Age-Dependent Effects of UCP2 Deficiency on Experimental Acute Pancreatitis in Mice

Reactive oxygen species (ROS) have been implicated in the pathogenesis of acute pancreatitis (AP) for many years but experimental evidence is still limited. Uncoupling protein 2 (UCP2)-deficient mice are an accepted model of age-related oxidative stress. Here, we have analysed how UCP2 deficiency affects the severity of experimental AP in young and older mice (3 and 12 months old, respectively) triggered by up to 7 injections of the secretagogue cerulein (50 μg/kg body weight) at hourly intervals. Disease severity was assessed at time points from 3 hours to 7 days based on pancreatic histopathology, serum levels of alpha-amylase, intrapancreatic trypsin activation and levels of myeloperoxidase (MPO) in lung and pancreatic tissue. Furthermore, in vitro studies with pancreatic acini were performed. At an age of 3 months, UCP2^-/- mice and wild-type (WT) C57BL/6 mice were virtually indistinguishable with respect to disease severity. In contrast, 12 months old UCP2^-/- mice developed a more severe pancreatic damage than WT mice at late time points after the induction of AP (24 h and 7 days, respectively), suggesting retarded regeneration. Furthermore, a higher peak level of alpha-amylase activity and gradually increased MPO levels in pancreatic and lung tissue were observed in UCP2^-/- mice. Interestingly, intrapancreatic trypsin activities (in vivo studies) and intraacinar trypsin and elastase activation in response to cerulein treatment (in vitro studies) were not enhanced but even diminished in the knockout strain. Finally, UCP2^-/- mice displayed a diminished ratio of reduced and oxidized glutathione in serum but no increased ROS levels in pancreatic acini. Together, our data indicate an aggravating effect of UCP2 deficiency on the severity of experimental AP in older but not in young mice. We suggest that increased severity of AP in 12 months old UCP2^-/- is caused by an imbalanced inflammatory response but is unrelated to acinar cell functions.     

2',4',6'-Tris(methoxymethoxy) chalcone (TMMC) attenuates the severity of cerulein-induced acute pancreatitis and associated lung injury

Acute pancreatitis (AP) is an inflammatory disease involving acinar cell injury and rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. 2',4',6'-Tris (methoxymethoxy) chalcone (TMMC), a synthetic chalcone derivative, displays potent anti-inflammatory effects. Therefore, we aimed to investigate whether TMMC might affect the severity of AP and pancreatitis-associated lung injury in mice. We used the cerulein hyperstimulation model of AP. Severity of pancreatitis was determined in cerulein-injected mice by histological analysis and neutrophil sequestration. The pretreatment of mice with TMMC reduced the severity of AP and pancreatitis-associated lung injury and inhibited several biochemical parameters (activity of amylase, lipase, trypsin, trypsinogen, and myeloperoxidase and production of proinflammatory cytokines). In addition, TMMC inhibited pancreatic acinar cell death and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 by inhibiting NF-κB and extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation. Neutralizing antibodies for TNF-α, IL-1β, and IL-6 inhibited cerulein-induced cell death in isolated pancreatic acinar cells. Moreover, pharmacological blockade of NF-κB/ERK1/2 reduced acinar cell death and production of TNF-α, IL-1β, and IL-6 in isolated pancreatic acinar cells. In addition, posttreatment of mice with TMMC showed reduced severity of AP and lung injury. Our results suggest that TMMC may reduce the complications associated with pancreatitis.     

L- 精氨酸和雨蛙素诱导急性胰腺炎模型的对比研究

目的:研究L-精氨酸和雨蛙素分别诱导SD大鼠急性胰腺炎(AP)模型的差异,为进一步研究急性胰腺炎提供可靠模型。方法:L-精氨酸采用3次腹腔注射,间隔1 h,雨蛙素采用7次腹腔注射,间隔1 h诱导急性胰腺炎模型。碘-淀粉比色法检测血清淀粉酶水平,血清脂肪酶测定试剂盒检测脂肪酶活性,胰腺组织切片观察组织的破坏情况,TUNEL法检测腺泡细胞凋亡。结果:①L-精氨酸诱导的大鼠模型血清淀粉酶和脂肪酶水平在诱导成功后6 h即显著升高,蛙皮素诱导的大鼠模型在12 h显著升高,与正常对照组比较均有统计学差异(P<0.05),提示急性胰腺炎建模成功。②L-精氨酸诱导的模型中胰腺组织结构破坏,有大片出血坏死灶、大量炎细胞浸润;而蛙皮素诱导的模型组织腺泡、间质水肿,炎性细胞浸润,少量散在出血坏死灶,血管变化常不明显,渗液清亮。结论:L-精氨酸和雨蛙素均能诱导SD大鼠急性胰腺炎模型,L-精氨酸诱导重症急性胰腺炎,雨蛙素诱导轻型急性胰腺炎,是研究急性胰腺炎的良好模型。     

