Trypsinogen Activation Peptide Research Articles

Trypsin and Trypsinogen Activation Peptide in the Prediction of Severity of Acute Pancreatitis.

To assess the predictive value of serum trypsin and trypsinogen activation peptide (TAP) for the severity of AP through a single center cohort study as well as a systematic review of the current literature. A literature search was conducted using Medline (PubMed), EMBASE and the Cochrane Central Register. A total of 142 patients with acute pancreatitis (AP) were included in the cohort study and parameters of the revised Atlanta criteria of 2012 and the APACHE II were assessed. The review showed promising results for the predictive value of serum trypsinogen-2 but conflicting results for serum TAP and trypsin. In the cohort study, patients were observed for 4 days after diagnosis of AP; 9 patients had severe AP, 35 patients had moderate AP and 81 patients had mild AP. The ratio of the geometric mean of severe vs. mild AP for trypsin was 0.72 (95% CI: 0.51-1.00), p = 0.053 and, for TAP, 0.74 (95% CI: 0.54-1.01), p = 0.055, respectively. The cohort study showed an inverse correlation of serum levels of TAP and trypsin with severity of AP. Serum TAP and trypsin have an inferior predictive value of severity of AP compared to the clinical APACHE II score.

Plasma biomarkers TAP, CPA1, and CPA2 for the detection of pancreatic injury in rat: the development of a novel multiplex IA-LC-MS/MS assay and biomarker performance evaluation

Drug-induced pancreatic injury (DIPI) is an issue seen in drug development both in nonclinical and clinical contexts. DIPI is typically monitored by measurement of lipase and/or amylase, however, both enzymes lack sensitivity and specificity. Although candidate protein biomarkers specific to pancreas exist, antibody-based assay development is difficult due to their small size or the rapid cleavage by proteolytic enzymes released during pancreatic injury. Here we report the development of a novel multiplexed immunoaffinity-based liquid chromatography mass spectrometric assay (IA-LC-MS/MS) for trypsinogen activation peptide (TAP) and carboxypeptidases A1 and A2 (CPA1, CPA2). This method is based on the enzymatic digestion of the target proteins, immunoprecipitation of the peptides with specific antibodies and LC-MS/MS analysis. This assay was used to detect TAP, CPA1, and CPA2 in 470 plasma samples collected from 9 in-vivo rat studies with pancreatic injury and 8 specificity studies with injury in other organs to assess their performance in monitoring exocrine pancreas injury. The TAP, CPA1, and CPA2 response was compared to histopathology, lipase, amylase and microRNA217. In summary, TAP, CPA1, and CPA2 proteins measured in rat plasma were sensitive and specific biomarkers for monitoring drug-induced pancreatic injury; outperforming lipase and amylase both by higher sensitivity of detection and by sustained increases in plasma observed over a longer time period. These protein-based assays and potentially others under development, are valuable tools for use in nonclinical drug development and as future translatable biomarkers for assessment in clinical settings to further improve patient safety.

Evolutionary expansion of polyaspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis.

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen paralogs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased the autoactivation of T7 trypsinogen threefold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side chains are preferred for maximal autoactivation, and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by NH2-terminal truncation with chymotrypsin C reduced the autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable with that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the polyaspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.NEW & NOTEWORTHY Unwanted autoactivation of the digestive protease trypsinogen can result in pancreatitis. The trypsinogen activation peptide contains a polyaspartate motif that suppresses autoactivation. This study demonstrates that evolutionary expansion of these aspartate residues in mouse cationic trypsinogen further inhibits autoactivation and enhances protection against pancreatitis.

Pancreas-specific SNAP23 depletion prevents pancreatitis by attenuating pathological basolateral exocytosis and formation of trypsin-activating autolysosomes

ABSTRACT Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKβ-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKβ inhibitors, and confirmed by the inability of IKBKB/IKKβ phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKβ’s involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy. Abbreviations: AL: autolysosome; AP: acute pancreatitis; AV: autophagic vacuole; CCK: cholecystokinin; IKBKB/IKKβ: inhibitor of nuclear factor kappa B kinase subunit beta; SNAP23: synaptosome associated protein 23; SNARE: soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; STX: syntaxin; TAP: trypsinogen activation peptide; VAMP: vesicle associated membrane protein; ZG: zymogen granule.

