Caerulein Research Articles

Dexamethasone inhibits NF‑кBp65 and HMGB1 expression in the pancreas of rats with severe acute pancreatitis.

Severe acute pancreatitis (SAP) starts as a local inflammation of pancreatic tissue that induces the development of multiple extra-pancreatic organ dysfunction; however, the underlying mechanisms remain unclear. The present study was designed to evaluate the effect of dexamethasone (DXM) on pancreatic damage and to investigate the role of high-mobility group box-1 (HMGB1) and nuclear factor-κB (NF-κBp65) in the development of SAP in animal and cell models. For the in vivo experiment, 35 Sprague-Dawley rats were randomly assigned to three groups: The sham-operation control group, the SAP group and the DXM treatment group. Histological analysis revealed that, when DXM was infused into SAP rats, edema formation and structural alterations with necrosis were reduced, and the number of apoptotic cells was markedly reduced. In addition, compared with the SAP group, the expression level of HMGB1 was significantly decreased in the nucleus and the expression level of NF-κBp65 was significantly decreased in the cytoplasm from rats treated with DXM. In vitro, DXM was able to suppress the apoptosis and cell death induced by caerulein (CAE), and DXM could suppress the expression of NF-κBp65 and HMGB1 induced by CAE, as demonstrated by western blotting and immunofluorescence analysis. Therefore, these results provide an experimental basis for investigating the underlying therapeutic mechanisms of DXM treatment for SAP.

The identification and roles of microRNA targeting protein kinase D1 in rat with acute pancreatitis

Objective To predict and verify the upstream regulatory microRNA (miRNA) of protein kinase D1 (PKD1), and to investigate its role in cerulein induced acute pancreatitis (AP) in rats. Methods Potential upstream regulatory miRNA of PKD1 was predicted by using bioinformatics software.Dual luciferase reporter gene system and Western blot were applied to verify the regulation of PKD1 by the selected miRNA.Experimental AP was induced by 6 intraperitoneal injection of cerulein (20 μg/kg) at hourly intervals after administration of the CY5-labeled notarget control (AP group, n=20) or selected miRNA (treatment group, n=20), respectively by intraperitoneal injection into rats.Other rats were divided randomly into a normal control group (n=10) without any treatment.Besides 10 rats in either AP or treatment group were sacrificed 6 hours after the first injection of cerulein, and the rats were all sacrificed 24 hours after the first injection.The blood samples and pancreatic tissues of each rat were collected to test serum amylase and lipase activities, or to make hematoxylin-eosin stain for AP pathological scores as well as PKD1 immunohistochemical staining, respectively. Results TargetScan 7.1 software analysis showed that miR-128-3p was the potential upstream regulatory miRNA of PKD1, which was verified by dual luciferase reporter gene system and Western blot detection.Compared to the normal control group, serum amylase and lipase activities after 6 h exposure to cerulein increased in both AP group and the treatment group[13 313.00(9 424.00-15 995.00) U/L, 13 552.00(10 399.50-18 408.25) U/L vs.1 430.50(1 214.25-1 543.25) U/L; 547.00 (515.00-627.00) U/L, 857.50(522.00-1 222.25) U/L vs.34.00(32.50-34.75) U/L], and the differences were significant(χ2=8.715, P<0.05; χ2=9.115, P<0.05), which indicated that the rat models of AP were successfully established.The immunohistochemical scores of PKD1 after 24 h exposure to cerulein decreased in the treatment group[0.50(0-2.75) scores], compared with the normal control group [4.00(4.00-8.00) scores]and the AP group [4.00(3.75-8.00) scores], and difference was significant(χ2=18.302, P<0.05). Accordingly, the total pathological scores of HE staining decreased significantly in the treatment group, as compared to the AP group (3.80±0.85 vs.6.90±1.15, t=4.481, P<0.01). The results showed that the inflammatory cell infiltration and tissue necrosis were significantly improved after miR-128-3p treatment. Conclusions miR-128-3p is the upstream regulatory microRNA of PKD1 which protects pancreata from necrotic injury and inflammatory cell infiltration in PKD1-mediated acute pancreatitis. Key words: Acute pancreatitis; miR-128-3p; Protein kinase D1; Necrosis