Chemokine receptor CCR5 deficiency exacerbates cerulein-induced acute pancreatitis in mice

Acute pancreatitis (AP) is an inflammatory disease involving the production of different cytokines and chemokines and is characterized by leukocyte infiltration. Because the chemokine receptor CCR5 and its ligands [the CC chemokines CCL3/MIP-1alpha, CCL4/MIP-1beta, and CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES)] regulate leukocyte chemotaxis and activation, we investigated the expression of CCR5 ligands and the role of CCR5 and its ligands in experimental AP in mice. AP was induced by hourly intraperitoneal injections of cerulein in CCR5-deficient (CCR5(-/-)) or wild-type (WT) mice. Induction of AP by cerulein resulted in an early increase of pancreatic CCL2, CCL3, and CCL4 mRNA expression, whereas CCL5 mRNA expression occurred later. CCR5(-/-) mice developed a more severe pancreatic injury than WT mice during cerulein-induced AP, as assessed by a more pronounced increase in serum amylase and lipase levels and by more severe pancreatic edema, inflammatory infiltrates (mainly neutrophils), and necrosis. CCR5(-/-) mice also exhibited increased production of CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-1beta during the course of cerulein-induced AP. In vivo simultaneous neutralization of CC chemokines with monoclonal antibodies in CCR5(-/-) mice reduced the severity of cerulein-induced AP, indicating a role of CC chemokines in exacerbating the course of AP in the absence of CCR5. Moreover, simultaneous neutralization of CCR5 ligands in WT mice also reduced the severity of cerulein-induced AP. In conclusion, lack of the chemokine receptor CCR5 exacerbates experimental cerulein-induced AP and leads to increased levels of CC chemokines and a more pronounced pancreatic inflammatory infiltrate, suggesting that CCR5 expression can modulate severity of AP.     

Fibroblast growth factor 21 alleviates acute pancreatitis via activation of the Sirt1‐autophagy signalling pathway

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein‐induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21‐treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein‐induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein‐induced pancreatic injury and improves cerulein‐induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up‐regulate Sirtuin‐1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein‐induced pancreatic injury and weakens the regulative effect in FGF21‐activated autophagy in mice. These results showed that FGF21 protects against cerulein‐induced AP by activation of Sirtuin‐1‐autophagy axis.     

A murine model of obesity implicates the adipokine milieu in the pathogenesis of severe acute pancreatitis

Obesity is clearly an independent risk factor for increased severity of acute pancreatitis (AP), although the mechanisms underlying this association are unknown. Adipokines (including leptin and adiponectin) are pleiotropic molecules produced by adipocytes that are important regulators of the inflammatory response. We hypothesized that the altered adipokine milieu observed in obesity contributes to the increased severity of pancreatitis. Lean (C57BL/6J), obese leptin-deficient (LepOb), and obese hyperleptinemic (LepDb) mice were subjected to AP by six hourly intraperitoneal injections of cerulein (50 microg/kg). Severity of AP was assessed by histology and by measuring pancreatic concentration of the proinflammatory cytokines IL-1beta and IL-6, the chemokine MCP-1, and the marker of neutrophil activation MPO. Both congenitally obese strains of mice developed significantly more severe AP than wild-type lean animals. Severity of AP was not solely related to adipose tissue volume: LepOb mice were heaviest; however, LepDb mice developed the most severe AP both histologically and biochemically. Circulating adiponectin concentrations inversely mirrored the severity of pancreatitis. These data demonstrate that congenitally obese mice develop more severe AP than lean animals when challenged by cerulein hyperstimulation and suggest that alteration of the adipokine milieu exacerbates the severity of AP in obesity.     

Pancreatic secretory trypsin inhibitor I reduces the severity of chronic pancreatitis in mice overexpressing interleukin-1β in the pancreas

IL-1β is believed to play a pathogenic role in the development of pancreatitis. Expression of human IL-1β in pancreatic acinar cells produces chronic pancreatitis, characterized by extensive intrapancreatic inflammation, atrophy, and fibrosis. To determine if activation of trypsinogen is important in the pathogenesis of chronic pancreatitis in this model, we crossed IL-1β transgenic [Tg(IL1β)] mice with mice expressing a trypsin inhibitor that is normally produced in rat pancreatic acinar cells [pancreatic secretory trypsin inhibitor (PTSI) I]. We previously demonstrated that transgenic expression of PSTI-I [Tg(Psti1)] increased pancreatic trypsin inhibitor activity by 190%. Tg(IL1β) mice were found to have marked pancreatic inflammation, characterized by histological changes, including acinar cell loss, inflammatory cell infiltration, and fibrosis, as well as elevated myeloperoxidase activity and elevated pancreatic trypsin activity, as early as 6 wk of age. In contrast to Tg(IL1β) mice, pancreatitis was significantly less severe in dual-transgenic [Tg(IL1β)-Tg(Psti1)] mice expressing IL-1β and PSTI-I in pancreatic acinar cells. These findings indicate that overexpression of PSTI-I reduces the severity of pancreatitis and that pancreatic trypsin activity contributes to the pathogenesis of an inflammatory model of chronic pancreatitis.     