LAP-like non-canonical autophagy and evolution of endocytic vacuoles in pancreatic acinar cells

ABSTRACT Activation of trypsinogen (formation of trypsin) inside the pancreas is an early pathological event in the development of acute pancreatitis. In our previous studies we identified the activation of trypsinogen within endocytic vacuoles (EVs), cellular organelles that appear in pancreatic acinar cells treated with the inducers of acute pancreatitis. EVs are formed as a result of aberrant compound exocytosis and subsequent internalization of post-exocytic structures. These organelles can be up to 12 μm in diameter and can be actinated (i.e. coated with F-actin). Notably, EVs can undergo intracellular rupture and fusion with the plasma membrane, providing trypsin with access to cytoplasmic and extracellular targets. Unraveling the mechanisms involved in cellular processing of EVs is an interesting cell biological challenge with potential benefits for understanding acute pancreatitis. In this study we have investigated autophagy of EVs and discovered that it involves a non-canonical LC3-conjugation mechanism, reminiscent in its properties to LC3-associated phagocytosis (LAP); in both processes LC3 was recruited to single, outer organellar membranes. Trypsinogen activation peptide was observed in approximately 55% of LC3-coated EVs indicating the relevance of the described process to the early cellular events of acute pancreatitis. We also investigated relationships between actination and non-canonical autophagy of EVs and concluded that these processes represent sequential steps in the evolution of EVs. Our study expands the known roles of LAP and indicates that, in addition to its well-established functions in phagocytosis and macropinocytosis, LAP is also involved in the processing of post-exocytic organelles in exocrine secretory cells. Abbreviations AP: acute pancreatitis; CCK: cholecystokinin; CLEM: correlative light and electron microscopy; DPI: diphenyleneiodonium; EV: endocytic vacuole; LAP: LC3-associate phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PACs: pancreatic acinar cells; PFA: paraformaldehyde; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; Res: resveratrol; TAP: trypsinogen activation peptide; TEM: transmission electron microscopy; TLC-S: taurolithocholic acid 3-sulfate; TRD: Dextran Texas Red 3000 MW Neutral; ZGs: zymogen granules.

Early trypsin activation develops independently of autophagy in caerulein-induced pancreatitis in mice.

Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60min and serum amylase within 2h, but fluorescent autophagosome formation only by 4h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile-Pro-Arg-AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease.

Investigation of non-pharmaceutical cures for acute pancreatitis induced by cerulein in rats

Purpose: To investigate the effect of N-acetylcysteine (NAC) alone or in combination with α-lipoic acid (ALA) on cerulein induced acute pancreatitis (AP) in rats.Methods: AP was induced in 40 healthy male albino rats using a daily dose of cerulein (40 μg/kg) for three days. The rats were divided into four groups: control, recovery (pancreatitis rats left without treatment), NAC (pancreatitis rats injected intraperitoneal (i.p.) with 25 mg NAC/kg daily) and NAC+ALA (pancreatitis rats treated with 25 mg NAC+100mg ALA/kg daily). The levels of carcinoembryonic antigen (CEA), cancer antigen242 (CA242), tumor necrosis factor-α (TNF-α), interlukin-1β (IL-1β), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as the enzyme activities (amylase, lipase and trypsinogen activation peptide) were determined after 2- and 4-weeks of treatment.Results: Treatment with NAC or NAC+ALA led to a marked decrease in the levels of tumor markers, inflammatory cytokines, enzymes activity, DNA and RNA. The best amelioration effect occurred in the NAC+ALA rats group.Conclusion: NAC induces a significant improvement in all studied parameters. Incorporation of NAC and ALA results in preeminent effects and it is considered an ideal cure for acute pancreatitis.Keywords: Acute pancreatitis, Cerulein, N-acetylcysteine, α-Lipoic acid

Novel Pathogenic PRSS1 Variant p.Glu190Lys in a Case of Chronic Pancreatitis.

