Activation of pancreatic stellate cells attenuates intracellular Ca2+ signals due to downregulation of TRPA1 and protects against cell death induced by alcohol metabolites.

Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca2+ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca2+ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-ß-activated PSCs, compare the expression of Ca2+ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca2+ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca2+-handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca2+ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca2+ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis.

The Role of Phosphate in Alcohol-Induced Experimental Pancreatitis.

BACKGROUND & AIMS: Heavy alcohol consumption is a common cause of acute pancreatitis; however, alcohol abuse does not always result in clinical pancreatitis. As a consequence, the factors responsible for alcohol-induced pancreatitis are not well understood. In experimental animals, it has been difficult to produce pancreatitis with alcohol. Clinically, alcohol use predisposes to hypophosphatemia, and hypophosphatemia has been observed in some patients with acute pancreatitis. Because of abundant protein synthesis, the pancreas has high metabolic demands, and reduced mitochondrial function leads to organelle dysfunction and pancreatitis. We proposed, therefore, that phosphate deficiency might limit adenosine triphosphate synthesis and thereby contribute to alcohol-induced pancreatitis. METHODS: Mice were fed a low-phosphate diet (LPD) before orogastric administration of ethanol. Direct effects of phosphate and ethanol were evaluated in vitro in isolated mouse pancreatic acini. RESULTS: LPD reduced serum phosphate levels. Intragastric administration of ethanol to animals maintained on an LPD caused severe pancreatitis that was ameliorated by phosphate repletion. In pancreatic acinar cells, low-phosphate conditions increased susceptibility to ethanol-induced cellular dysfunction through decreased bioenergetic stores, specifically affecting total cellular adenosine triphosphate and mitochondrial function. Phosphate supplementation prevented ethanol-associated cellular injury. CONCLUSIONS: Phosphate status plays a critical role in predisposition to and protection from alcohol-induced acinar cell dysfunction and the development of acute alcohol-induced pancreatitis. This finding may explain why pancreatitis develops in only some individuals with heavy alcohol use and suggests a potential novel therapeutic approach to pancreatitis. Finally, an LPD plus ethanol provides a new model for studying alcohol-associated pancreatic injury.

Lipopolysaccharide enhances TGF-ß1 signalling pathway and rat pancreatic fibrosis.

Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor-beta1 (TGF-ß1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF-ß1 signalling and pancreatic fibrosis. Sprague-Dawley rats fed with a Lieber-DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP-2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF-ß1 which was paralleled by an increased number of TLR4-positive or TGF-ß1-positive Mφs or PSCs in ALC-fed rats. In vitro, TLR4 or TGF-ß1 production in Mφs or RP-2 cells was up-regulated by LPS. LPS alone or in combination with TGF-ß1 significantly increased type I collagen and α-SMA production and Smad2 and 3 phosphorylation in serum-starved RP-2 cells. TGF-ß pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si-TLR4 RNA or by inhibitors of MyD88/NF-kB. Additionally, knockdown of Bambi with Si-Bambi RNA significantly increased TGF-ß1 signalling in RP-2 cells. These findings indicate that LPS increases TGF-ß1 production through paracrine and autocrine mechanisms and that LPS enhances TGF-ß1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF-kB activation.

Alcohol and cigarette smoke components activate human pancreatic stellate cells: implications for the progression of chronic pancreatitis.