Effects of resolvin D1 on the autophagy in acute pancreatitis in mice

Objective To investigate the effects of resolvin D1 on autophagy in the prevention of acute pancreatitis (AP) in mice. Methods Thirty C57BL/6 mice were divided into control group, AP group and resolvin D1 group. AP model was established by intraperitoneal injection of cerulein at 50 μg·kg-1·h-1. Resolvin D1 was intraperitoneally given at 50 μg/kg one hour before and four hours after modeling. The mice of control group were intraperitoneally injected the same volume of 0.9% sodium chloride solution. The serum levels of amylase and lipase were measured by colorimetric method. The pathological injury of the lung and pancreatitis were observed under optical microscope. Autophagic vacuoles in acinar cells of pancreas of mice were evaluated by transmission electron microscope. And the expressions of autophagy related markers Beclin-1, p62 and LC3-Ⅱ at the mRNA and protein levels in pancreas of mice were detected by real time quantitative polymerase chain reaction (RT-qPCR) and Western blotting method. One-way analysis of variance and SNK-q were performed for statistical analysis. Results There were statistically significant differences in serum amylase and lipase levels between control group, AP group and resolvin D1 group (F=62.99 and 149.69, both P<0.01). The serum amylase and lipase levels of mice in resolvin D1 group were lower than those of AP group ((525.08±41.12) U/L vs. (752.62±42.03) U/L, (758.24±134.77) U/L vs. (1 201.06±112.53) U/L), and the differences were both statistically significant (both SNK-q test and P<0.01). In addition, there were statistically significant differences in the ratio of the pancreas and lung wet mass to body mass between control group, AP group and resolvin D1 group (F=11.36 and 18.51, both P<0.05). Pathological injury scores of pancreas and lung of resolvin D1 group were both lower than those of AP group (3.3±0.6 vs. 5.6±0.6, 5.4±0.5 vs. 8.8±0.4), and the differences were statistically significant (both SNK-q test and P<0.05). The results of transmission electron microscopy observation revealed that the number of autophagic vacuole of resolvin D1 group was less than that of AP group, and the size was smaller. Moreover, there were statistically significant differences in Beclin-1, p62 and LC3-Ⅱ mRNA between control group, AP group and resolvin D1 group (F=270.95, 151.83 and 124.77, all P<0.05). The relative expression mRNA levels of Beclin-1, p62 and LC3-Ⅱ of resolvin D1 group were 1.59±0.12, 2.75±0.27 and 1.34±0.14, respectively, which were lower than those of AP group (2.68±0.13, 3.32±0.30 and 3.37±0.26, respectively), and the differences were statistically significant (all SNK-q test and P<0.05). There were statistically significant differences in the expressions of Beclin-1, p62 and LC3-Ⅱat the protein level between control group, AP group and resolvin D1 group (F=116.63, 384.40 and 192.45, all P<0.05). The expressions of Beclin-1, p62 and LC3-Ⅱ at protein level of resolvin D1 group were 0.98±0.03, 0.57±0.04 and 0.31±0.03, respectively, which were lower than those of AP group (1.34±0.07, 1.02±0.03 and 0.48±0.04, respectively), and the differences were statistically significant (all SNK-q test and P<0.05). Conclusion Resolvin D1 ameliorates the severity of AP by attenuating the impaired autophagy and restoring autophagic flux in AP mice. Key words: Autophagy; Acute pancreatitis; Resolvin D1; Mice