Mutations in the PRSS1 (serine protease 1) gene encoding human cationic trypsinogen cause hereditary pancreatitis or may be associated with sporadic chronic pancreatitis. The mutations exert their pathogenic effect either by increasing intra-pancreatic trypsinogen activation (trypsin pathway) or by causing proenzyme misfolding and endoplasmic reticulum stress (misfolding pathway). Here we report a novel heterozygous c.568G>A (p.Glu190Lys) variant identified in a case with chronic pancreatitis. The parents of the index patient had no history of pancreatitis but were unavailable for genetic testing. Functional characterization revealed 2.5-fold increased autoactivation of the mutant trypsinogen relative to wild type. Unlike many other clinically relevant PRSS1 mutations, p.Glu190Lys did not alter the chymotrypsin C (CTRC)-dependent degradation of trypsinogen nor did it increase CTRC-mediated processing of the trypsinogen activation peptide. Cellular secretion of the mutant protein was unchanged indicating normal folding behavior. Based on the genetic and functional evidence, we classify the p.Glu190Lys PRSS1 variant as likely pathogenic, which stimulates autoactivation of cationic trypsinogen independently of CTRC.

Establishment and evaluation of a Beagle model of grade III pancreatic trauma.

Pancreatic trauma (PT) is a severe abdominal injury that is often combined with multiple organ injury. It is a severe disease that is difficult to diagnose and has a high mortality rate, particularly for grade III PT. The pathogenesis, disease progress and complications have not been fully investigated due to the lack of a reliable animal model. To address this, a Beagle model of grade III PT was established in the present study using a procedure involving rupture of the main pancreatic duct. Peripancreatic effusions and the degree of pancreatic damage were examined by routine ultrasound and contrast-enhanced ultrasound (CEUS). Also, ascites were collected for the examination of amylase and lipase levels, and whole blood samples were collected for the analysis of amylase, lipase, C-reactive protein (CRP), interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels in the serum. Urine samples were also collected for the examination of trypsinogen activation peptide (TAP). In addition, the pancreas was sectioned and stained with hematoxylin and eosin. In comparison with routine ultrasound, CEUS showed a large area of focal trauma, with a depth greater than half of the anteroposterior diameter of the pancreas, with a clear boundary, clear capsular rupture and trauma induced by active bleeding. The volume of ascites peaked at 48 h post-trauma and decreased thereafter. Amylase and lipase levels in the ascites were elevated at 24 h post-trauma and significantly decreased at 48 and 72 h post-trauma (P<0.01). In addition, serum amylase and lipase levels increased to peak levels at 48 h post-trauma and then decreased (P<0.05), while serum CRP, IL-6 and TNF-α levels peaked at 24 h post-trauma and then decreased (P<0.05). Urinary TAP levels also peaked at 24 h post-trauma and subsequently decreased (P<0.05). At 72 h post-trauma, the pancreatic cells were loosely distributed, with damaged acini, hyperchromatic nuclei and severe inflammatory cell invasion. These results indicated that the Beagle model of grade III PT was satisfactorily established, and that CEUS is potentially useful as an auxiliary diagnosis method for PT. This animal model may be useful for studying the pathogenesis, disease progress and complications of PT.