BACKGROUND: Chronic pancreatitis, a known complication of alcohol abuse, is characterized histopathologically by prominent fibrosis. Pancreatic stellate cells (PSCs) are responsible for producing this fibrous tissue in chronic pancreatitis and are activated by alcohol. Progression of alcoholic chronic pancreatitis (as assessed by calcification and fibrosis) is thought to be facilitated by concurrent smoking, but the mechanisms are unknown. This study aimed to (a) determine whether human PSCs (hPSCs) and rat PSCs express nicotinic acetylcholine receptors (nAChRs), which are known to bind 2 important components of cigarette smoke, namely nicotine and nicotine-derived nitrosamine ketone (NNK), and (b) examine the effects of cigarette smoke components in the presence and absence of alcohol on PSC activation in vitro. METHODS: Western blotting was used to detect the presence of nAChRs in primary cultures of PSCs. Clinically relevant concentrations of cigarette smoke components (either cigarette smoke extract [CSE], NNK, or nicotine) ± ethanol (EtOH) were used to treat primary cultures of PSCs, and stellate cell activation was assessed by cell migration, proliferation, collagen production, and apoptosis. RESULTS: We demonstrate, for the first time, that PSCs express nAChRs (isoforms α3, α7, ß, ε) and that the expression of the α7 isoform in hPSCs is induced by CSE + EtOH. We also provide novel findings that PSCs are activated by CSE and NNK (both alone and in combination with EtOH) as evidenced by an increase in cell migration and/or proliferation. Further, we demonstrate that activation of PSCs by CSE + EtOH and NNK + EtOH may be mediated via nAChRs on the cells. CONCLUSIONS: PSCs are activated by clinically relevant concentrations of cigarette smoke components (CSE and NNK), alone and in combination with EtOH. Thus, in alcoholics who smoke, progression of pancreatic fibrosis may be facilitated by the combined effects of alcohol and cigarette smoke components on hPSC behavior.

Regulation of pancreatic inflammation by connective tissue growth factor (CTGF/CCN2).

Pancreatitis is caused by long-term heavy alcohol consumption, which results in injury and death of pancreatic acinar cells (PAC). The PAC play a pivotal role in mediating early inflammatory responses but the underlying mechanisms remain poorly understood. Treatment of C57BL/6 mice with ethanol and cerulein resulted in increased staining for acinar interleukin- 1b (IL-1b), chemokine (C-C motif) ligand 3 (CCL3), or connective tissue growth factor (CTGF/CCN2) by Day 16 and this was associated with increased infiltration of F4/80-positive macrophages and increased expression of pancreatic CTGF/CCN2 mRNA. Compared with wild-type Swiss Webster mice, ethanol treatment of pan-green fluorescent protein (GFP)-CTGF/CCN2 transgenic mice caused enhanced acinar staining for GFP or CTGF/CCN2 and a significant increase in pancreatic infiltration of F4/80-positive macrophages or NIMP-R14-positive neutrophils. Treatment of primary mouse PAC or the rat AR42J PAC line with ethanol or CTGF/CCN2 resulted in enhanced expression of IL-1b or CCL3. Conditioned medium from CTGF/CCN2-treated AR42J cells induced chemotaxis in NR8383 macrophages and this response was abrogated in a dose dependent manner by addition of BX471, an inhibitor of chemokine (C-C motif) receptor 1. These results reveal that acinar CTGF/CCN2 plays a novel role in alcohol-induced inflammatory processes in the pancreas by increasing infiltration of macrophages and neutrophils and increasing acinar production of inflammatory mediators such as IL-1b or CCL3. The early production of CTGF/CCN2 by PAC to drive inflammation is distinct from its previously reported production by pancreatic stellate cells to drive fibrosis at later stages of pancreatic injury.

Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis.

OBJECTIVE: Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. DESIGN: Intracellular calcium ([Ca(2+)](C)), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. RESULTS: Inhibition of OME with 4-MP converted predominantly transient [Ca(2+)](C) rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. CONCLUSIONS: A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation.

[Inhibition of pancreatic stellate cell activation by lisinopril for prevention fibrogenesis in experimental chronic alcoholic pancreatitis].

Lisinopril (angiotensin-converting enzyme inhibitor) attenuates fibrotic changes in pancreas after distal pancreatectomy in rats with experimental alcohol induced chronic pancreatitis. Lisinopril was administered after distal pancreatectomy in rats with experimental alcohol induced chronic pancreatitis. The animals were treated with lisinopril at the dose of 10 mg/kg body weight per day for 21 days after operation. To estimate the efficacy of the treatment on activity and number of pancreatic stellate cells the immunohistochemical investigation was made with alpha-smooth muscle actin (alpha-SMA), desmin, vimentin, glial fibrillary acidic protein (GFAP), matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2) using. The treatment of rats after operation with lisinopril inhibite activity of pancreatic stellate cells and characterized by significant decrease of the alpha-SMA, desmin, GFAP, vimentin and TIMP-2 expression. The ratio of MMP-1/TIMP-2 was greater in the group with treatment then in the control group. This therapy had a trend to alleviate the fibrotic changes in pancreas.

