NFV reduces active caspase 3 staining and cytochrome c (Cyto.c) leakage into the cytosol during pancreatitis. Pancreas was harvested 1 h after the last of a series of 12 hourly intraperitoneal injections of saline (A), 50 μg/kg caerulein (B), animals pretreated with water before administration of caerulein (C), and animals pretreated with NFV/ritonovir (RTV) (N/R; D) or Z-VAD-fmk (ZVAD; E) before administration of caerulein. These were fixed and stained for active caspase-3 and accessed as described in materials and methods. Representative ×400 images are shown. F: % active caspase-3 positive cells expressed as means ± SE. Immunoblots of cytoplasmic fractions (G) and mitochondrial fractions (H) blotted for cytochrome c (top) and then stripped and blotted for HSP70 (G, middle and H, bottom) which was used a control for lane loading of the mitochondrial fractions. Since there was no leakage of heat shock protein (HSP)70 into the cytosolic fractions, PCNA was used as a control for lane loading of the cytosolic fractions as described in materials and methods (G, bottom). Each column represents pancreatic protein amounts in the respective fractions of pancreatic tissue after the different treatments. The 4 different treatments were control animals, 3-h caerulein pancreatitis, 3-h pancreatitis prophylaxed with vehicle, and 3-h pancreatitis prophylaxed with NFV/RTV.

NFV/RTV reduce serum amylase without affecting the increase in trypsin, pancreatic water content, or neutrophil infiltration. Serum amylase (A), pancreatic water content (B), or pancreatic MPO levels (C) were measured in control animals or those which received 50 μg/kg caerulein intraperitoneal every hour for 12 h, caerulein after pretreatment with vehicle (Veh) before the induction of pancreatitis, or pretreatment with Z-VAD-fmk NFV/RTV. *P value <0.05 compared with vehicle-treated group. D: intrapancreatic trypsin activity measured in control mice or 30 min after receiving 50 μg/kg ip caerulein, or those pretreated with vehicle, Z-VAD-fmk, or NFV/RTV before the induction of pancreatitis. *P value <0.05 compared with vehicle-treated group.

Nelfinavir (NFV) stabilizes mitochondrial membrane potential in acinar cells. Acini were preincubated with saline (CON) or 30 micromolar NFV for 30 min (solid bars) followed by addition of 10 nM caerulein (Cer) for 3 h as indicated. 3, 3′-Dihexyloxacarbocyanine (DiOC6) retention was measured as described in materials and methods and expressed as means ± SE from 3 different experiments. *P value <0.05 compared with vehicle-treated group. There was no difference induced by vehicle treatment compared with caerulein alone.

Terminal deoxynucleotide dUTP transferase nick-end labeling (TUNEL) staining and histology [hematoxylin and eosin (H&E)] showing pancreatic acinar apoptosis and injury are reduced by NFV pretreatment. Pancreas was harvested 1 h after the last of a series of 12 hourly intraperitoneal injections of saline (A), 50 μg/kg caerulein (B), animals pretreated with water before administration of caerulein (C), and animals pretreated with Z-VAD-fmk (D) or NFV/RTV (E) before administration of caerulein. These were fixed and stained for TUNEL as described in materials and methods. F: quantitation of these apoptotic nuclei depicted as means ± SE. G–L: acinor injury as determined by H & E staining as indicated. *Significant (P < 0.05) reduction compared with untreated caerulein- or vehicle-treated groups.

Lung inflammation as seen on histology and neutrophil infiltration as measured by MPO are unaffected by NFV/RTV. Representative ×250 images of lungs harvested, fixed, and stained with H&E 1 h after the last of a series of 12 hourly intraperitoneal injections of saline (A), 50 μg/kg caerulein (B), animals pretreated with vehicle before administration of caerulein (C), and animals pretreated with NFV/RTV (D) or Z-VAD-fmk (E) before administration of caerulein. F: bar graph of MPO activity per mg protein. The different groups are saline (CON), 50 μg/kg caerulein, animals pretreated with vehicle before administration of caerulein, and animals pretreated with Z-VAD-fmk or NFV/RTV before administration of caerulein.