Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.     

Autophagy and acute pancreatitis: a novel autophagy theory for trypsinogen activation

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. Although lysosomal hydrolases, such as cathepsin B, play a key role in intrapancreatic trypsinogen activation, it remains unclear where and how trypsinogen meets these lysosomal enzymes. Autophagy is an intracellular bulk degradation system in which cytoplasmic components are directed to the lysosome/vacuole by a membrane-mediated process. To analyze the role of autophagy in acute pancreatitis, we produced a conditional knockout mouse that lacks the autophagy-related (Atg) gene Atg5 in the pancreatic acinar cells. The severity of acute pancreatitis induced by cerulein is greatly reduced in these mice. In addition, Atg5-deficient acinar cells show a significantly decreased level of trypsinogen activation. These data suggest that autophagy exerts a detrimental effect in pancreatic acinar cells by activation of trypsinogen to trypsin. We propose a theory in which autophagy accelerates trypsinogen activation by lysosomal hydrolases under acidic conditions, thus triggering acute pancreatitis in its early stage.     

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.     

Protection of cerulein-induced pancreatic fibrosis by pancreas-specific expression of Smad7

Pancreatic fibrosis is the hallmark of chronic pancreatitis, currently an incurable disease. Pancreatitis fibrosis is caused by deposition of extracellular matrix (ECM) and the underlying pathological mechanism remains unclear. In addition to its broad biological activities, TGF-β is a potent pro-fibrotic factor and many in vitro studies using cell systems have implicated a functional role of TGF-β in the pathogenesis of pancreatic fibrosis. We analyzed the in vivo role of TGF-β pathway in pancreatic fibrosis in this study. Smad7, an intracellular inhibitory protein that antagonizes TGF-β signaling, was specifically expressed in the pancreas using a transgenic mouse model. Chronic pancreatitis was induced in the mouse with repeated administration of cerulein. Smad7 expression in the pancreas was able to significantly inhibit cerulein-induced pancreatic fibrosis. Consistently, the protein levels of collagen I and fibronectin were decreased in the Smad7 transgenic mice. In addition, α-smooth muscle actin, a marker of activated pancreas stellate cells, was reduced in the transgenic mice. Taken together, these data indicate that inhibition of TGF-β signaling by Smad7 is able to protect cerulein-induced pancreatic fibrosis in vivo.     

The Novel Cytokine Interleukin-33 Activates Acinar Cell Proinflammatory Pathways and Induces Acute Pancreatic Inflammation in Mice

Background

Acute pancreatitis is potentially fatal but treatment options are limited as disease pathogenesis is poorly understood. IL-33, a novel IL-1 cytokine family member, plays a role in various inflammatory conditions but its role in acute pancreatitis is not well understood. Specifically, whether pancreatic acinar cells produce IL-33 when stressed or respond to IL-33 stimulation, and whether IL-33 exacerbates acute pancreatic inflammation is unknown.

Methods/Results

In duct ligation-induced acute pancreatitis in mice and rats, we found that (a) IL-33 concentration was increased in the pancreas; (b) mast cells, which secrete and also respond to IL-33, showed degranulation in the pancreas and lung; (c) plasma histamine and pancreatic substance P concentrations were increased; and (d) pancreatic and pulmonary proinflammatory cytokine concentrations were increased. In isolated mouse pancreatic acinar cells, TNF-α stimulation increased IL-33 release while IL-33 stimulation increased proinflammatory cytokine release, both involving the ERK MAP kinase pathway; the flavonoid luteolin inhibited IL-33-stimulated IL-6 and CCL2/MCP-1 release. In mice without duct ligation, exogenous IL-33 administration induced pancreatic inflammation without mast cell degranulation or jejunal inflammation; pancreatic changes included multifocal edema and perivascular infiltration by neutrophils and some macrophages. ERK MAP kinase (but not p38 or JNK) and NF-kB subunit p65 were activated in the pancreas of mice receiving exogenous IL-33, and acinar cells isolated from the pancreas of these mice showed increased spontaneous cytokine release (IL-6, CXCL2/MIP-2α). Also, IL-33 activated ERK in human pancreatic tissue.

Significance

As exogenous IL-33 does not induce jejunal inflammation in the same mice in which it induces pancreatic inflammation, we have discovered a potential role for an IL-33/acinar cell axis in the recruitment of neutrophils and macrophages and the exacerbation of acute pancreatic inflammation.

Conclusion

IL-33 is induced in acute pancreatitis, activates acinar cell proinflammatory pathways and exacerbates acute pancreatic inflammation.     