Hepatic alcohol dehydrogenase deficiency induces pancreatic injury in chronic ethanol feeding model of deer mice

The single most common cause of chronic pancreatitis (CP, a serious inflammatory disease) is chronic alcohol abuse, which impairs hepatic alcohol dehydrogenase (ADH, a major ethanol oxidizing enzyme). Previously, we found ~5 fold greater fatty acid ethyl esters (FAEEs), and injury in the pancreas of hepatic ADH deficient (ADH−) vs. hepatic normal ADH (ADH+) deer mice fed 3.5g% ethanol via liquid diet daily for two months. Therefore, progression of ethanol-induced pancreatic injury was determined in ADH− deer mice fed ethanol for four months to delineate the mechanism and metabolic basis of alcoholic chronic pancreatitis (ACP). In addition to a substantially increased blood alcohol concentration and plasma FAEEs, significant degenerative changes, including atrophy and loss of acinar cells in some areas, ultrastructural changes evident by such features as swelling and disintegration of endoplasmic reticulum (ER) cisternae and ER stress were observed in the pancreas of ethanol-fed ADH− deer mice vs. ADH+ deer mice. These changes are consistent with noted increases in pancreatic injury markers (plasma lipase, pancreatic trypsinogen activation peptide, FAEE synthase and cathepsin B) in ethanol-fed ADH− deer mice. Most importantly, an increased levels of pancreatic glucose regulated protein (GRP) 78 (a prominent ER stress marker) were found to be closely associated with increased phosphorylated eukaryotic initiation factor (eIF) 2α signaling molecule in PKR-like ER kinase branch of unfolded protein response (UPR) as compared to X box binding protein 1S and activating transcription factor (ATF)6 - 50kDa protein of inositol requiring enzyme 1α and ATF6 branches of UPR, respectively, in ethanol-fed ADH− vs. ADH+ deer mice. These results along with findings on plasma FAEEs, and pancreatic histology and injury markers suggest a metabolic basis of ethanol-induced pancreatic injury, and provide new avenues to understand metabolic basis and molecular mechanism of ACP.

Effects of Calcium Channel Blockers on Trypsinogen Activation and Severity of Cerulein-induced Acute Pancreatitis in Rats

Acute pancreatitis is an inflammatory disease of the pancreas associated with high mortality but without a specific therapy. Intrapancreatic protease activation appears to be an early event in the development of acute pancreatitis but its association with pathogenesis has not been established. Recently it has been demonstrated that an enormous rise in the cytoplasmic Ca2+ levels of pancreatic acinar cells could be associated with the development of acute pancreatitis, thereby suggesting that Ca2+ channel could be a potential target for treatment. In this study, the protective effects of calcium channel blockers such as 2-aminoethoxydiphenyl borate, dantrolene and verapamil were investigated in cerulein-induced acute pancreatitis in vivo. Rats were divided into five groups. Group 1 (control), group 2 (cerulein, 100 µg/kg), group 3 (cerulein+ 2-aminoethoxydiphenyl borate, mg/kg), group 4 (cerulein+dantrolene, 10 mg/kg) and group 5 (cerulein+verapamil, 2.5 mg/kg). Activities of amylase, lipase, cathepsin B, pancreatic secretory trypsin inhibitor and trypsin and levels of trypsinogen activation peptide and trypsinogen were measured addition to histological examination of the sections of pancreas. The results showed that cerulein increased the amylase, lipase and trypsin activities and trypsinogen activation peptide levels significantly. Administration of calcium channel blockers significantly protected the pancreas from histological damage (p<0.05). The results of this study demonstrated that calcium channel blockers can mitigate early protease activation and pancreas injury. It was suggested regulation of calcium channels would be relevant to maintain pancreatic acinar cell homeostasis and further research is necessary to understand the protective effects of calcium channel blockers.

Trypsinogen Activation Peptide Induces HMGB1 Release from Rat Pancreatic Acinar Cells.