Preventive role of gallic acid on alcohol dependent and cysteine protease-mediated pancreas injury.

In order to investigate an association between alcohol consumption and lysosomal cysteine protease induced pancreatic injury and preventive effect of gallic acid as dose-dependent, we determined myeloperoxidase and malondialdehyde levels, serum amylase activities and cathepsin B and L activities in the cytosolic and lysosomal fractions of pancreatic tissue in the ethanol (8 g/kg) and ethanol plus gallic acid (at different doses 50, 100 and 200 mg/kg) given rats. Absolute ethanol (8 g/kg) was given by oral gavage. Gallic acid was dissolved in the saline (2 ml/kg) and administered before 30 min the oral administration of ethanol. Pancreatic myeloperoxidase and also malondialdehyde levels and serum amylase activities were measured. Besides, histological investigations were made. Cathepsin B activities in the cytosolic fraction were decreased by gallic acid (200 mg/kg) and increased in ethanol given rats. Cytosolic/lysosomal ratio of cathepsin B and L were found to be low in the all doses of gallic acid as compared to ethanol group. Serum amylase, pancreatic myeloperoxidase activities and malondialdehyde levels in the ethanol group were higher than in the control group. These were not statistically significant for myeloperoxidase and malondialdehyde. Also, our histopathologic results indicated that ethanol administration increased pancreatic tissue injury. Gallic acid especially at 200 mg/kg improved ethanol-mediated pancreatic tissue damage.In conclusion, gallic acid treatments were decreased release of lysosomal cathepsin B and L enzymes into cytoplasmic fraction and prevented alcohol mediated pancreatic tissue injury. Preventive effect of gallic acid might be dose-dependent.

Withdrawal of alcohol promotes regression while continued alcohol intake promotes persistence of LPS-induced pancreatic injury in alcohol-fed rats.

BACKGROUND AND AIMS: Administration of repeated lipopolysaccharide (LPS) injections in alcohol-fed rats leads to significant pancreatic injury including fibrosis. However, it remains unknown whether alcoholic (chronic) pancreatitis has the potential to regress when alcohol is withdrawn. The aims of the study were (1) to compare the effect of alcohol withdrawal/continuation on pancreatic acute injury and fibrosis; and (2) to assess the effects of alcohol ± LPS on pancreatic stellate cell (PSC) apoptosis in vivo and in vitro. METHODS: Rats fed isocaloric Liebere-De-Carli liquid diets ± alcohol for 10 weeks were challenged with LPS (3 mg/kg/week for 3 weeks) and then either switched to control diet or maintained on an alcohol diet for 3 days, 7 days or 3 weeks. Pancreatic sections were assessed for acute tissue injury, fibrosis, PSC apoptosis and activation. Cultured rat PSCs were exposed to 10 mM ethanol 6 1 mg/ml LPS for 48 or 72 h and apoptosis was assessed (Annexin V, caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)). RESULTS: Withdrawal of alcohol led to resolution of pancreatic lesions including fibrosis and to increased PSC apoptosis. Continued alcohol administration perpetuated pancreatic injury and prevented PSC apoptosis. Alcohol and LPS significantly inhibited PSC apoptosis in vitro, and the effect of LPS on PSC apoptosis could be blocked by Toll-like receptor 4 small interfering RNA. CONCLUSIONS: Induction of PSC apoptosis upon alcohol withdrawal is a key mechanism mediating the resolution of pancreatic fibrosis. Conversely, continued alcohol intake perpetuates pancreatic injury by inhibiting apoptosis and promoting activation of PSCs. Characterisation of the pathways mediating PSC apoptosis has the potential to yield novel therapeutic strategies for chronic pancreatitis.