Effect and mechanism of Bafiomycin A1 on acute pancreatitis in mice

Objective To investigate the effect and mechanism of Bafiomycin A1 on acute pancreatitis in mice. Methods 60 BALB/c mice were randomly divided into control group, model group, low dose group and high dose group (15 mice in each group). Model group, low dose group and high dose group of mice by intraperitoneal injection of caerulein 50 g/kg, 1/h, a total of 7 times to establish the model of acute pancreatitis. Mice in low dose group and high dose group were intraperitoneally injected with 25 and 75 μg/kg Bafiomycin A1. Mice in the control group and model group were injected with equal volume of normal saline intraperitoneally. Mice in four groups were sacrificed in 24 h after treatment. Serum amylase in mice (AMY), inflammatory factor tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were analyzed by enzyme linked immunosorbent assay (ELISA). The mouse pancreatic lesions and score (Schmidt score) were analyzed by hematoxylin and eosin (HE) staining. autophagy substrate p62 and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blotting. The pancreatic tissue p62 were analyzed by immunofluorescence. Results Compared with the model group [(4 910.98±349.43) U/L, (109.43±10.43) pg/L, (329.16±20.91) pg/L, 9.43±3.98], the AMY, TNF-α, IL-6 and Schmidt scores of the low dose group [(3 109.13±301.44) U/L, (80.18±9.34) pg/L, (215.32±18.43) pg/L, 6.48±3.18] and the high dose group [(1 432.33±187.43) U/L, (45.19±8.14) pg/L, (153.22±12.63) pg/L, 3.14±1.02] significantly decreased (P=0.000). Compared with the low dose group, the AMY, TNF-α, IL-6 and Schmidt scores of the high dose group more significantly decreased. Compared with model group (0.15±0.06, 0.56±0.06), p62 and LC3 in pancreatic tissue of mice in low dose group (0.39±0.09, 0.75±0.09) and high dose group (0.61±0.07, 0.99±0.08) obviously enhanced (P=0.000). Compared with the low dose group, the accumulation of p62 and LC3 in the pancreatic tissue of the high dose group more obviously accumulated (P=0.000). Conclusion Bafiomycin A1 can significantly inhibit the level of autophagy in pancreatic tissue of mice with acute pancreatitis and play a protective role in pancreatic tissue. Key words: Acute pancreatitis; Autophagy; Bafiomycin A1; Inflammation

Exendin-4 dose not evoke pancreatitis or pancreatitis-associated histopathological and genetic changes in high-fat diet induced diabetic mice

Background: Clinical reports have suggested the potential link of Glucagon-Like Peptide 1 Receptor Agonists (GLP-1RAs) with the development of pancreatitis. We investigated the effects of long-term exposure of Exendin-4 (Ex-4), a kind of GLP-1RA, on biochemical, histological and genetic markers of pancreatitis in High-Fat Diet (HFD) induced mice. Methodology: HFD induced mice received subcutaneous twice-daily injections of Ex-4 (3 and 30 μg/kg/d) or vehicle for 12 w. Pancreatitis was induced with Caerulein (CRN) in Ex-4 treated mice or wild mice. Changes in body weight, food consumption, serum amylase, serum lipase, glucose, and insulin concentrations were measured in each group. An extended histopathological and ultralstructural by transmission electron microscopy evaluation of exocrine pancreas was performed. The expressions of pancreatitis-associated microRNAs and genes were assessed by real-time PCR. Results: Ex-4 improved physical condition, glucose concentrations, decreased food intake, and increased serum insulin sensitivity in HFD induced mice. No deleterious effects on serum amylase and lipase were observed after Ex-4 treatment. The histopathological and ultralstructural findings did not reveal adverse effects of Ex-4. Ex-4 administration did not significantly modify the level of pancreatitisassociated microRNAs (miR-181, miR-148, miR-210 and miR-216a) or pancreatitis-associated genes (RegIII, MCP-1 and IL-6). However, we noted slightly pancreatitis ultralstructural changes in one mouse from 30 μg/kg/d Ex-4-treated group. Conclusion: Ex-4 does not affect biochemical, histopathological or genetic markers of pancreatitis in HFD induced mice. However, surveillance for a possible increased risk of pancreatitis under excess GLP-1RAs administration is warranted to ensure at least benefits far outweigh potential risks.

