OBJECTIVE: To investigate the expression of Ca²⁺/calmodulin-dependent protein kinase II (CaMK Ⅱ) in pancreatic tissues of mice with severe acute pancreatitis (SAP) and explore the protective effect of KN93, a CaMK Ⅱ inhibitor, against pancreatic injury in SAP and the possible mechanism. METHODS: Thirty-six healthy male C57 mice were randomly divided into sham operation group, SAP group, KN93 group and SAP + KN93 group (n=9). Serum and pancreatic tissue samples were collected 24 h after modeling. The pathological changes in the pancreatic tissues were observed using HE staining. Serum lipase and amylase activities and the levels of inflammatory factors were detected using ELISA. Western blotting was used to detect the expressions of CaMK Ⅱ, p-CaMK Ⅱ, p-NF-κB, MAPK and p-MAPK in mouse pancreas. RESULTS: Compared with those in sham operation group, the expressions of p-CaMK Ⅱ, p-NF-κB and p-MAPK were significantly increased in SAP group (P < 0.05). KN93 treatment obviously alleviated pathological injuries of the pancreas in SAP mice, and significantly lowered serum levels of lipase, amylase and inflammatory factors (TNF-α and IL-6) and phosphorylation levels of NF-κB, ERK and MAPK proteins (P < 0.05). CONCLUSION The activity of CaMK Ⅱ is significantly increased in the pancreatic tissue of SAP mice. KN93 can alleviate pancreatic injury and inflammation in SAP mice possibly through the ERK/MAPK signaling pathway.
Acute Disease ; Animals ; Inflammation/metabolism* ; Male ; Mice ; NF-kappa B/metabolism* ; Pancreas/pathology* ; Pancreatitis/pathology*

Acute pancreatitis (AP) is a common and potentially life-threatening pancreatic inflammatory disease. Although it is usually self-limiting, up to 20% of patients will develop into severe AP. It may lead to systemic inflammatory response syndrome and multiple organ dysfunction, affecting the lungs, kidneys, liver, heart, etc. Surviving patients usually have sequelae of varying degrees, such as chronic hyperglycemia after AP (CHAP), pancreatic exocrine insufficiency, and chronic pancreatitis. Lacking specific target treatments is the main reason for high mortality and morbidity, which means that more research on the pathogenesis of AP is needed. Ferroptosis is a newly discovered regulated cell death (RCD), originally described in cancer cells, involving the accumulation of iron and the depletion of plasma membrane polyunsaturated fatty acids, and a caspase-independent RCD. It is closely related to neurological diseases, myocardial infarction, ischemia/reperfusion injury, cancer, etc. Research in the past years has also found the effects of ferroptosis in AP, pancreatic cancer, and AP complications, such as acute lung injury and acute kidney injury. This article reviews the research progress of ferroptosis and its association with the pathophysiological mechanisms of AP, trying to provide new insight into the pathogenesis and treatment of AP, facilitating the development of better-targeted drugs.
Humans ; Pancreatitis/pathology* ; Acute Disease ; Ferroptosis ; Pancreas/pathology* ; Acute Lung Injury/metabolism*

BACKGROUNDIndoleamine 2,3-dioxygenase (IDO), an enzyme for tryptophan metabolism through the kynurenine pathway, exhibits an immunosuppressive effect and induces immune tolerance in tumor cells. The effects of IDO on pancreatic cancer are poorly understood. This study aimed to investigate the expression and prognostic significance of IDO in pancreatic cancer.

METHODSWe evaluated the protein expression of IDO in PANC-1, CFPAC-1, and BxPC-3 cell lines with or without 48 h treatment by 500 U/ml interferon-γ (IFN-γ). We performed immunohistochemical staining and Western blot analysis for IDO expression in both pancreatic cancer and normal pancreas tissues obtained from Chinese PLA General Hospital from July 2012 to December 2013. Survival analysis was performed to correlate IDO expression and histopathologic parameters with overall survival. The Kaplan-Meier method and Cox proportional hazards regression model were conducted.

RESULTSPANC-1, CFPAC-1, and BxPC-3 cell lines expressed IDO at the protein level, and the relative expression amount increased after stimulation with 500 U/ml IFN-γ. Immunohistochemical analysis results revealed that high IDO expression was observed in 59% of pancreatic adenocarcinoma tissues. Compared with normal pancreatic tissues, pancreatic adenocarcinoma showed significantly higher IDO expression levels, especially among patients with high tumor node metastasis (TNM) stages (χ2 = 4.550, P = 0.030), poor histological differentiation (χ2 = 5.690, P = 0.017), and lymph node metastasis (χ2 = 4.340 P = 0.037). Kaplan-Meier survival curves showed that high IDO expression was correlated with low survival rates (hazard ratio [HR] = 0.49 P = 0.009). Multivariate analysis using Cox proportional hazards model indicated that lymph node metastasis (HR = 0.35 P = 0.010) and IDO expression (HR = 0.42 P = 0.020) were two independent prognostic predictors of pancreatic adenocarcinoma.