Chronic alcohol intake increases the severity of pancreatitis induced by acute alcohol administration, hyperlipidemia and pancreatic duct obstruction in rats.

BACKGROUND: The mechanism of alcoholic pancreatitis is still unknown. It is of special interest why only about 5% of all alcoholics develop an episode of pancreatitis. We evaluated the role of long-term alcohol intake in the pathogenesis of alcoholic pancreatitis in rats. METHODS: To evaluate the effect of long-term alcohol intake, rats were fed either a Lieber-DeCarli control diet (CD) or a Lieber-DeCarli alcohol diet (AD) for 6 weeks. Then, rats were infused over 2 h with either Ringer's solution (CO) or ethanol (E). In additional animals, alcoholic pancreatitis was induced by ethanol combined with hyperlipidemia and temporary pancreatic duct obstruction (EFO). Controls received Ringer's solution combined with hyperlipidemia and temporary pancreatic duct obstruction (RFO). Intravital microscopy (pancreatic perfusion and leukocyte adhesion), alcohol concentrations, amylase, lipase, cholesterine and triglyceride levels in plasma, myeloperoxidase activity and histology were evaluated at different time intervals. RESULTS: In those animals which received the Lieber-DeCarli control diet, capillary perfusion was reduced in the E group and further reduced in the EFO group as compared to the controls (CO, RFO; p < 0.01). Leukocyte adhesion was significantly increased in rats receiving E (p < 0.01), and was further increased in the combination group EFO (p < 0.01). EFO induced histologically evident acute pancreatitis. The additional administration of a long-term alcohol diet further increased microcirculatory disturbances and pancreatic injury significantly (EFO-AD > EFO-CD). CONCLUSIONS: This study shows that alcoholic pancreatitis is induced by the combination of ethanol and individual cofactors. Chronic alcohol abuse intensifies these changes. Therefore, long-term alcohol intake seems to be a major factor in the pathogenesis of alcoholic pancreatitis.

Cholinergic mediation of alcohol-induced experimental pancreatitis.

OBJECTIVES: The mechanisms initiating pancreatitis in patients with chronic alcohol abuse are poorly understood. Although alcohol feeding has been previously suggested to alter cholinergic pathways, the effects of these cholinergic alterations in promoting pancreatitis have not been characterized. For this study, we determined the role of the cholinergic system in ethanol-induced sensitizing effects on cerulein pancreatitis. METHODS: Rats were pair-fed control and ethanol-containing Lieber-DeCarli diets for 6 weeks followed by parenteral administration of 4 hourly intraperitoneal injections of the cholecystokinin analog, cerulein at 0.5 µg/kg. This dose of cerulein was selected because it caused pancreatic injury in ethanol-fed but not in control-fed rats. Pancreatitis was preceded by treatment with the muscarinic receptor antagonist atropine or by bilateral subdiaphragmatic vagotomy. Measurement of pancreatic pathology included serum lipase activity, pancreatic trypsin, and caspase-3 activities, and markers of pancreatic necrosis, apoptosis, and autophagy. In addition, we measured the effects of ethanol feeding on pancreatic acetylcholinesterase activity and pancreatic levels of the muscarinic acetylcholine receptors m1 and m3. Finally, we examined the synergistic effects of ethanol and carbachol on inducing acinar cell damage. RESULTS: We found that atropine blocked almost completely pancreatic pathology caused by cerulein administration in ethanol-fed rats, while vagotomy was less effective. Ethanol feeding did not alter expression levels of cholinergic muscarinic receptors in the pancreas but significantly decreased pancreatic acetylcholinesterase activity, suggesting that acetylcholine levels and cholinergic input within the pancreas can be higher in ethanol-fed rats. We further found that ethanol treatment of pancreatic acinar cells augmented pancreatic injury responses caused by the cholinergic agonist, carbachol. CONCLUSION: These results demonstrate key roles for the cholinergic system in the mechanisms of alcoholic pancreatitis.

Effects of gadolinium chloride and glycine on hepatic and pancreatic tissue damage in alcoholic pancreatitis.