Angiotensin 1-7 ameliorates caerulein-induced inflammation in pancreatic acinar cells by downregulating Toll-like receptor 4/nuclear factor-κB expression.

The present study aimed to investigate the effects of angiotensin (Ang) 1–7 on caerulein (CAE)-stimulated nuclear factor (NF)-κB, Toll-like receptor (TLR4) and cytokine expression using pancreatic acinar AR42J cells. AR42J cells were treated with 10 nmol/l CAE for various durations. In addition, cells were pretreated with various concentrations of Ang 1–7 or A779, a specific antagonist of Ang 1–7, and were stimulated with CAE for 12 h. Control cells were treated with vehicle (F-12K complete medium with 2% fetal bovine serum, 10 U/ml penicillin and 100 mg/ml streptomycin) alone. The mRNA and protein expression levels of TLR4, NF-κB, interleukin (IL)-6, IL-8, IL-10 and tumor necrosis factor-α (TNF-α) were determined by western blotting, immunofluorescence and reverse transcription-quantitative polymerase chain reaction. CAE treatment stimulated TLR4 and NF-κB expression within AR42J cells. Immunofluorescence indicated that TLR4 was expressed on the membranes and in the cytoplasm of AR42J cells, whereas NF-κB expression accumulated in the cytoplasm and nuclei. CAE-induced expression of TLR4 and NF-κB within AR42J cells was abrogated by 10−5 mmol/l Ang 1–7; however, TLR4 and NF-κB expression was enhanced with the addition of A779, particularly 10−5 mmol/l. In addition, treatment with 10−6 and 10−5 mmol/l Ang 1–7 significantly mitigated CAE-induced expression of IL-6, IL-8 and TNF-α, whereas it enhanced IL-10 expression. Conversely, A779 treatment enhanced the CAE-induced expression of IL-6, IL-8 and TNF-α, and reduced IL-10 expression in AR42J cells. In conclusion, these results suggested that Ang 1–7 may attenuate CAE-induced inflammation by downregulating TLR4, NF-κB and proinflammatory cytokine expression within AR42J cells. Therefore, Ang 1–7 may exert protective effects against the pathological progression of AP in a cell model of AP induced by CAE and may be considered in the development of treatments for this disease.

Angiotensin-converting enzyme 2-angiotensin (1-7)-Mas axis prevents pancreatic acinar cell inflammatory response via inhibition of the p38 mitogen-activated protein kinase/nuclear factor-κB pathway.

The aim of the present study was to investigate the role of the angiotensin-converting enzyme (ACE)2-angiotensin‑(Ang)-(1-7)-Mas axis in the pathogenesis of pancreatitis and the association between this axis and the p38mitogen-activated protein kinase (p38MAPK)/nuclear factor(NF-κB) signaling pathway in pancreatic acinar cells. Mouse pancreatic acinar cancer (MPC-83) cells were stimulated with 10nM caerulein (CAE) to create an invitro model of acute pancreatitis, and collected for analysis at 2, 6, 12, 24 and 48h post stimulation. In addition, cells were pretreated with different concentrations of Ang‑(1‑7), Ang‑(1‑7) antagonist A779, p38MAPK inhibitor SB203580 or ACE2 inhibitor DX600 for 30min, and then stimulated with CAE for 24h. The ACE2, Mas receptor, p38MAPK, phosphorylated (p)-p38MAPK and NF-κB expression levels were evaluated using western blotting and immunofluorescence. p38MAPK, NF-κB, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8 and IL-10 mRNA expression levels were assessed using reverse transcription-quantitative polymerase chain reaction. The results of the immunofluorescence assay demonstrated that ACE2 and p38MAPK were present mainly in the cytoplasm, while the Mas receptor was located mainly in the cell membrane. ACE2, p38MAPK and p-p38MAPK protein levels were significantly increased (P<0.05) following stimulation with CAE compared with those in the control group and peaked at 24h. Mas receptor protein levels were significantly upregulated (P<0.05) between 6 and 24h, peaking at 12h. Ang‑(1‑7) and SB203580 downregulated p-p38MAPK and NF-κB expression and the mRNA levels of inflammatory factors IL-6, TNF-α and IL-8, but upregulated the mRNA level of inflammatory factor IL-10 compared with those treated with CAE alone. These results were supported by the opposite outcomes observed for cells treated with A779 or DX600. Therefore, it was concluded that the ACE2-Ang‑(1‑7)-Mas axis significantly inhibits pancreatitis by inhibition of the p38MAPK/NF-κB signaling pathway.