Expression profiling in pancreas during the acute phase of pancreatitis using cDNA microarrays

Most attacks of acute pancreatitis are self-limiting, suggesting that the pancreatic cells adapt their phenotype to prevent progression of the disease. Such phenotypic change must involve a coordinated modification in the expression of numerous genes. To identify differentially expressed genes, high-density mouse cDNA microarrays were hybridized with cDNA probes from both healthy pancreas and pancreas affected by acute pancreatitis. From the 7981 mouse genes analyzed, 239 showed significant changes in their expression during the acute phase of pancreatitis. Among them, 107 genes were up-regulated whereas 132 were down-regulated. They include genes whose function was not previously related to pancreatitis, suggesting that they are involved in some way into the acute pancreatic response. Finally, 40% of differentially expressed genes corresponded to ESTs. Demonstration that a large quantity of unexpected or yet uncharacterized genes showed altered expression during acute pancreatitis underscores the interest of a genome-based investigation. Some of these genes are certainly involved in the cellular defense against pancreatitis and, as such, deserve being studied further.     

白细胞介素-6在急性胰腺炎中的作用及其机制研究

目的:明确白细胞介素-6(IL-6)在小鼠急性胰腺炎中的作用及其机制研究。方法:通过胰胆管结扎的方法诱导小鼠急性胰腺炎;分离小鼠胰腺腺泡细胞。采用ELISA方法检测胰腺组织或腺泡细胞裂解物中的细胞因子;通过western blot分析检测组织或细胞中IL-6或ERK表达。结果:IL-6浓度在胰腺组织和腺泡细胞中显著增加(P0.05)。在离体原代小鼠腺泡细胞,TNF-α刺激增加IL-6释放(P0.05);与此同时,IL-6刺激可增加其它促炎性细胞因子的释放,两者都涉及ERK MAP激酶通路。黄酮类化合物木犀草素抑制IL-6刺激引起白细胞介素-6(IL-6)和人巨嗜细胞激活蛋白-1(CCL2/MCP-1)释放。最后进一步证实,IL-6激活人胰腺组织中的ERK。结论:IL-6在急性胰腺炎中增加,激活炎症通路并加重急性胰腺炎。     

A novel mammalian trans-membrane protein reveals an alternative initiation pathway for autophagy

Autophagy is an early cellular event during acute pancreatitis, a disease defined as pancreas self-digestion. The Vacuole Membrane Protein 1 (VMP1) is a trans-membrane protein highly activated in acinar cells early during pancreatitis-induced autophagy and it remains in the autophagosomal membrane. We have shown that VMP1 expression is able to trigger autophagy in mammalian cells, even under nutrient-replete conditions. VMP1 is induced by autophagy stimuli and its expression is required for autophagosome development. VMP1 interacts with Beclin 1 through its hydrophilic C-terminal region, which we named Atg domain, as it is essential for autophagy. Remarkably, VMP1 pancreas-specific transgenic expression in mice promotes autophagosome formation. Most of the autophagy-related proteins were described in yeast or have a yeast homologue. VMP1 does not have any known homologue in yeast but its expression is required to start the autophagic process in mammalian cells. These findings support the hypothesis that mammalian cells may regulate autophagy in a different way. We propose that VMP1 is a novel autophagy related trans-membrane protein, which may lead the way in the search for alternative mechanisms of autophagosome formation.     

P53-dependent expression of the stress-induced protein (SIP)

The mouse stress-induced protein (SIP) mRNA is activated in the pancreas with acute pancreatitis and in several cell lines in response to various stress agents. The SIP gene is alternatively spliced, generating two proteins (SIP'8 and SIP27). Both proteins, located mainly in the nucleus, promote cell death when overexpressed in vitro. We show that induction by stress agents of the expression of SIP18 and SIP27 mRNAs, observed in human- and mouse-derived cell lines, is absent from cells with deleted, mutated or inactive p53, suggesting that regulation of SIP gene expression is dependent on p53. That hypothesis is consistent with the presence of a functional p53-response element within the promoter region of the mouse SIP gene and confirmed by the induction of SIP mRNA expression in mouse embryo fibroblasts upon activation of a p53-dependent pathway by transfection with rasV12 or rasV12/E1A. In conclusion, SIP being a proapoptotic gene induced through p53 activation could be a stress-induced gene with antitumour properties.     