OBJECTIVE: Systemic complications in alcoholic pancreatitis are supposed to be aggravated by inflammatory liver damage. Resident macrophages including hepatic Kupffer cells play a pivotal role in mediating systemic complications in severe necrotizing pancreatitis (SNP). The aim of this study was to evaluate the effects of Kupffer cell inhibition on the inflammatory liver damage in experimental alcoholic pancreatitis. METHODS: Rats were fed with either alcohol or control diet for 6 weeks before induction of SNP. Animals were allocated into 4 groups: healthy controls, controls with SNP, SNP with gadolinium chloride or glycine (permanent vs temporary inhibition of hepatic Kupffer cells) prophylaxis. Hepatic microcirculation and morphologic damage of the liver and pancreas were assessed. RESULTS: Alcohol feeding and SNP increased hepatic and pancreatic injury compared with SNP alone. Gadolinium chloride and glycine improved hepatic microcirculation. In contrast, pancreatic and hepatic morphological damage was reduced by gadolinium chloride but not by glycine. CONCLUSIONS: Alcohol exposure aggravates hepatic and pancreatic injury in SNP. Gadolinium chloride reduces both microcirculatory and morphological damage, whereas glycine did not improve histological damage.

Pancreas and liver injury are associated in individuals with increased alcohol consumption.

BACKGROUND & AIMS: Although chronic pancreatitis and liver cirrhosis are common sequelae of excess alcohol consumption, the 2 conditions are rarely associated. We studied the prevalence of simultaneous liver cirrhosis and chronic pancreatitis in alcoholics. METHODS: Postmortem autopsy data from 620 individuals with a history of excess alcohol consumption and 100 nonalcoholics (controls) were analyzed. The individuals were classified into groups based on macroscopic observations of pancreas (no injury, acute pancreatitis, fibrosis, and chronic pancreatitis) and liver (no injury, moderate steatosis, severe steatosis, and cirrhosis). The same classification system was used for histological data, which was used to confirm and correlate macroscopic results. RESULTS: Out of the 183 patients with liver cirrhosis, 33 (18%) had chronic pancreatitis and 93 (51%) had pancreatic fibrosis. Out of the 230 patients with severe steatosis, 37 (16%) had chronic pancreatitis and 97 (42%) were found to have a pancreatic fibrosis. Thirty-three (39%) with chronic pancreatitis also showed liver cirrhosis and 37 (44%) showed severe steatosis. Thirty-eight percent of the patients with a pancreatic fibrosis were found also to have liver cirrhosis and in another 40% severe steatosis. Thirty-five patients showed neither hepatic or pancreatic injury. We found no chronic pancreatitis or liver cirrhosis in the control group (n = 100). CONCLUSIONS: Contrary to common belief there is a close association between pancreatic and hepatic injury in patients with increased alcohol consumption, and the degree of organ damage between the 2 organs correlate.

Preparation and in vivo efficacy study of pancreatin microparticles as an enzyme replacement therapy for pancreatitis.

Deficiency manifestations because of pancreatic insufficiency are treated by oral administration of pancreatic enzymes. As pancreatic enzymes get denatured in hostile acidic conditions of stomach, this investigation was aimed at formulating multiparticulates of pancreatic enzymes coated with enteric polymers such as eudragit L100, cellulose acetate phthalate, and hydroxyl propyl methyl cellulose phthalate, which will circumvent gastric inactivation in addition to providing optimal mixing with chyme. Pancreatin microspheres were prepared by emulsification phase separation by nonsolvent addition and solvent evaporation techniques. This process was optimized for core : coat ratio (1:0.5), stirrer speed (350-400 rpm), dispersant concentration, and amount of nonsolvent added to precipitate microspheres. Optimized formulations were assessed for % enzyme content, acid resistance, flow properties, particle size, particle morphology (by standard electron microscopy), compatibility of drug and polymer in formulation (by differential scanning calorimetry), in vitro release kinetics, and in vivo efficacy study in pancreatitis-induced animal model. Capsules containing enteric-coated pancreatin microspheres offered adequate protection to enzymes and prevented their denaturation in acidic environment. Developed multiparticulate dosage forms promoted effective mixing, instant and complete in vitro release compared with marketed tablets.

