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Dissecting the effect of cyclosporine on the sequence of events leading to hepatocellular apoptosis


Gottschalk,  S
Department High-Field Magnetic Resonance, Max Planck Institute for Biological Cybernetics, Max Planck Society;

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Gottschalk, S., Hohnholt M, Raymond VA, Zwingmann, C., & Bilodeau, M. (2008). Dissecting the effect of cyclosporine on the sequence of events leading to hepatocellular apoptosis. Talk presented at Canadian Digestive Diseases Week CASL Winter Meeting (CDDW 2008). Montreal, Canada.

INTRODUCTION: The immunosuppressant cyclosporine (Cyclosporin A, CsA) is known to have the capacity to prevent cellular apoptosis by inhibition of the mitochondrial permeability transition (MPT). We have recently shown that Fas receptor-mediated apoptosis in mouse liver involves early upregulations of specific glucose metabolic pathways and that pre-treatment with CsA prevented these early metabolic events. AIMS: A) In order to further characterize the protective effect of CsA on apoptotic liver injury, we analyzed the sequence of cellular events leading to hepatocyte cell death following anti-Fas injection. B) We then investigated the effects of pre-treatment with CsA on these characteristics that lead to hepatocellular cell death. METHODS: BALB/C mice were injected with anti-Fas antibody (0.5 µg/g, ip). CsA (50 mg/kg; Sandimmune®, ip) was injected 45 min prior to anti-Fas. Animals were sacrificed at five time points from 45min up to 7.5hrs after anti-Fas injection. Standard enzymatic assays were used for serum-ALT/AST and caspase-3 determinations. BID/tBID was analyzed by Western blot and GSH/GSSG by HPLC. RESULTS: A) Anti-Fas-induced liver injury followed a characteristic sequence of cellular events leading to death of the mice at around 8 hrs after anti-Fas injection. First the cleavage of BID was identified 2 hrs after injection, followed by elevation in caspase-3 activity at 3 hrs (26.8±5.3 U, P<0.001 vs. saline-treated controls). Liver cell damage was evident at 5 hrs. At this time serum ALT/AST were increased (7584±2240/5835±1158 U/L) and hepatocyte apoptosis and tissue hemorrhage were identified on histology. Intracellular GSH and GSSG started to decrease at 5 hrs but the ratio of GSH/GSSG remained constant at all time points. B) Pre-treatment with CsA significantly delayed the increase in caspase-3 activity (3 hrs: 7.6±2.1 U, P<0.01 vs. anti-Fas-only; 5 hrs: 43±6.4 U). Serum ALT were also significantly lower at 5 hrs (3100±1104 U/L, P<0.01 vs. anti-Fas-only). Histological evidence of apoptosis was markedly reduced at 7.5hrs. Interestingly, pre-treatment with the vehicle of the CsA formulation, Cremophor® EL (CrEL), offered a very similar protective effect on liver injury. CONCLUSIONS: Our results demonstrate that the protective effect of the CsA formulation occurs at all levels of the injury process. However, they also suggest that the inhibition of hepatocellular apoptosis observed with CsA has to be attributed mainly to the vehicle CrEL.