BackgroundThe development of acute pancreatitis (AP) is associated with intracellular events in pancreatic cells, as well as with early and late inflammatory responses; however, their underlying mechanisms remain unclear. This study investigated trypsinogen activation peptide (TAP)-induced release of high mobility group box-l (HMGB1) from pancreatic acinar cells and how ethyl pyruvate (EP) affects this release.MethodologyPancreatic acinar cells from Sprague Dawley rats were divided into control, TAP (administered TAP), and EP (administered TAP and EP) groups. Cells were collected at 3, 6, 12, and 24 hours after TAP administration to detect HMGB1 mRNA and protein levels using quantitative PCR (qPCR) and Western blotting, respectively.ResultsThe TAP and EP groups exhibited higher levels of HMGB1 mRNA and protein expression (P<0.05) than the control group. The HMGB1 mRNA and protein expression levels also increased with prolonged TAP activity (P<0.05)–especially at 12 and 24 hours (P<0.01)–and showed positive correlations with TAP activity duration (3, 6, 12, and 24 hours) (r=0.971, P<0.01; r=0.966, P<0.01, respectively).ConclusionTAP induces HMGB1 release from pancreatic acinar cells. A positive temporal link exists between early TAP activity and late HMGB1 expression in AP, and EP inhibits HMGB1 release.

Interferon-γ Aggravated L-Arginine-Induced Acute Pancreatitis in Sprague-Dawley Rats and Its Possible Mechanism: Trypsinogen Activation and Autophagy Up-regulation.

It has been confirmed that the initiation of acute pancreatitis (AP) involves intracellular trypsinogen activation and local cytokines release during its early stage. The former is related to autophagic disorder, and the latter is resulting from nuclear factor-κB activation. Although great efforts have been exerted, there is still nonspecific treatment currently. Recent data showed that immunomodulatory therapy is always promising. However, the effects of interferon-γ (IFN-γ) on AP are controversial. This study is designed to elucidate the effects of IFN-γ on AP severity and explore its impacts on the major mechanisms of AP. Sprague-Dawley rats were used to establish AP model by intraperitoneal injection of 20% L-arginine (4 g/kg) twice with an interval of 1 hour. The effects of IFN-γ on the severity of AP, trypsinogen activation peptide, and tumor necrosis factor α, Interleukin-1, Interleukin-6 levels, and autophagy activity were detected. Compared with AP rats without IFN-γ administration, AP rats with IFN-γ administration had more severe pathological changes in pancreata, greater levels of trypsinogen activation concomitant with autophagy up-regulation, and higher levels of cytokine release. Interferon-γ aggravated L-arginine-induced AP in Sprague-Dawley rats and led to intracellular trypsinogen activation and inflammatory response. The former may be related to autophagy up-regulation.

Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis

The human pancreas expresses two major trypsinogen isoforms, cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2). Mutations in PRSS1 cause hereditary pancreatitis by altering cleavage of regulatory nick sites by chymotrypsin C (CTRC) resulting in reduced trypsinogen degradation and increased autoactivation. Despite 90% identity with PRSS1 and a strong propensity for autoactivation, mutations in PRSS2 are not found in hereditary pancreatitis suggesting that activation of this isoform is more tightly regulated. Here, we demonstrated that CTRC promoted degradation and thereby markedly suppressed autoactivation of human anionic trypsinogen more effectively than previously observed with cationic trypsinogen. Increased sensitivity of anionic trypsinogen to CTRC-mediated degradation was due to an additional cleavage site at Leu-148 in the autolysis loop and the lack of the conserved Cys-139-Cys-206 disulfide bond. Significant stabilization of anionic trypsinogen against degradation was achieved by simultaneous mutations of CTRC cleavage sites Leu-81 and Leu-148, autolytic cleavage site Arg-122, and restoration of the missing disulfide bridge. This stands in stark contrast to cationic trypsinogen where single mutations of either Leu-81 or Arg-122 resulted in almost complete resistance to CTRC-mediated degradation. Finally, processing of the trypsinogen activation peptide at Phe-18 by CTRC inhibited autoactivation of anionic trypsinogen, although cationic trypsinogen was strongly stimulated. Taken together, the observations indicate that human anionic trypsinogen is controlled by CTRC in a manner that individual natural mutations are unlikely to increase stability enough to promote intra-pancreatic activation. This unique biochemical property of anionic trypsinogen explains the lack of association of PRSS2 mutations with hereditary pancreatitis.