Relationships among iron, protein oxidation and lipid peroxidation levels in rats with alcohol-induced acute pancreatitis.

It has been previously shown that alcohol induces the damage of pancreatic parenchyma tissue, but the mechanism of this damage is still poorly understood. Assuming that oxygen radical damage may be the involved, we measured markers of oxidative damage in pancreatic tissue, blood serum, plasma, and whole blood of rats with early-stage alcohol-induced acute pancreatitis. Thirty-eight male Wistar rats were divided into three groups: the control group (group 1), the acute pancreatitis group 1 day (group 2), and 3 days (group 3) after the injection of ethyl alcohol into the common biliary duct, respectively. The levels of Fe in tissue and serum, whole blood viscosity, plasma viscosity, fibrinogen and homocysteine (Hcy) levels, erythrocyte and plasma malondialdehyde (MDA), and tissue and plasma protein carbonyl levels were found to be significantly higher in groups 2 and 3 than in group 1. However, the levels of reduced glutathione (GSH) in tissue and erythrocytes were significantly lower in groups 2 and 3 than in group 1. These results suggest that elevated Fe levels in serum and pancreatic tissue in rats with early-stage alcohol-induced acute pancreatitis is associated with various hemorheological changes and with oxidative damage of the pancreas.

A rat model reproducing key pathological responses of alcoholic chronic pancreatitis.

Although alcohol abuse is the major cause of chronic pancreatitis, the pathogenesis of alcoholic chronic pancreatitis (ACP) remains obscure. A critical obstacle to understanding the mechanism of ACP is lack of animal models. Our objective was to develop one such model. Rats were pair-fed for 8 wk ethanol or control Lieber-DeCarli liquid diet. For the last 2 wk, they received cyclosporin A (CsA; 20 mg/kg once daily) or vehicle. After 1 wk on CsA, one episode of acute pancreatitis was induced by four 20 microg/kg injections of cerulein (Cer); controls received saline. Pancreas was analyzed 1 wk after the acute pancreatitis. CsA or Cer treatments alone did not result in pancreatic injury in either control (C)- or ethanol (E)-fed rats. We found, however, that alcohol dramatically aggravated pathological effect of the combined CsA+Cer treatment on pancreas, resulting in massive loss of acinar cells, persistent inflammatory infiltration, and fibrosis. Macrophages were prominent in the inflammatory infiltrate. Compared with control-fed C+CsA+Cer rats, their ethanol-fed E+CsA+Cer counterparts showed marked increases in pancreatic NF-kappaB activation and cytokine/chemokine mRNA expression, collagen and fibronectin, the expression and activities of matrix metalloproteinase-2 and -9, and activation of pancreatic stellate cells. Thus we have developed a model of alcohol-mediated postacute pancreatitis that reproduces three key responses of human ACP: loss of parenchyma, sustained inflammation, and fibrosis. The results indicate that alcohol impairs recovery from acute pancreatitis, suggesting a mechanism by which alcohol sensitizes pancreas to chronic injury.

Bacterial endotoxin: a trigger factor for alcoholic pancreatitis? Evidence from a novel, physiologically relevant animal model.

