Measuring Autophagy in Pancreatitis.

Acute pancreatitis is one of the first pathological processes where autophagy has been described in a human tissue. Autophagy, autodigestion, and cell death are early cellular events in acute pancreatitis. Recent advances in understanding autophagy highlight its importance in pathological conditions. However, methods for monitoring autophagic activity during complex diseases, involving highly differentiated secretory cells, are complicated, and the results are sometimes misinterpreted. Here, we describe methods used to identify autophagic structures and to measure autophagic flux in cultured cell models and animal models of pancreatitis. We also briefly describe the pancreas specific autophagy mouse model that was useful to understand the actual role of autophagy in pancreatitis and to identify a novel selective autophagy pathway named zymophagy. Lastly, we describe the immunomagnetic isolation of autophagosomes and the detection of autophagy in pancreatic tissue samples derived from humans.

1,8-cineole (eucalyptol) ameliorates cerulein-induced acute pancreatitis via modulation of cytokines, oxidative stress and NF-κB activity in mice.

AIMS: Acute pancreatitis (AP) is an inflammatory condition wherein pro-inflammatory mediators, oxidative stress, and NF-κB signaling play a key role. Currently, no specific therapy exists and treatment is mainly supportive and targeted to prevent local pancreatic injury and systemic inflammatory complications. This study was aimed to examine whether 1,8-cineole, a plant monoterpene with antioxidant and anti-inflammatory properties could ameliorate cerulein-induced acute pancreatitis. MAIN METHODS: AP was induced in Swiss mice by six one hourly injections of cerulein (50 µg/kg, i.p.). 1,8-cineole (100, 200 and 400mg/kg, p.o.) was administered 1h prior to first cerulein injection, keeping vehicle and thalidomide treated groups as controls. Blood samples were taken 6-h later to determine serum levels of amylase and lipase, and cytokines. The pancreas was removed for morphological examination, myeloperoxidase (MPO) and malondialdehyde (MDA) assays, reduced glutathione (GSH) levels, and for nuclear factor (NF)-κB immunostaining. KEY FINDINGS: 1,8-cineole effectively reduced the cerulein-induced histological damage, pancreatic edema and NF-κB expression, levels of MPO activity and MDA, and replenished the GSH depletion. Cerulein increased serum levels of amylase and lipase, and pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 were also decreased by 1,8-cineole pretreatment, similar to thalidomide, a TNF-α inhibitor. The anti-inflammatory IL-10 cytokine level was, however, enhanced by 1,8-cineole. SIGNIFICANCE: These findings indicate that 1,8-cineole can attenuate cerulein-induced AP via an anti-inflammatory mechanism and by combating oxidative stress. Further studies are needed to clearly elucidate its benefits in patients on acute pancreatitis.

The natural flavonoid quercetin ameliorates cerulein-induced acute pancreatitis in mice.

Many plant-derived flavonoids including quercetin exhibit antioxidant and antiinflammatory properties. Proinflammatory cytokines and oxidative stress play an important role in acute pancreatitis. This study aimed to evaluate the effect of quercetin on cerulein-induced acute pancreatitis in mice. Animal groups were pretreated with quercetin (25, 50, 100 mg/kg, per os (p.o.)), thalidomide (200 mg/kg, p.o.) or vehicle (2% dimethyl sulfoxide (DMSO)) 1 h before hourly (x5) intraperitoneal injections of cerulein. A saline (0.9%, NaCl)-treated control group was included for comparison. Cerulein significantly enhanced the serum levels of amylase and lipase, and pancreatic myeloperoxidase activities, malondialdehyde and the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6, as well as the pancreatic wet weight/body weight ratio. Cerulein significantly reduced the serum levels of IL-10. Histological assessment of the pancreas showed tissue edema, neutrophil infiltration, acinar vacuolization, and cell necrosis and a marked increase in the immunoreactivity staining for TNF-alpha. Pretreatment with quercetin or thalidomide significantly attenuated the severity of cerulein-induced acute pancreatitis as evidenced by effective reductions in the pancreatic wet weight/body weight ratio, biochemical indices, proinflammatory cytokines, myeloperoxidase activity, malondialdehyde formation, and an increase in antiinflammatory cytokine IL-10. Quercetin treatment also markedly suppressed the histological changes such as pancreatic edema, inflammatory cell infiltration, acinar cell necrosis, and the expression of TNF-alpha. Taken together, these results indicate that quercetin ameliorates the severity of cerulein-induced acute pancreatitis by acting as an antiinflammatory and antioxidant agent.