A novel thermosensitive in-situ gel of gabexate mesilate for treatment of traumatic pancreatitis: An experimental study

Gabexate mesilate (GM) is a trypsin inhibitor, and mainly used for treatment of various acute pancreatitis, including traumatic pancreatitis (TP), edematous pancreatitis, and acute necrotizing pancreatitis. However, due to the characteristics of pharmacokinetics, the clinical application of GM still needs frequently intravenous administration to keep the blood drug concentration, which is difficult to manage. Specially, when the blood supply of pancreas is directly damaged, intravenous administration is difficult to exert the optimum therapy effect. To address it, a novel thermosensitive in-situ gel of gabexate mesilate (GMTI) was developed, and the optimum formulation of GMTI containing 20.6% (w/w) P-407 and 5.79% (w/w) P188 with different concentrations of GM was used as a gelling solvent. The effective drug concentration on trypsin inhibition was examined after treatment with different concentrations of GMTI in vitro, and GM served as a positive control. The security of GMTI was evaluated by hematoxylin-eosin (HE) staining, and its curative effect on grade II pancreas injury was also evaluated by testing amylase (AMS), C-reactive protein (CRP) and trypsinogen activation peptide (TAP), and pathological analysis of the pancreas. The trypsin activity was slightly inhibited at 1.0 and 5.0 mg/mL in GM group and GMTI group, respectively (P<0.05 vs. P-407), and completely inhibited at 10.0 and 20.0 mg/mL (P<0.01 vs. P-407). After local injection of 10 mg/mL GMTI to rat leg muscular tissue, muscle fiber texture was normal, and there were no obvious red blood cells and infiltration of inflammatory cells. Furthermore, the expression of AMS, CRP and TAP was significantly increased in TP group as compared with control group (P<0.01), and significantly decreased in GM group as compared with TP group (P<0.01), and also slightly inhibited after 1.0 and 5.0 mg/mL GMTI treatment as compared with TP group (P<0.05), and significantly inhibited after 10.0 and 20.0 mg/mL GMTI treatment as compared with TP group (P<0.01). HE staining results demonstrated that pancreas cells were uniformly distributed in control group, and they were loosely arranged, partially dissolved, with deeply stained nuclei in TP group. Expectedly, after gradient GMTI treatment, pancreas cells were gradually restored to tight distribution, with slightly stained nuclei. This preliminary study indicated that GMTI could effectively inhibit pancreatic enzymes, and alleviate the severity of trauma-induced pancreatitis, and had a potential drug developing and clinic application value.

Changes and its significance of autophagy in rats with acute necrosis pancreatitis

Objective To investigate the changes and significance of autophagy in rats with experimental acute necrosis pancreatitis (ANP). Methods According to method of random number, 18 rats were randomly divided into control group, ANP group, ANP+ rapamycin (RAP) group. The ANP rat model was established by intraperitoneal injection of 20% L–arginine. The rats of ANP+ RAP group were intraperitoneal injected with RAP 1.2 mg/kg at 30 minutes before modeling. The rats of control group were intraperitoneal injected with 0.9% NaCl solution. The blood was drawed from the hearts nine hours after modeling for subsequent experiments. Serum levels of trypsinogen activation peptide (TAP), interleukin (IL–1), IL–6 and tumor necrosis factor (TNF)α were measured with enzyme–linked immunosorbent assay. The pancreatic tissues were pathologically scored. Autophagy–related structures in rat pancreatic acinar cells were observed by transmition electron microscopy. The expression of autophagy marker microtuble assciated protein 1 light chain 3 (LC3)–Ⅱ and Beclin–1 at mRNA and protein level were measured by quantitative real–time polymerase chain reaction (qRT–PCR), Western bloting and immunohistochemistry. The single factor analysis of variance was used for mean comparison among groups. Results A rat model of ANP was successfully established.Histopathological score of pancreas acinar cell necrosis of ANP+ RAP group (2.19±1.38) was higher than that of ANP group (0.97±0.68), and the difference was statistically significant(F=33.75, P 0.05). Conclusions Autophagy increased in rats with ANP. Promoting autophagy could significantly activate trypsinogen, aggravate pancreatic injury and increase inflammation reaction, which indicated that autophagy might involve in the pathogenesis of ANP through trypsinogen activation. Key words: Pancreatitis acute necrotizing; Autophagy; Trypsinogen