CONCLUSIONSThe study confirmed that high IDO expression in pancreatic adenocarcinoma was related to poor prognosis of patients. These findings provided evidence that IDO was involved in pancreatic adenocarcinoma progression and might serve as a relevant therapeutic target.

Adenoma ; enzymology ; mortality ; pathology ; Blotting, Western ; Cell Line, Tumor ; Female ; Humans ; Immunohistochemistry ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Pancreas ; enzymology ; metabolism ; pathology ; Pancreatic Neoplasms ; enzymology ; mortality ; pathology ; Prognosis ; Survival Rate

OBJECTIVETo study the clinicopathologic and immunohistochemical features, and the prognosis of intraductal papillary mucinous neoplasms (IPMN) of the pancreas.

METHODSThe clinical findings, morphologic features, immunophenotype and prognosis were investigated in 61 cases of IPMN.

RESULTSOf these 61 cases, 33 were in the pancreatic head and 14 were in the body and tail, and 14 in the entire pancreas. The average patients' age was 61.8 years. The initial symptom was abdominal pain in 37 cases, and the tumors were detected at routine checkup in 14 cases. The imaging examination showed dilated ducts and/or cystic and solid masses. Grossly, 32 cases were multi-loculated cystic masses containing mucin and papillary areas; 13 cases were solid. Microscopically, the IPMN showed four patterns, including gastric-type (16 cases), intestinal-type (21 cases), pancreatobiliary-type (21 cases) and eosinophilic-type (3 cases). The IPMN cohort included 13, 13 and 6 IPMN with low, intermediate and high-grade dysplasia respectively, and 29 IPMN associated with invasive carcinoma. The IPMN associated carcinomas were mainly ductal adenocarcinoma (23/29, 79.3%), followed by colloid carcinoma (4/29, 13.8%) and undifferentiated carcinoma (2/29, 6.9%). Immunohistochemically, IPMN expressed MUC5AC (51/57, 89.4%), MUC2 (21/57, 36.8%), and MUC1 (13/46, 28.3%). The mean postoperative follow-up period was 32 months (range 12-112 months). Six of 61 patients were lost to follow-up. Overall 5-year survival rate was 76%. The 5-year survival rate of IPMN with low, intermediate or high-grade dysplasia was 100%, and recurrence was local in 3 patients. The 3-year survival rate of IPMN associated with invasive carcinoma was 55%. 12 of 13 patients died within 2 years after operation.

CONCLUSIONSIPMN is a common cystic neoplasm of the pancreas located in the ducts. The pathologic types and classifications are clearly defined. MUC stains are helpful for the diagnosis and papillary typing. IPMN with invasive carcinoma was associated with significantly worse survival than IPMN with dysplasia.

Adenocarcinoma, Mucinous ; diagnosis ; pathology ; Carcinoma, Pancreatic Ductal ; diagnosis ; pathology ; Humans ; Middle Aged ; Mucins ; metabolism ; Neoplasm Recurrence, Local ; Pancreas ; pathology ; Pancreatic Neoplasms ; diagnosis ; pathology ; Prognosis ; Survival Rate

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.
Amylases ; metabolism ; Animals ; C-Reactive Protein ; metabolism ; Delayed-Action Preparations ; chemical synthesis ; pharmacokinetics ; pharmacology ; Gabexate ; chemistry ; pharmacokinetics ; pharmacology ; Gels ; Male ; Muscle, Skeletal ; drug effects ; enzymology ; Oligopeptides ; metabolism ; Pancreas ; drug effects ; enzymology ; pathology ; Pancreatitis ; drug therapy ; enzymology ; etiology ; pathology ; Poloxamer ; chemistry ; Rats ; Rats, Sprague-Dawley ; Serine Proteinase Inhibitors ; chemistry ; pharmacokinetics ; pharmacology ; Temperature ; Wounds, Penetrating ; complications ; drug therapy ; enzymology ; pathology

OBJECTIVETo investigate the effect of MSX2 interference on epithelial-mesenchymal transitions (EMT) of pancreatic cancer cell line PANC-1.

METHODSThree vectors containing short hairpin RNAs (shRNAs) of MSX2 (shMSX2-1, shMSX2-2, and shMSX2-3) and the empty vector (negative control) were transfected separately into PANC-1 cell line with Lipofectamine2000. Real-time RT-PCR and Western blotting were used to observe changes in the expressions of MSX2, E-cadherin, and vimentin in the cells. CCK-8 assay was used to assess the changes in the cell growth, and wound scratch assay and Transwell assay were employed to evaluate the cell invasion and metastasis after the transfection.