BACKGROUND & AIMS: This study examined the possible role of endotoxinemia (from increased gut permeability) as an additional trigger factor for overt pancreatic disease and as a promoter of chronic pancreatic injury in alcoholics by using a rat model of chronic alcohol feeding and in vitro experiments with cultured pancreatic stellate cells (PSCs), the key mediators of pancreatic fibrosis. METHODS: In the in vivo model, Sprague-Dawley rats fed isocaloric Lieber-DeCarli liquid diets +/- alcohol for 10 weeks were challenged with a single dose or 3 repeated doses of the endotoxin lipopolysaccharide (LPS) and the pancreas was examined. In the in vitro studies, rat PSCs were assessed for activation on exposure to LPS +/- ethanol. The expression of LPS receptors TLR4 and CD14 also was assessed in rat and human PSCs. RESULTS: In the in vivo model, single or repeated LPS challenge resulted in significantly greater pancreatic injury in alcohol-fed rats compared with rats fed the control diet without alcohol. Notably, repeated LPS injections caused pancreatic fibrosis in alcohol-fed rats, but not in rats fed the control diet. In the in vitro studies, PSCs were activated by LPS. Alcohol + LPS exerted a synergistic effect on PSC activation. Importantly, both rat and human PSCs expressed TLR4 and CD14. CONCLUSIONS: This study describes, for the first time, a clinically relevant animal model of alcohol-related pancreatic injury and provides strong in vivo and in vitro evidence that suggests that LPS is a trigger factor in the initiation and progression of alcoholic pancreatitis.

Alcohol redirects CCK-mediated apical exocytosis to the acinar basolateral membrane in alcoholic pancreatitis.

The molecular mechanism of clinical alcohol-induced pancreatitis remains vague. We had reported that experimental high-dose cholecystokinin (CCK)-induced pancreatitis is in part because of excessive aberrant basolateral exocytosis. High-dose CCK caused Munc18c on basolateral plasma membrane (BPM) to dissociate from syntaxin (Syn)-4, activating Syn-4 to complex with plasma membrane (PM)-SNAP-23 and granule-VAMP to mediate basolateral exocytosis. We now hypothesize that alcohol could render the acinar cell BPM conducive to exocytosis by a similar mechanism. Weakly stimulating postprandial doses of alcohol (20-50 mM) inhibited postprandial low-dose CCK-stimulated secretion by blocking physiologic apical exocytosis and redirecting exocytosis to less-efficient basal PM (visualized by FM1-43 fluorescence imaging) and lateral PM sites (electron microscopy). Alcohol or low-dose CCK had no effect on PM-Munc18c, but alcohol preincubation enabled low-dose CCK to displace Munc18c from BPM, leading to SNARE complex assembly in the BPM. Similarly, alcohol diet-fed rats did not exhibit morphologic defects in the pancreas nor affected PM-Munc18c behavior, but subsequent intraperitoneal injections of low-dose CCK analog cerulein caused Munc18c displacement from BPM and cytosolic degradation, which contributed to pancreatitis. We conclude that alcohol induces BPM-Munc18c to become receptive to postprandial CCK-induced displacement into the cytosol, a process which facilitates SNARE complex assembly that in turn activates restricted BPM sites to become available for aberrant exocytosis into the interstitial space, where zymogen activation would take place and cause pancreatitis.

Long-term ethanol consumption alters pancreatic gene expression in rats: a possible connection to pancreatic injury.

OBJECTIVES: Long-term ethanol consumption does not cause acute pancreatitis but rather sensitizes the pancreas to subsequent insults. The mechanisms responsible for this sensitization are unknown. To determine whether alterations in pancreatic gene expression might participate in ethanol-mediated sensitization, we performed gene-profiling analysis. METHODS: Animals were fed ethanol-containing Lieber-DeCarli or control diet (pair-fed). After 8 weeks, pancreatic RNA expression was analyzed using Affimetrix GeneChips. Changes in specific genes were verified using quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Long-term ethanol feeding caused a significant alteration of pancreatic gene expression. Selection criteria of changes more than 3-fold and P < 0.05 yielded 114 probe sets. Activating transcription factor 3, heat shock protein 70, heat shock protein 27, and mesotrypsinogen were increased, whereas pancreatitis associate protein, folate carrier, and metallothionein were decreased. CONCLUSIONS: Ethanol had a profound effect on pancreatic gene expression. The genes identified as elevated and reduced in this study may contribute to pancreatic sensitivity to stress. This study indicates for the first time the identities of multiple genes whose expression levels are dramatically influenced by long-term ethanol feeding. The identified genes may help explain the relationship between long-term ethanol abuse and pancreatic disease and lead to possible preventative or therapeutic approaches to ethanol-induced pancreatic disease.