Liver Injury during Acute Pancreatitis: The Role of Pancreatitis-Associated Ascitic Fluid (PAAF), P38-MAPK and Caspase-3 in Hepatocyte Apoptosis
Abstracts
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Background: Liver injury is a clinical prognostic indicator in acute pancreatitis. We have demonstrated that pancreatitis-associated ascitic fluid (PAAF) contributes to liver injury and hepatocyte death during acute pancreatitis; a phenomenon independent of PAAF's enzymatic content and Kupffer cell-derived cytokines. Aim: To characterize the mechanisms of PAAF-induced hepatocyte death. Methods: Pathogen-free PAAF was obtained from rats with acute pancreatitis induced by intraductal injection of Na-taurocholate. NIH mice were injected intraperitoneally with 50uL/g of PAAF: 24 hr later, serum AST, ALT, LDH and apoptosis in the liver (TUNEL) were measured (n=5 each). Hepatocytes from a human cell line (CCL-13) were cultured and treated with increasing doses of PAAF (6-50% v/v): Apoptosis was measured by flow cytometery after dual labeling with Annexin-V/7-AAD, and confirmed by TUNEL stain. Phosphorylation of P38-MAPK and cleavage of Caspase-3 (western) were quantitated using densitometry. Data are mean?SEM of triplicates. Results: In-vivo, PAAF increased serum AST (338?49 vs 86?9), ALT (292?8 vs 40?2), LDH (12101?1658 vs 1842?76), and apoptotic cells (36?5 vs 18?2/hpf, TUNEL), all p <0.03 vs sham. In-vitro, PAAF induced a time and dose dependant increase in dual labeled CCL-13 cells by flow cytometry that peaked at 3hr and 20% v/v (data not shown, p<0.01, vs control), and increased apoptotic cells from 9.8?0.4 to 22.3?0.8/hpf (TUNEL, p<0.001 vs control). PAAF increased phosphorylated P38-MAPK (744?46 vs 436?40; p<0.02 vs control) and Caspase-3 cleavage (88?6 vs 62?2; p=0.03 vs control). In addition, PAAF increased Caspase-3 activity (69?5 vs 12 ?0.8; p<0.001 vs control, immunofluorescence: CaspaTag), and decreased mitochondrial DioC6 staining (11?0.3 vs 27?0.1; p<0.01 vs control). SB203580 (10uM) decreased apoptotic cells by 20% from baseline (flow cyto: p<0.001) but did not affect DioC6 staining. On the other hand, Caspase-3 inhibitor II, attenuated the PAAF-induced mitochondrial damage, thereby increasing DioC6 staining (44?4 vs 21?4; p=0.05 vs PAAF). Conclusion: PAAF induces liver injury during acute pancreatitis by activating P38-MAPK and Caspase-3-dependant pro-apoptotic pathways. These data suggest that P38-MAPK and Caspase-3 may be divergent since inhibiting P38-MAPK reduces apoptosis independent of mitochondrial injury. |