Effects of Erdosteine on Experimental Acute Pancreatitis Model.

To create acute pancreatitis condition experimentally in rats using cerulein, and to reveal histopathological effects in pancreatic tissue with erdosteine. An experimental study. Department of General Surgery, Duzce University, Turkey, from June to October 2014. Thirty male Wistar albino rats were divided into three groups. No procedures were applied to Group 1. The rats in Group 2 and Group 3 were injected cerulein, to establish an experimental pancreatitis model and the blood amylase and lipase values were examined. The rats in Group 3 were given 10 mg/kg erdosteine. This treatment was continued for another 2 days and the rats were sacrificed. The pancreatic tissues were examined histopathologically for edema, inflammation, acinar necrosis, fat necrosis, and vacuolization. The lipase and amylase values and the histopathological examination of pancreatic tissues evidenced that the experimental acute pancreatitis model was established and edema, inflammation, acinar necrosis, fat necrosis, and vacuolization were observed in the pancreatic tissues. The statistical results suggest that erdosteine can decrease the edema, inflammation, acinar necrosis, fat necrosis and vacuolization scores in the tissues. The severity of acute pancreatitis, induced by cerulein in rats, is reduced with the use of erdosteine.

Angiotensin‑(1‑7) attenuates caerulein‑induced pancreatic acinar cell apoptosis.

Extensive apoptosis of pancreatic acinar cells frequently occurs in acute pancreatitis (AP), and has been identified to be closely associated with the decrease of pancreatic parenchymal cells and pancreatic damage. The present study aimed to investigate the possible effect of angiotensin (Ang)‑(1‑7) on caerulein (CAE)‑induced pancreatic acinar cell apoptosis. Mouse pancreatic acinar cancer cells (MPC‑83) were divided into 4 groups: Control group; CAE group; CAE + Ang‑(1‑7) group; and CAE + Ang‑(1‑7) antagonist (A779) group. The control group consisted of normal MPC‑83 cells without special treatment. The CAE group was stimulated with 10nmol/l CAE and harvested at 2, 6, 12, 24 and 48h. For the CAE + Ang‑(1‑7) group and CAE + A779 group, the CAE‑induced pancreatic acinar cells were mock pretreated or pretreated with different concentrations of Ang‑(1‑7) or A779 (10‑7, 10‑6 or 10‑5mol/l) for 30min. Caspase‑3 is a critical executioner of apoptosis, as it is either partly or completely responsible for the proteolytic cleavage of numerous key proteins including the nuclear enzyme poly (ADP‑ribose) polymerase. Activation of caspase‑3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Thus, the present study investigated the apoptotic markers, including cleaved caspase‑3, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑like protein 4 (Bax) and renin‑angiotensin system (RAS) pathway related proteins (ACE2 and Mas receptor). The results demonstrated that the cleaved caspase‑3 levels were increased in the CAE group (P<0.05), peaking at 24h, and declined when incubated with Ang‑(1‑7). Following treatment with Ang‑(1‑7), levels of the anti‑apoptotic protein Bcl‑2 rose dramatically in a dose‑dependent manner. The ratio of the pro‑apoptotic protein Bax to the anti‑apoptotic protein Bcl‑2 dropped notably, which demonstrated a tendency towards curbing apoptosis. In addition, the cleaved caspase‑3 levels, and the ratio of Bax to Bcl‑2 in the CAE + A779 group presented a significant rise compared with the CAE group. It was concluded that Ang‑(1‑7) may possess an inhibitory effect on CAE‑induced pancreatic acinar cell apoptosis and that appropriate interventions in RAS may attenuate pancreatic injury during AP.