RESULTSAmong the 3 shRNA, shMSX2-1 showed the highest interference efficiency. MSX2 knockdown by the specific shRNA of MSX2 significantly increased E-cadherin expressions, lowered vimentin expressions, and suppressed the invasion, metastasis and proliferation of the cells (P<0.05). MSX2 knockdown also resulted in morphological changes of the cells into cobblestone-like cells in close contact. RT-PCR results revealed significantly reduced mRNA expressions of the transcription factors snail and twist (P<0.05) without affecting slug and zeb1 expressions in the cells with MSX2 knockdown. Conclusion MSX2 knockdown can reverse EMT and induce MET in PANC1 cells, in which process the transcription factors snail and twist may play a role.

Cadherins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Epithelial-Mesenchymal Transition ; Homeodomain Proteins ; metabolism ; Humans ; Nuclear Proteins ; metabolism ; Pancreas ; Pancreatic Neoplasms ; pathology ; RNA, Small Interfering ; Snail Family Transcription Factors ; Transcription Factors ; metabolism ; Transfection ; Twist-Related Protein 1 ; metabolism ; Vimentin ; metabolism

OBJECTIVETo explore the role and mechanism of oxidative inflammatory cascade in pancreatic fibrosis progression of chronic pancreatitis (CP) in mice induced by dibutyltin dichloride (DBTC) plus ethanol.

METHODSThirty-six KM mice were randomly divided into 2 groups (n = 18): control group and model group (DBTC combined with ethanol). The mice in model group were intravenously injected with DBTC (8 mg/kg) in tail vein and drink 10% ethanol. After modeling 2 weeks, 4 weeks and 8 weeks, the mice were anesthetized and sacrificed, the pathological changes and the degree of fibrosis in the pancreas were observed by HE and Masson staining, the F4/80 expression level were detected by immunohistochemistry, the content of superoxide dismutase (SOD), malondialdehyde(MDA) and myeloperoxidase (MPO) were measured in the pancreatic homogenates.

RESULTSThe fibroblasts and macrophages (f4/80 positive staining) could be seen obviously in pancreas of model group at 2 weeks. At 4 weeks and 8 weeks, macrophages infiltration increased and pancreatic tissue was substituted by the proliferation of fibrosis significantly. At every time-point, in pancreatic homogenates SOD was decreased, MDA and MPO markedly increased. There was significant differences between two groups (P < 0.05).

CONCLUSIONDBTC injection joint ethanol drinking can successfully establish the model of chronic pancreatitis and pancreatic fibrosis in mice. Oxidative inflammatory cascade plays an important role in the progression of pancreatic fibrosis.

Animals ; Disease Progression ; Ethanol ; adverse effects ; Fibrosis ; Immunohistochemistry ; Malondialdehyde ; metabolism ; Mice ; Organotin Compounds ; adverse effects ; Oxidative Stress ; Pancreas ; pathology ; Pancreatitis, Chronic ; chemically induced ; physiopathology ; Peroxidase ; metabolism ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the effect of Chaiqin Chengqi Decoction (,CQCQD) on cholecystokinin receptor 1 (CCKR1)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis (ANP).

METHODSTwenty-seven Sprague-Dawley rats were randomized into three groups: the control group, the ANP group, and the CQCQD group (9 in each group). ANP rats were induced by two intraperitoneal injections of 8% L-arginine (pH=7.0, 4.4 g/kg) over a 2-h period. Rats were treated with 1.5 mL/100 g body weight of CQCQD (CQCQD group) or physiological saline (control and ANP groups) at 2 h interval. And 6 h after induction, pancreatic tissues were collected for histopathological examination. Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression, phospholipase C (PLC) and inositol-1,4,5-triphosphate (IP3), and determination of fluorescence intensity (FI) as a measure of intracellular calcium ion concentration [Ca(2+)]i.

RESULTSThe pancreatic histopathological score (6.2 ± 1.1) and the levels of PLC (1,187.2 ± 228.2 μg/mL) and IP3 (872.2 ± 88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control (2.8 ± 0.4, 682.5 ± 121.8 μg/mL, 518.4 ± 115.8 μg/mL) and the CQCQD (3.8 ± 0.8, 905.3 ± 78.5 μg/mL, 611.0 ± 42.5 μg/mL) groups (P<0.05). [Ca(2+)]i FI for the ANP group (34.8±27.0) was higher than that in the control (5.1 ± 2.2) and CQCQD (12.6 ± 2.5) groups (P<0.05). The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated (expression ratio=1.761; P=0.024) compared with the control group. The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated (expression ratio=0.311; P=0.035) compared with the ANP group. The ratio of gray values of the CCKR1 and β-actin in the ANP group (1.43 ± 0.17) was higher than those in the control (0.70 ± 0.15) and CQCQD (0.79 ± 0.11) groups (P<0.05).

CONCLUSIONSPancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein. CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells, relieving the calcium overload and reducing the pathological changes in rats with ANP.

Acinar Cells ; drug effects ; metabolism ; Animals ; Blotting, Western ; Calcium ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fluorescence ; Gene Expression Regulation ; drug effects ; Inositol 1,4,5-Trisphosphate ; metabolism ; Pancreas ; pathology ; Pancreatitis, Acute Necrotizing ; drug therapy ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Receptors, Cholecystokinin ; genetics ; metabolism ; Signal Transduction ; drug effects ; Type C Phospholipases ; metabolism

BACKGROUND/AIMS: To investigate the expression of Toll-like receptor 4 (TLR4) in the pancreases of rats with acute necrotizing pancreatitis (ANP) and any changes upon treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB (NF-kappaB), as well as to determine the relationship between TLR4 and NF-kappaB in ANP pathogenesis. METHODS: A total of 72 SD rats were randomly divided into three groups, namely, the control (sham-operation), ANP, and ANP with PDTC pretreatment groups. The PDTC-pretreated group was intraperitoneally injected with PDTC at a dose of 100 mg/kg 1 hour before the induction of ANP. The expressions of TLR4 and NF-kappaB in pancreatic tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA levels of cytokines tumor necrosis factor alpha, interleukin (IL)-1beta, and IL-6 were measured by reverse transcription polymerase chain reaction. RESULTS: The expressions of TLR4, NF-kappaB, and cytokine (NF-kappaB target) genes in the pancreatic tissue increased more significantly in the ANP groups than in the sham-operation group at 3, 6, and 12 hours. Pretreatment with PDTC alleviated the inflammatory activation in the pancreas with ANP, causing a significant decrease in the expressions of TLR4, NF-kappaB, and cytokine genes in the pancreatic tissue. CONCLUSIONS: The expressions of TLR4 and NF-kappaB were increased in the pancreases of rats with ANP. PDTC not only inhibits NF-kappaB but also suppresses the expression of TLR4 and downregulates the expression of the related cytokine genes.
Animals ; Antioxidants/*pharmacology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Male ; NF-kappa B/*drug effects/metabolism ; Pancreas/metabolism/pathology ; Pancreatitis, Acute Necrotizing/chemically induced/*drug therapy ; Pyrrolidines/*pharmacology ; RNA, Messenger/metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates/*pharmacology ; Toll-Like Receptor 4/*drug effects/metabolism ; Tumor Necrosis Factor-alpha/genetics/metabolism

BACKGROUND/AIMS: To investigate the expression of Toll-like receptor 4 (TLR4) in the pancreases of rats with acute necrotizing pancreatitis (ANP) and any changes upon treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB (NF-kappaB), as well as to determine the relationship between TLR4 and NF-kappaB in ANP pathogenesis. METHODS: A total of 72 SD rats were randomly divided into three groups, namely, the control (sham-operation), ANP, and ANP with PDTC pretreatment groups. The PDTC-pretreated group was intraperitoneally injected with PDTC at a dose of 100 mg/kg 1 hour before the induction of ANP. The expressions of TLR4 and NF-kappaB in pancreatic tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA levels of cytokines tumor necrosis factor alpha, interleukin (IL)-1beta, and IL-6 were measured by reverse transcription polymerase chain reaction. RESULTS: The expressions of TLR4, NF-kappaB, and cytokine (NF-kappaB target) genes in the pancreatic tissue increased more significantly in the ANP groups than in the sham-operation group at 3, 6, and 12 hours. Pretreatment with PDTC alleviated the inflammatory activation in the pancreas with ANP, causing a significant decrease in the expressions of TLR4, NF-kappaB, and cytokine genes in the pancreatic tissue. CONCLUSIONS: The expressions of TLR4 and NF-kappaB were increased in the pancreases of rats with ANP. PDTC not only inhibits NF-kappaB but also suppresses the expression of TLR4 and downregulates the expression of the related cytokine genes.
Animals ; Antioxidants/*pharmacology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Male ; NF-kappa B/*drug effects/metabolism ; Pancreas/metabolism/pathology ; Pancreatitis, Acute Necrotizing/chemically induced/*drug therapy ; Pyrrolidines/*pharmacology ; RNA, Messenger/metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates/*pharmacology ; Toll-Like Receptor 4/*drug effects/metabolism ; Tumor Necrosis Factor-alpha/genetics/metabolism