A1/
Bfl-1, but not iNOS, to the control level.
NF-kappaB activation coincided with the change of A1/
Bfl-1.
Survivin, cIAP1, cIAP2, XIAP, and A1/
Bfl-1, but not iNOS, were down-regulated by pan-
caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl
ketone. In addition, benzyloxycarbonyl-VAD-fluoromethyl
ketone inhibited release of
cytochrome c and suppressed
NF-kappaB activation. Our data suggested that
caspase pathway is an important regulatory factor during hepatocyte apoptosis. GCDC-induced
caspase response is reversible, which may activate anti-apoptotic genes to protect hepatocytes from apoptosis.