2005 Abstracts: The Role of P65 Nf-kB/ReIA in Pancreatitis-Induced Kupffer Cell Apoptosis
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The Role of P65 Nf-kB/ReIA in Pancreatitis-Induced Kupffer Cell Apoptosis
Yanhua Peng, Scott F. Gallagher, University of South Florida Health Science Center, Tampa, FL; Regine Landman, University Hospital Basel Switzerland, Tampa, FL; Krista Haines, Michel Murr, University of South Florida Health Science Center, Tampa, FL
Introduction: Acute pancreatitis (AP) induces liver injury by upregulating Kupffer cell-derived cytokine production and Fas/FasL. AP also induces apoptosis of Kupffer cells via NF-κB-dependant upregulation of Fas/FasL. This balance between upregulation of Fas/FasL and Fas/FasL-induced apoptosis of its originator cell may determine the severity of organ injury.
Aim: To determine the role of p65 NF-κB/RelA in pancreatitis-induced Kupffer cell apoptosis Methods: AP was induced in c57BL mice by CDE diet (n=4). Mouse Kupffer cell line clone3 (MKC) was transfected with p65 siRNA to inhibit p65 NF-κB/RelA expression and block NF-κB function, or transfected with control siRNA, and treated with pancreatic elastase to mimic pancreatitis. Total p65 NF-κB/RelA, Fas/FasL and activated caspase-3 (immunoblot), nuclear translocation of p65 NF-κB/RelA (ELISA) and DNA fragmentation were determined. Gels were quantified by densitometry. Experiments were in triplicates. Data are mean±SEM. Results: CDE pancreatitis upregulated nuclear translocation of p65 NF-κB/RelA, Fas/FasL, Caspase-3 and DNA fragmentation in mice livers (*all p<0.001).In-vitro, pancreatic elastase mimicked CDE-pancreatitis by upregulating nuclear translocation of p65 NF-κB/RelA, Fas/FasL, Caspase-3 and DNA fragmentation in Kupffer cells (§ all p<0.001 vs. control). Transfection with p65 siRNA blocked p65 NF-κB/RelA expression and attenuated the elastase-induced nuclear translocation of p65 NF-κB/RelA and upregulation of Fas/FasL, caspase-3 and DNA fragmentation in Kupffer cells (¶ all p<0.001 vs. control siRNA+E). Conclusion: Acute pancreatitis activates p65 NF-κB/RelA and induces apoptosis. Inhibition of p65NF-κB/RelA attenuates elastase-induced upregulation of pro-apoptotic death receptor/ligand pathways and apoptosis. The ability of Kupffer cells to autoregulate their stress response by inducing self apoptosis may have important implications in the pathophysiology of organ injury.| p65 NF-κB expression | p65 NF-κB nuclear trans. | Fas | FasL | Caspase-3 | DNA frag. (%) | |
| Mice | 3301±152 | 10±0.47 | 1025±44 | 834±29 | 1254±44 | 7±1 |
| Mice+ CDE | 3204±160 | 32±2* | 4824±207* | 4024±170* | 3201±144* | 39±2* |
| MKC (Mouse Kupffer cell) | 3408±153 | 22±1 | 575±27 | 721±30 | 964±43 | 5±1 |
| MKC+ Elastase (E) | 3391±139 | 77±4§ | 2889±105§ | 3821±164§ | 3327±149§ | 36±2§ |
| MKC+ control siRNA | 3478±146 | 29±1 | 1235±58 | 1159±48 | 1357±61 | 8±1 |
| MKC+ p65 siRNA | 1105±55 | 10±1 | 848±35 | 742±30 | 1023±48 | 5±1 |
| MKC+ control siRNA + E | 3305±125 | 85±4 | 4980±209 | 4321±133 | 4503±202 | 41±2 |
| MKC+ p65 siRNA +E | 1098±43¶ | 45±3¶ | 2505±100¶ | 2401±80¶ | 2541±106¶ | 29±1¶ |
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