Cell-cycle distribution of pancreatic cells from rats with acute pancreatitis induced by bile-pancreatic obstruction

The aim of this study was to analyze, using electron microscopy, the morphological alterations that progressively appear in the pancreas of rats with acute pancreatitis induced by bile-pancreatic obstruction over 48 h. In addition, in order to ascertain the capability of pancreas regeneration at different stages of pancreatitis, the distribution of pancreatic cells throughout the different phases of the cell cycle was also analyzed by flow cytometry using propidium iodide staining. Interstitial edema, macrophage infiltration, vacuolization, and dilatation of endoplasmic reticulum were observed from 1.5 h after obstruction onward. Interestingly, cell cycle studies showed an increased proportion of S-phase cells at early stages of pancreatitis (1.5 h after obstruction), which leads to a significant increase in cells in G2/M phase 12 h after pancreatic obstruction. Histological studies revealed severe alterations in pancreas of rats with obstruction maintained over 48 h which affects the nuclear structure. Intracellular disorganization, apoptosis, and focal necrosis were observed at this stage. Furthermore, flow-cytometric analysis of cell DNA contents showed a significant decrease in the proportion of S and G2/M cells and a significant increase in G0/G1 cells, suggesting an arrest of almost all cells in quiescent states. These results suggest that rat pancreas cells are able to recover during the first 12 h after pancreatic obstruction. However, the gland would lose its ability to regenerate if the obstruction was maintained for longer periods.     

Alteration in inflammatory/apoptotic pathway and histone modifications by nordihydroguaiaretic acid prevents acute pancreatitis in swiss albino mice

Reactive oxygen radicals, pro-inflammatory mediators and cytokines have been implicated in caerulein induced acute pancreatitis. Nordihydroguaiaretic acid (NDGA), a plant lignin, has marked anti-inflammatory properties. The present study aimed to investigate the possible protective effect of NDGA against caerulein induced pancreatitis. Acute pancreatitis was induced by intraperitoneal administration of eight doses of caerulein in male swiss albino mice. NDGA was administered after 9 h of acute pancreatitis induction. Pancreatic damage and the protective effect of NDGA were assessed by oxidative stress parameters and histopathology of pancreas. The mRNA expression of heat shock proteins (DNAJ C15 and HSPD1) was examined by real-time RT-PCR analysis. Expression of HSP 27, NF-κB, TNF-α, p-p38, Bcl-2, p-PP2A, procaspase-3, caspase-3 and histone modifications were examined by western blotting. NDGA attenuated the oxidative stress, led to increased plasma α-amylase and decreased IGF-1 in AP mice. It modulated the mRNA and protein levels of heat shock proteins and reduced the expression of NF-κB, TNF-α and p-p38. It increased the number of TUNEL positive apoptotic cells in the pancreas of AP mice. In addition, NDGA prevented the changes in modifications of histone H3 in acute pancreatitis. To best of our knowledge, this is the first report which suggests that NDGA prevents the progression of acute pancreatitis by involving alteration of histone H3 modifications and modulating the expression of genes involved in inflammatory/apoptotic cascade, which may be responsible for decreased necrosis and increased apoptosis in this model of acute pancreatitis.     

Autophagy and acute pancreatitis: A novel autophagy theory for trypsinogen activation

Auto-digestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. Although lysosomal hydrolases, such as cathepsin B, play a key role in intrapancreatic trypsinogen activation, it remains unclear where and how trypsinogen meets these lysosomal enzymes. Autophagy is an intracellular bulk degradation system in which cytoplasmic components are directed to the lysosome/vacuole by a membrane-mediated process. To analyze the role of autophagy in acute pancreatitis, we produced a conditional knockout mouse that lacks the autophagy-related (Atg) gene Atg5 in the pancreatic acinar cells. The severity of acute pancreatitis induced by cerulein is greatly reduced in these mice. In addition, Atg5-deficient acinar cells show a significantly decreased level of trypsinogen activation. These data suggest that autophagy exerts a detrimental effect in pancreatic acinar cells by activation of trypsinogen to trypsin. We propose a theory in which autophagy accelerates trypsinogen activation by lysosomal hydrolases under acidic conditions, thus triggering acute pancreatitis in its early stage.

Addendum to: Hashimoto D, Ohmuraya M, Hirota M, Yamamoto A, Suyama K, Ida S, Okumura Y, Takahashi E, Kido H, Araki K, Baba H, Mizushima N, Yamamura K. Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells. J Cell Biol 2008; 181:1065-72.     

Phytic acid exerts protective effects in cerebral ischemia-reperfusion injury by activating the anti-oxidative protein sestrin2

ABSTRACT

Cerebral ischemia reperfusion (I/R) is a therapeutic strategy for ischemia; however, it usually causes injury by the aspect of inflammation and neuron apoptosis. This investigation aims to investigate the protective effects of phytic acid (IP6) for cerebral I/R injury in vitro. PC-12 cells under Oxygen and glucose deprivation/reperfusion (OGD/R) were performed to mimic cerebral I/R. IP6 was pretreated before PC-12 cells under OGD/R treatment. The data showed that IP6 activated the expression of sestrin2 in OGD/R injured PC-12 cells. IP6 inhibited OGD/R induced inflammation, oxidative stress, and apoptosis by activating sestrin2. Besides, p38 MAPK may mediate the effects of sestrin2 activated by IP6. Therefore, IP6 can be a potential drug to prevent neurological damage in cerebral I/R injury.