Chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in rats

Abstract

Alcohol consumption is a risk factor for chronic pancreatitis (CP), but the mechanism in humans remains obscure because prolonged alcohol consumption in most humans and animal models fails to produce alcoholic chronic pancreatitis (ACP). We hypothesize that the process leading to ACP is triggered by a sentinel acute pancreatitis (AP) event; this event causes recruitment of inflammatory cells, which initiates fibrosis driven by the anti-inflammatory response to recurrent AP and/or chronic oxidative stress. The aim was to determine whether chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in the rat. Wistar male rats were pair-fed control (C) or 5% ethanol (E) Lieber-DeCarli liquid diets. Animals were studied without pancreatitis (P0), with cerulein pancreatitis induced once (P1), or with cerulein-induced pancreatitis weekly for 3 weeks (P3). AP markers, inflammation, and fibrosis were measured histologically, by gene expression profiling and protein expression. Macrophage infiltration was reduced in EP0 versus CP0 rats, but the pattern was reversed after AP. Microabscess, severe necrosis, and early calcification were only induced in the EP3 rats. Fibrosis was significantly induced in the EP3 rats versus EP1, CP1, and CP3 by histology, hydroxyproline content, and mRNA expression for collagen alpha1(1) and procollagen alpha2(1). Proinflammatory cytokine mRNAs were up-regulated shortly after induction of AP, while the anti-inflammatory cytokines (interleukin-10 and transforming growth factor-beta) were strongly up-regulated later and in parallel with fibrogenesis, especially in the EP3 rats. Pancreatic fibrosis develops after repeated episodes of AP and is potentiated by alcohol. Expression of fibrosis-associated genes was associated with expression of anti-inflammatory cytokines in alcohol-fed rats.

Figure 1

Time frame of experimental design. Nine days, ramp time for gradually increasing ethanol concentration in ethanol diet-fed group. Cer, cerulein. ♦, Selected sacrifice time for that group of rats.

Figure 2

The alteration of serum amylase and pancreas wet weight versus body weight induced by cerulein in control-fed rats (pilot study). a: Alteration of serum amylase. b: Pancreas wet weight versus body weight. **, P < 0.01; ***, P < 0.001, as compared to the values in the vehicle group.

Figure 3

Body weight gain throughout the experimental period.

Figure 4

The daily average intake of control and alcohol-liquid diets consumed by control-fed and alcohol-fed rats (ml/day/rat). First, second, and third episode, demonstrated the four injections of the cerulein at that specific day.

Figure 5

Comparison of one episode and three episodes of cerulein pancreatitis on serum amylase levels. C, Control diet-fed rats; E, alcohol liquid diet-fed rats. *, P < 0.05; **, P < 0.01, as compared to the respective vehicle-treated group.

Figure 6

Comparison of one episode and three episodes of cerulein pancreatitis on pancreas wet weight versus body weight. C, Control diet-fed rats; E, alcohol-liquid diet-fed rats. *, P < 0.05; **, P < 0.01, as compared to their corresponding vehicle-treated group (control-fed cerulein/control-fed vehicle, alcohol-fed cerulein/alcohol-fed vehicle).

Figure 7

Representative pancreatic tissue sections from vehicle-treated control-fed rats (A, CP0); vehicle-treated alcohol-fed rats (B, EP0); 24-hours one episode of cerulein pancreatitis control-fed rats (C, CP1) (*, typical vacuolization in acinar cells); 24-hours one episode of cerulein pancreatitis alcohol-fed rats (D, EP1) (*, typical vacuolization in acinar cells); 96-hours three episodes cerulein pancreatitis control-fed rats (E, CP3); 96-hours three episodes cerulein pancreatitis alcohol-fed rats (F, EP3). G–J: Findings in EP3 rats. G: Hemorrhage (labeled as *); H: white blood cell infiltration (labeled as #) and acinar cell necrosis; I: severe white blood cell infiltration around the necrosis and calcification; and J: calcification (labeled as *) and early fibrosis (labeled as #) in parenchyma of pancreas in EP3 rats. H&E stain. Original magnifications, ×200.

Figure 8

The alterations of pancreatic hydroxyproline levels at 96 hours after the last injection in vehicle-treated, one or three episodes cerulein pancreatitis. C, Control-fed group; E, alcohol-fed group. **, P < 0.01, as compared to the vehicle-treated group.

Figure 9

Representative tissue sections stained by Sirius Red. a: Control vehicle-treated group (CP0); b: ethanol vehicle-treated group (EP0); c: 96-hours three episodes of cerulein pancreatitis in control-fed group (CP3); and d: 96-hours three episodes of cerulein pancreatitis in alcohol-fed group (EP3). Sirius Red stain. Original magnifications, ×100.

Figure 10

The relative mRNA expression levels of collagen-α1(1) in vehicle-treated and cerulein-induced pancreatitis in both one and three episodes pancreatitis groups. a: The mRNA expression levels relative to the 18s RNA internal control, The bar represents the values of the mRNA expression in the ethanol-fed rats as compared to the respective control-fed rats. *, P < 0.05; **, P < 0.01, as compared to the values in the vehicle-treated groups; B: the real-time amplification plots of collagen-α1(1) and 18s RNA.

Figure 11

Alteration of macrophage infiltration in pancreas of control- and alcohol-fed rats. a: The relative changes of resident macrophages (ED2-immunoreactive positive cells) counting per high-power field in control-fed rats. **, P < 0.01, as compared to the values of the corresponding control diet-fed groups. B: Representative tissue sections stained by ED2 immunohistochemical technique. a: Vehicle-treated control-fed group (CP0); b: vehicle-treated alcohol-fed group (EP0); c: 24-hours one episode of cerulein-treated control-fed rats (CP1); d: 24-hours one episode of cerulein-treated alcohol-fed rats (EP1); e: 24-hours three episodes cerulein-treated control-fed rats (CP3); and f: 24-hours three episodes cerulein-treated alcohol-fed rats (EP3). *, Resident macrophages. ED2 immunohistochemical stain. Original magnifications, ×100.

Figure 12

Comparison of the distribution of α-SMA immune-reactive positive cells with ED2 immune-reactive positive cells in pancreas of one episode of pancreatitis in alcohol-fed rats on consecutive tissue sections at 24-hour group. Left: α-SMA immunoblots (top) and α-SMA immunochemical stain (bottom). Right: ED1 and ED2 immunoblots (top) and ED2 immunochemical stain (bottom). α-SMA and ED2 immunohistochemical stain. Original magnifications, ×100.