Polyethylene glycol 35 ameliorates pancreatic inflammatory response in cerulein-induced acute pancreatitis in rats

Abstract

Background: Acute pancreatitis (AP) is a sudden inflammatory process of the pancreas that may also involve surrounding tissues and/or remote organs. Inflammation and parenchymal cell death are common pathological features of this condition and determinants of disease severity. Polyethylene glycols (PEGs) are non-immunogenic, non-toxic water-soluble polymers widely used in biological, chemical, clinical and pharmaceutical settings.

Aim: To evaluate the protective effect of a 35-kDa molecular weight PEG (PEG35) on the pancreatic damage associated to cerulein-induced acute pancreatitis in vivo and in vitro.

Methods: Wistar rats were assigned at random to a control group, a cerulein-induced AP group and a PEG35 treatment group. AP was induced by five hourly intraperitoneal injections of cerulein (50 μg/kg/bw), while the control animals received saline solution. PEG35 was administered intraperitoneally 10 minutes before each cerulein injection in a dose of 10 mg/kg. After AP induction, samples of pancreatic tissue and blood were collected for analysis. AR42J pancreatic acinar cells were treated with increasing concentrations of PEG35 prior to exposure with tumor necrosis factor α (TNFα), staurosporine or cerulein. The severity of AP was determined on the basis of plasma levels of lipase, lactate dehydrogenase activity, pancreatic edema and histological changes. To evaluate the extent of the inflammatory response, the gene expression of inflammation-associated markers was determined in the pancreas and in AR42J-treated cells. Inflammation-induced cell death was also measured in models of in vivo and in vitro pancreatic damage.

Results: Administration of PEG35 significantly improved pancreatic damage through reduction on lipase levels and tissue edema in cerulein-induced AP rats. The increased associated inflammatory response caused by cerulein administration was attenuated by a decrease in the gene expression of inflammation-related cytokines and inducible nitric oxide synthase enzyme in the pancreas. In contrast, pancreatic tissue mRNA expression of interleukin 10 was markedly increased. PEG35 treatment also protected against inflammation-induced cell death by attenuating lactate dehydrogenase activity and modulating the pancreatic levels of apoptosis regulator protein BCL-2 in cerulein hyperstimulated rats. Furthermore, the activation of pro-inflammatory markers and inflammation-induced cell death in pancreatic acinar cells treated with TNFα, cerulein or staurosporine was significantly reduced by PEG35 treatment, in a dose-dependent manner.

Conclusion: PEG35 ameliorates pancreatic damage in cerulein-induced AP and AR42J-treated cells through the attenuation of the inflammatory response and associated cell death. PEG35 may be a valuable option in the management of AP.

Keywords: AR42J cells; Acute pancreatitis; Cell death; Cytokines; Inflammation; Polyethylene glycols.

Figure 1

Effect of PEG35 treatment on plasma lipase activity, pancreatic edema and histological changes in experimental cerulein-induced acute pancreatitis. A: Plasma lipase levels in U/L; B: Pancreatic wet-to-dry weight ratio. Bars represent mean values of each group ± SEM. ^aP < 0.05 vs control, ^cP < 0.05 vs Cerulein-induced acute pancreatitis (CerAP). Each determination was carried out in triplicate; C: Representative images of hematoxylin and eosin-stained pancreatic sections for each experimental group. Control group showed normal pancreas structure. CerAP group presented areas of necrosis, infiltrated polymorphonuclear neutrophils, interstitial edema and vacuolation of the acinar cells. Administration of 35-kDa polyethylene glycol notably reduced these features. Scale bar, 100 and 50 μm. CerAP: Cerulein-induced acute pancreatitis; PEG35: 35-kDa polyethylene glycol.

Figure 2

Role of PEG35 on the modulation of inflammation-associated cytokines and inducible nitric oxide synthase enzyme expression in cerulein-induced acute pancreatitis. Pancreatic tissue gene expression of tumor necrosis factor α, interleukin (IL) 1β, IL6, inducible nitric oxide synthase and IL10 by real-time qRT-PCR. Bars represent mean values of each group ± SEM. ^aP < 0.05 vs control, ^cP < 0.05 vs cerulein-induced acute pancreatitis. Each determination was carried out in triplicate. CerAP: Cerulein-induced acute pancreatitis; PEG35: 35-kDa polyethylene glycol.

Figure 3

Gene expressions of inflammatory markers in AR42J-treated cells. A: Gene expression by real-time qRT-PCR of tumor necrosis factor α (TNFα) and IL1β in cerulein-treated AR42J cells subjected to increasing concentrations of 35-kDa polyethylene glycol (PEG35); B: Gene expression by real-time qRT-PCR of TNFα and inducible nitric oxide synthase in TNFα-treated AR42J cells subjected to increasing concentrations of PEG35. In both cases, mRNA induction levels were normalized to GAPDH mRNA expression. Bars represent mean values of each group ± SEM. ^aP < 0.05 vs control, ^cP < 0.05 vs cerulein or TNFα. Each determination was carried out in triplicate. Cer: Cerulein; PEG35: 35-kDa polyethylene glycol; TNFα: Tumor necrosis factor α.

Figure 4

Effect of 35-kDa polyethylene glycol on inflammation-induced cell death in cerulein-induced acute pancreatitis and cultured pancreatic acinar AR42J cells. A: Plasma lactate dehydrogenase (LDH) activity after cerulein-induced acute pancreatitis expressed as mU/mL; B: Pancreatic protein expression of cleaved caspase-3 and BCL-2 assessed by western blot analysis. β-actin expression was used as loading control. Data shown are representative blots for each group; C: Densitometry quantification of western blot for cleaved caspase-3 and BCL-2 in pancreatic tissue; D: Cell death rate measured through LDH activity. AR42J cells pre-treated with increasing concentrations of PEG35 (0.5%, 1%, 2%, 4% or 6%) for 30 min and then co-incubated with 10nM cerulein for another 24 h or 100 ng/mL of tumor necrosis factor α (TNFα) for another 2.5 h; E: Cell viability rate determined by MTT assay. AR42J cells were pre-treated with increasing concentrations of PEG35, as indicated, for 30 min and then incubated with or without 2 µM or 4 µM staurosporine for another 24 h. The values shown represent the mean ± SEM. ^aP < 0.05 vs control, ^cP < 0.05 vs cerulein-induced acute pancreatitis, cerulein, TNFα or staurosporine. Each determination was carried out in triplicate. CerAP: Cerulein-induced acute pancreatitis; Cer: Cerulein; PEG35: 35-kDa polyethylene glycol; TNFα: Tumor necrosis factor α; ST: Staurosporine.