Effect of Janus activated kinase 2/signal transducer and activators of transcription 3 signaling pathway on the release of high mobility group box 1 in trypsinogen activation peptide-induced rat pancreatic acinar cells

Objective To research the effect of Janus activated kinase 2/signal transducer and activators of transcription 3 (JAK2/STAT3) signaling pathway on the release of high mobility group box 1 (HMGB1) in trypsinogen activation peptide (TAP)-induced rat pancreatic acinar cells.Methods The rat pancreatic acinar cells were isolated,and randomly divided imo normal control group,TAP promotion group (3 nmol/L),AG490 treatment group (25 μ mol/L) and RPM treatment group (40 μg/L).Within 3-24 h the HMGB1 mRNA and protein expression levels were detected by using PCR and Western blotting.Results In comparison with the normal control group (1.01 ± 0.04),HMGB1 mRNA expression was significantly increased at 6,12 and 24 h in TAP promotion group (2.87 ±0.08,32.85 ± 1.48 and 38.43 ±1.60,P ＜0.01 for all),that was dramatically inhibited at 12 and 24 h in AG490 treatment group as compared with TAP promotion group (23.40 ± 1.38 and 19.44 ± 0.89,P ＜ 0.01 for all),and that was dramatically inhibited at 12 and 24 h in RPM treatment group as compared with TAP promotion group (21.14 ± 1.32 and 17.76 ± 0.57,P＜ 0.01 for all).In comparison with the normal control group (0.39 ±0.02),the HMGB1 expression was significantly increased at 3,6,12 h and 24 h in TAP promotion group (0.46 ± 0.02,0.67 ± 0.03,0.72 ± 0.02 and 0.90 ± 0.03,P ＜ 0.01 for all),that was dramatically inhibited at 12 and 24 h in AG490 treatment group as compared with TAP promotion group (0.60 ±0.02 and 0.54 ±0.01,P ＜0.01 for all),and that was dramatically inhibited at 12 and 24 h in RPM treatment group as compared with TAP promotion group (0.59 ± 0.02 and 0.58 ± 0.01,P ＜ 0.01 for all).Conclusion TAP induces rat pancreatic acinar cells to release HMGB1,which is related to intracellular JAK2/STAT3 signaling pathway. Key words: Pancreatic acinar cells; Trypsinogen activation peptide; Highly-mobility group box-1 ; Janus activated kinase 2 ; Rapamycin

Identification of a Novel Frame-Shift Mutation in PRSS1 Gene in Han Patients with Autoimmune Pancreatitis

To detect mutations of trypsinogen gene (PRSS1) in patients with autoimmune pancreatitis (AIP) and to determine the underlying pathogenesis. DNA sequencing was used to detect full-length of PRSS1, cystic fibrosis transmembrane conductance regulator (CFTR), and pancreatic secretory trypsin inhibitor (SPINK1) genes mutations in an AIP family and a sporadic case and 520 normal controls. Furthermore, a mutant-expressing system was constructed for functional confirmation. For the first time, we report a deletion mutation at exon 2 of PRSS1 gene (IVS 2 +56_60 del CCCAG) which encoded a truncated PRSS1 protein without trypsinogen activation peptide (TAP). Vitro functional study suggested the identified mutation would result in loss of PRSS1 activity. Mutant trypsinogen activated at a faster rate than wild-type trypsinogen in the autoactivation experiment. Histopathologic examination revealed the ratio of IgG4/IgG-positive plasma cells exceeded 0.455 in pancreas, and the patients responded to glucocorticoids. PRSS1: IVS 2 +56_60 del CCCAG is a noval mutant which may contribute to AIP pathogenesis.

Structure basis for the unique specificity of medaka enteropeptidase light chain

Structure basis for the unique specificity of medaka enteropeptidase light chain

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.