Role of parathyroid hormone-related protein in the pro-inflammatory and pro-fibrogenic response associated with acute pancreatitis

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1-36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1-36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7-34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.     

Pro- and anti-inflammatory response of acinar cells during acute pancreatitis. Effect of N-acetyl cysteine

We investigate the ability of acinar cells to produce tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) at different stages of acute pancreatitis (AP). Since oxidative stress is involved in the inflammatory response, the effect of N-acetyl cysteine (NAC) has also been evaluated. AP was induced in rats by bile-pancreatic duct obstruction (BPDO). NAC (50 mg/kg) was administered 1h before and 1h after BPDO. Acinar cells were incubated for 4 h at 37 degrees C in 5% CO2 atmosphere in absence and presence of 24-h BPDO-PAAF (20%, v/v) as stimulant agent. Acinar production of TNF-alpha and IL-10 was analysed by flow cytometry. Plasma amylase activity and histological studies of the pancreas indicated the severity of AP. PAAF significantly stimulated the acinar production of TNF-alpha and IL-10 in control rats. TNF-alpha production was also significantly stimulated in acinar cells of rats with AP, although a decrease in the pro-inflammatory response was found from 6 h after BPDO onwards. However, acinar cells failed to produce IL-10 from 3 h after BPDO. The protective effect of NAC treatment against oxidative cell damage reduced the pancreatic injury and maintained and enhanced the ability of acinar cells to produce IL-10 at early AP stages. As long as acinar cells were not severely damaged in the course of AP, greater ability to produce cytokines in response to PAAF was found in those with higher forward scatter (R2 cells). We suggest that the capability of acinar cells to maintain an appropriate balance between the production of pro- and anti-inflammatory mediators could contribute to determine the degree of severity of AP.     

Acinar inflammatory response to lipid derivatives generated in necrotic fat during acute pancreatitis

Lipids play a role in acute pancreatitis (AP) progression. We investigate the ability of pancreatic acinar cells to trigger inflammatory response in the presence of lipid compounds generated in necrotic areas of peripancreatic adipose tissue (AT) during AP induced in rats by 5% sodium taurocholate. Lipid composition of AT was analyzed by HPLC–mass spectrometry. Acinar inflammatory response to total lipids as well as to either the free fatty acid (FFA) fraction or their chlorinated products (Cl-FFAs) was evaluated. For this, mRNA expression of chemokine (C-C motif) ligand 2 (CCL2) and P-selectin as well as the activation of MAPKs, NF-κB and STAT-3 were analyzed in pancreatic acini. Myeloperoxidase (MPO) activity, as an inducer of Cl-FFA generation, was also analyzed in AT. MPO activity significantly increased in necrotic (AT-N) induced changes in lipid composition of necrotic fat, such as increase in FFA and phospholipid (PL) content, generation of Cl-FFAs and increases in saturated FFAs and in the poly-:mono-unsaturated FFA ratio. Total lipids from AT-N induced overexpression of CCL2 and P-selectin in pancreatic acini as well as MAPKs phosphorylation and activation of NF-κB and STAT3. FFAs, but not Cl-FFAs, up-regulated CCL2 and P-selectin in acinar cells. We conclude that FFAs are capable of up-regulating inflammatory mediators in pancreatic acini and given that they are highly produced during AP, mainly may contribute to the inflammatory response triggered in acinar cells by fat necrosis. No role is played by Cl-FFAs generated as a result of neutrophil infiltration.     

Involvement of thrombopoietin in acinar cell necrosis in l-arginine-induced acute pancreatitis in mice

Thrombopoietin (TPO) plays an important role in injuries of different tissues. However, the role of TPO in acute pancreatitis (AP) is not yet known. The aim of the study was to determine the involvement of TPO in AP. Serum TPO was assayed in necrotizing pancreatitis induced by l-arginine in mice. Recombinant TPO and anti-TPO antibody were given to mice with necrotizing pancreatitis. Amylase, lipase, lactate dehydrogenase, myeloperoxidase activity and pancreatic water content were assayed in serum and tissue samples. Pancreas and lung tissue samples were also collected for histological evaluation. Immunohistochemistry of amylase α and PCNA were applied for the study of acinar regeneration and TUNEL assay for the detection of apoptosis in the pancreas. Increased levels of serum TPO were found in necrotizing pancreatitis. After TPO administration, more severe acinar necrosis was found and blockade of TPO reduced the acinar necrosis in this AP model. Acinar regeneration and apoptosis in the pancreas were affected by TPO and antibody treatment in necrotizing pancreatitis. The severity of pancreatitis-associated lung injury was worsened after TPO treatment, but attenuated after Anti-TPO antibody treatment. In conclusion, serum TPO is up-regulated in the necrotizing pancreatitis induced by l-arginine in mice and may be a risk factor for the pancreatic acinar necrosis in AP. As a pro-necrotic factor, blockade of TPO can attenuate the acinar necrosis in AP and may be a possible therapeutic intervention for AP.     