Endogenous hypertriglyceridemia intensifies the course of cerulein-induced pancreatitis in rat: relation with changes in the VLDL composition.

AIMS: To study if the course of cerulein-induced pancreatitis in rats changes in a state of triglyceride-rich lipoprotein metabolism alteration. METHODS: Two groups of rats received control diet during a 90-day period (A) and sucrose-rich diet to induce endogenous hypertriglyceridemia (B). Subgroups A2 and B2 received i.p. 45 microg cerulein/kg body weight (to induce acute pancreatitis). Histological examination of pancreas tissue, serum pancreatic lipase, lipoprotein profile and VLDL chemical composition were assessed. Then, pancreatic lipase hydrolytic activity on VLDL-triglycerides was evaluated in vitro. RESULTS: Cellular vacuolization was observed in all of the cerulein-injected rats, but only in subgroup B2 fat necrosis was present. Serum triglycerides were higher in subgroup B1 than in subgroup A1 (mean +/- SEM, mg/dl 123,77 +/- 25.7 vs. 65.8 +/- 7, p < 0.01). Triglycerides from rats fed with sucrose-rich diet, decreased after cerulein-induced pancreatitis (80.38 +/- 11.3 vs. 123,77 +/- 25.7, p < 0.02). Moreover, the endogenous hypertriglyceridemic rats showed an increment of VLDL triglyceride content, which decreased when rats were injected with cerulein. A negative correlation was found between VLDL-triglyceride content and serum pancreatic lipase activity (r = 0.58, p < 0.02). The in vitro assay showed a decrease in VLDL-triglyceride content post incubation with pancreatic lipase enriched serum (mean +/- SD: 59.2 +/- 27.7%, p < 0.01). CONCLUSIONS: The endogenous hypertriglyceridemia intensifies the course of cerulein-induced pancreatitis and it could be related to the decrease in VLDL-triglycerides as a consequence of pancreatic lipase hydrolytic activity.

Cloning of IP15, a pancreatitis-induced gene whose expression inhibits cell growth.

We describe the cloning and expression of the mouse gene interferon-inducible-protein 15 (IP15), whose activation is related to the acute phase of experimental pancreatitis. Analysis of its structure indicates that it encodes a putative transmembrane protein of 137 amino acids. This gene contains a predicted IFN-stimulable-response element. In vivo studies showed that IP15 is strongly activated in pancreas early during caerulein-induced pancreatitis. In situ hybridization of IP15 mRNA showed that its expression is restricted to acinar cells. IP15 was also induced in pancreas under systemic-lipopolysaccharide treatment and in intestine under Salmonella infection. In vitro studies using NIH3T3 fibroblasts showed that IP15 is induced by IFN-alpha. Growth rate was significantly lower in cells transfected with pcDNA4/IP15 plasmid. In addition, cells expressing IP15 showed less capacity to develop colonies after antibiotic selection. In conclusion, we identified a new interferon-inducible gene that is activated early in pancreas with pancreatitis and whose expression inhibits cell growth.

Effect of PAF antagonists on cerulein-induced pancreatitis.

The present study was undertaken to investigate the involvement of PAF in acute pancreatitis induced by cerulein in rats. Cerulein (two doses of 20 micrograms/rat, the first s.c. and the second i.v., 1 h apart) induced a significant increase in vascular permeability in the pancreas, evaluated by the Evans blue (EB) extravasation method. Plasma amylase levels were also significantly increased in this group. The PAF antagonists, BN-52021 (5 mg/kg) and WEB-2170 (1 and 10 mg/kg), both significantly reduced the extravasation of EB in the pancrease induced by i.v. injection of PAF (1 microgram/kg). At these concentrations, BN-52021 was effective at inhibiting cerulein-induced pancreatitis (60-70% of inhibition) whereas WEB-2170 had no significant effect. Although the inhibition induced by BN-52021 suggests the involvement of PAF in cerulein-pancreatitis, the lack of effect of WEB-2170 reported here does not allow a definite conclusion. Further studies are needed to elucidate the differential effect of the PAF antagonists.