Evaluation of miR-216a and miR-217 as Potential Biomarkers of Acute Exocrine Pancreatic Toxicity in Rats.

Detecting and monitoring exocrine pancreatic damage during nonclinical and clinical testing is challenging because classical biomarkers amylase and lipase have limited sensitivity and specificity. Novel biomarkers for drug-induced pancreatic injury are needed to improve safety assessment and reduce late-stage attrition rates. In a series of studies, miR-216a and miR-217 were evaluated as potential biomarkers of acute exocrine pancreatic toxicity in rats. Our results revealed that miR-216a and miR-217 were almost exclusively expressed in rat pancreas and that circulating miR-216a and miR-217 were significantly increased in rats following administration of established exocrine pancreatic toxicants caerulein (CL) and 1-cyano-2-hydroxy-3-butene (CHB) as well as in rats administered a proprietary molecule known to primarily affect the exocrine pancreas. Conversely, neither microRNA was increased in rats administered a proprietary molecule known to cause a lesion at the pancreatic endocrine-exocrine interface (EEI) or in rats administered an established renal toxicant. Compared with amylase and lipase, increases in miR-216a and miR-217 were of greater magnitude, persisted longer, and/or correlated better with microscopic findings within the exocrine pancreas. Our findings demonstrate that in rats, miR-216a and miR-217 are sensitive and specific biomarkers of acute exocrine pancreatic toxicity that may add value to the measurement of classical pancreatic biomarkers.

Effects of Astragaloside IV on tumor necrosis factor-α-induced expressions of matrix metalloproteinases in rat vascular smooth muscle cells

Objective To investigate the effect of Astragaloside IV (AS-IV) on tumor necrosis factor-α (TNF-α)-induced expressions of matrix metalloproteinases (MMPs) in a rat vascular smooth muscle cells (VSMCs) proliferation model and its mechanism. Methods VSMCs were prepared from the thoracoabdominal aorta of rats by using issue-sticking method. Morphology of cells was observed by inverted microscope, and identified by immunohistochemical methods with antibody against SM-α-actin. The model of VSMCs proliferation and migration was established by TNF-α inducer in vitro, and randomly divided into the following groups: the control group, the TNF-α group, the TNF-α+ AS-IV (0.5 μg/ml) group, the TNF-α+ AS-IV (5 μg/ml) group, the TNF-α+ AS-IV (25 μg/ml) group, and the TNF-α+ AS-IV (50 μg/ml) group. The effect of AS-IV on TNF-α-induced VSMCs proliferation activity was detected by the caerulein and cholecystokinin octapeptide (CCK-8) method. The quantitative real-time polymerase chain reaction (real-time PCR) and Western blotting were used to examine the effects of AS-IV on the VSMCs-secreted mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2), respectively. Results The proliferative activity, migratory distance and invasive capacity of VSMCs were obviously increased in TNF-α stimulation group versus in control group (all P<0.01), which suggested that TNF-α can promote VSMCs proliferation and migration, and that the rat model of VSMCs proliferation in vitro was successfully established. The results of CCK-8 tests showed that VSMCs proliferation was obvious and the optical density (OD) value was elevated (P<0.01) after a preset time incubation with TNF-α. VSMCs proliferation was inhibited in each AS-IV treatment group, and the OD value was decreased as compared with the TNF-α group. And the inhibitive effect was increased along with the increments of AS-IV concentration and the acting time, which indicated that AS-IV can inhibit TNF-α-induced VSMCs proliferation in a time- and dose-dependent manner. The results of real-time PCR and Western blotting assays indicated that TNF-α changed the ratio of MMPs to the tissue inhibitors of metalloproteinases (TIMPs) by down-regulating active MMP-2 expression without influencing proMMP-2 and TIMP-2 expressions, and thus promoted the degradation of ECM. AS-IV (0.5-50 μg/ml) inhibited VSMCs proliferation and migration by down-regulating the TNF-α-induced MMP-2 overexpression in a dose-dependent manner, up-regulating the mRNA and protein expressions of TIMP-2, and modulating the ratio of MMPs to TIMPs, thereby inhibited the degradation of ECM. Conclusions AS-IV inhibits VSMCs proliferation and migration in a time- and dose-dependent manner. AS-IV inhibits VSMCs proliferation and migration by down-regulating TNF-α-induced MMP-2 overexpression, up-regulating TIMP-2 expressions, and normalizing the ratio of MMPs to TIMPs. Therefore, AS-IV inhibits the degradation of ECM, which may play a role in the prevention and treatment of in-stents restenosis after PCI. Key words: Astragalus; Muscle, smooth, vascular; Tumor necrosis factor-alpha; Matrix