Signal transduction of MCP-1 expression induced by pancreatitis-associated ascitic fluid in pancreatic acinar cells

Pancreatitis-associated ascitic fluid (PAAF) is known to contribute to the progression of acute pancreatitis (AP). We have investigated the capability of PAAF to activate the expression of MCP-1 in pancreatic acinar cells and the involvement of MAPK, NF-κB and STAT3 as downstream signalling transduction pathways. The actions of dexamethasone (Dx) and N-acetylcysteine (NAC) on the PAAF's acinar effects have also been evaluated. Acinar cells were incubated for 1 hr with PAAF collected from rats with severe AP induced by sodium taurocholate in the absence or presence of Dx (10⁻⁷ M) or NAC (30 mM). MCP-1 mRNA expression, phospho-p38-MAPK, IκBα, nuclear p65 levels and nuclear translocation of STAT3 were analysed. In response to PAAF, overexpression of MCP-1, phosphorylation of p38-MAPK, degradation of IκBα and increases in p65 nuclear levels and STAT3 activity were found in acinar cells. PAAF-mediated MCP-1 up-regulation was completely suppressed by Dx and NAC. MAPK activation was only inhibited by NAC, NF-κB activation was repressed by Dx and NAC, and STAT3 pathway was strongly blocked by Dx and significantly reduced by NAC. In conclusion, acinar cells were activated by PAAF to produce MCP-1, mainly via NF-κB and STAT3 pathways. Both downstream pathways were targeted by Dx and NAC to repress the PAAF-mediated acinar MCP-1 up-regulation.     

Changes in the expression of LIMP-2 during cerulein-induced pancreatitis in rats: Effect of inhibition of leukocyte infiltration,cAMP and MAPKs early on in its development

Lysosomal integral membrane protein-2 (LIMP-2) is an important protein in lysosomal biogenesis and function and also plays a role in the tissue inflammatory response. It is known that lysosomes play a central role in acute pancreatitis, with inflammatory cell infiltration triggering the disease early on. In this study we report increases in pancreatic LIMP-2 protein and mRNA levels as early events that occur during the development of cerulein (Cer)-induced acute pancreatitis (AP) in rats. GdCl₃, a macrophage inhibitor, but not FK506, a T lymphocyte inhibitor, was able to reverse the increase in LIMP-2 expression after Cer treatment, although such reversion was abolished if the animals were depleted of neutrophils due to a vinblastine sulfate pre-treatment. Immunostaining revealed that the cellular source of LIMP-2 was mainly acinar cells. Additionally, pre-treatments with the MAPKs inhibitors SP600125 and PD98059, inhibitors of JNK and ERK½ activation, respectively, but not of rolipram, a type IV phosphodiesterase inhibitor, suppressed the increase in the expression of LIMP-2 after Cer administration. Together, these results indicate that neutrophils are able to drive a macrophage activation that would regulate the increase in LIMP-2 expression during the early phase of Cer-induced AP, with the stress kinases JNK and ERK½ also playing a coordinated role in the increase of LIMP-2 expression due to Cer.     

Inhibition of cyclooxygenase-2 ameliorates the severity of pancreatitis and associated lung injury

Cyclooxygenase-2 (COX-2), a widely distributed enzyme, plays an important role in inflammation. We have studied the role of COX-2 in acute pancreatitis and pancreatitis-associated lung injury using both the pharmacological inhibition of COX-2 and genetic deletion of COX-2. Pancreatitis was induced in mice by 12 hourly injections of cerulein. The severity of pancreatitis was assessed by measuring serum amylase, pancreatic trypsin activity, intrapancreatic sequestration of neutrophils, and acinar cell necrosis. The severity of lung injury was evaluated by measuring lactate dehydrogenase levels in the bronchoalveolar lavage fluid and by quantitating neutrophil sequestration in the lung. In both the pharmacologically inhibited and genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was reduced compared with the noninhibited strains of COX-2-sufficient mice. This reduction in injury indicates that COX-2 plays an important proinflammatory role in pancreatitis and its associated lung injury. Our findings support the concept that COX-2 inhibitors may play a beneficial role in the prevention of acute pancreatitis or in the reduction of its severity.