Effects of pioglitazone pre-treating on acinar cells apoptosis induced by caerulein in acute pancreatitis

Objective To investigate the effects of pioglitazone pre-treating on pancreatic acinar cell (AR42J cells) apoptosis induced by caerulein. Methods AR42J cells were divided into blank control group (with normal culture), pioglitazone group (40 μmol/L), caerulein control group (1×10-8 mol/L), pioglitazone+ caerulein group (40 μmol/L pioglitazone+ 1×10-8 mol/L caerulein) and pioglitazone+ GW9662+ caerulein group (40 μmol/L pioglitazone+ 5 μmol/L GW9662+ 1×10-8 mol/L caerulein). Pioglitazone and GW9662 were added 30 minutes earlier than caerulein. Cell proliferation rate of each group was determined by MTT assay at three, six, 12 and 24 hour. The cell apoptosis rate was detected by flow cytometry with Annexin Ⅴ/PI staining and terminal dexynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining. The activity of Caspase 3, 8 and 9 of each group was measured. Mitochondrial membrane potential (MMP) was detected by flow cytometry with JC-1 staining. Single factor analysis of variance and LSD test were performed for data analysis. Results At six, 12 and 24 hour, the cell proliferation rate of pioglitazone group and pioglitazone+ caerulein group was 0.19±0.02, 0.22±0.02, 0.36±0.02 and 0.20±0.04, 0.23±0.02, 0.38±0.02, respectively, which were significantly lower than those of blank control group (0.25±0.04, 0.28±0.03 and 0.46±0.02) and caerulein group (0.23±0.02, 0.29±0.01 and 0.46±0.05, tcontrol group=-3.16, -4.61 and -6.25, tcaerulein group=-1.58, -4.61 and -6.15, all P 0.05), but cell apoptosis of these two groups were higher than those of control group ((5.52±0.64)%, (5.30±0.97)% and (5.47±0.88)%) and caerulein group ((5.98±1.21)%, (7.47±0.58)% and (8.11±1.32)%) respectively, and the differences were statistically significant (tcontrol group=9.81, 4.45 and 10.74, tcaerulein group=4.38, 7.62 and 6.98, all P <0.05). There was no significant difference in apoptosis rate between pioglitazone+ GW9662+ caerulein group ((5.82±0.26)%, (6.05±0.83)% and (9.23±0.90)%) and caerulein group; while significantly higher when compared with those of pioglitazone+ caerulein group (t=-4.63, -10.07 and -5.70, all P<0.05). At 12 hour, the apoptosis rate detected by TUNEL staining of pioglitazone group ((3.93±0.40)%) was significantly higher than that of control group ((2.73±0.68)%), the apoptosis rate of pioglitazone+ caerulein group ((8.43±1.65)%) was significantly higher than that of caerulein group ((2.80±0.56)%), the apoptosis rate of pioglitazone+ GW9662+ caerulein group ((3.87±0.35)%) was lower than that of pioglitazone+ caerulein group (t=7.93, 8.92, -5.35, all P<0.05). At 24 hour, the activity of Caspase 3, 8 and 9 of pioglitazone+ caerulein group (1.28±0.05, 1.38±0.04 and 1.53±0.09) significantly increased compared with those of caerulein group (1.12±0.88, 1.22±0.02 and 0.53±0.07, t=3.20, 8.62 and 1.29, all P<0.05). After treated with GW9662, part of activity of Caspase enzymes recovered. The number of cells with potential change of mitochondrial membrane in pioglitazone group and pioglitazone+ caerulein group was more than that of caerulein group (28.50±0.91)% and (28.20±2.56)% vs (15.00±3.67)%) and part of membrane potential recovered after GW9662 added ((20.67±2.20)%). Conclusions Pioglitazone might promote AR42J cell apoptosis through the activation of caspases enzymes and changing membrane potential. And the antagonist GW9662 would partially inhibit the apoptosis induced by pioglitazone. Key words: Pioglitazone; Caerulein; AR42J cells; Acute pancreatitis

The dose-dependent relationship between caerulein and the severity of pancreatitis

Objective To explore the severity of acute pancreatitis induced by intraperitoneal injection of 50μg/kg caerulein at different doses in mice. Methods Acute pancreatitis(AP)was induced by intraperitoneal infection of 50μg/kg caerulein at 3,6,9,12times, respectively.We evaluated the severity of AP biochemically and morphologically. Results Conventional doses of caerulein(50μg/kg) were injected with 3, 6, 9, 12 times, the level of serum amylase in experimental group were significantly higher than that in control group(P< 0.05), peaked in the 9 times[(25 966.67±3 787.02)U/L]; pancreatic water content in the experimental group was significantly higher than that in control group(P< 0.05), peaked in the 6 times[(81.13±1.03)%]; pancreatic tissue hematoxylin and eosin(HE)staining showed that pancreas was slightly edema and infiltration of inflammatory cells at 3 times, which showed a large amount of neutrophil infiltration and obvious edema at the 6 times, and pancreatic leaflet margin appeared a small amount necrosis at the 9 times, and the acinar necrosis foci could be seen in large range an the 12 times(histopathological score 8.20±0.84), the microscope exhibited altered necrotizing pancreatitis. Conclusion Conventional caerulein doses(50μg/kg)may induce acute edematous pancreatitis by 6 times of intraperitoneal injection in mice, and 12 times may induce acute necrotizing pancreatitis. Key words: Acute pancreatitis; Caerulein; Model, animal

Protective effects of Commiphora myrrha on acute pancreatitis

ABSTRACT Objectives : Commiphora myrrha (CM) has been used in traditional medicine for treating diseas e such as obesity, hyperlipidemia, atherosclerosis, diabetes and osteoart hritis. However, the protective effects of CM on acute pancreatitis (AP) has not been reported. Thus, the aim of this study was to evaluate the protective effects of CM water extract on cerulein-induced AP.Methods : AP was induced in mice via intraperitoneal injection of supram aximal concentrations of the stable cholecystokinin analogue cerulein (50 µg/kg) every hour for 6 times. Water extract of CM (0.1, 0.2, or 0.5 g/kg) was administrated intraperitoneally 1 h prior to the first injection of cerulein. The mice were killed at 6 h after the final cerulein injection. Pancreas was rapidly remove d for morphologic and histochemical examination, myeloperoxidase (MPO) assay. Blood samples were taken to determ ine serum amylase and lipase activities.Results : Administration of CM significantly inhibited pancreatic weight /body weight ratio, pancreas histological injury. And CM administration inhibited the serum digestive enz yme elevation such as amylase and lipase on cerulein-induced pancreatitis. In addition, Pancreas MPO activi ty which indicates neutrophil infiltration was inhibited by CM extract on cerulein-induced pancreatitis.Conclusions : In conclusion, our results could suggest that pre-treatment of CM reduces the severity of cerulein-induced AP. Therefore, CM could be used as a protectiv e agent against AP. Also, this study could give a clinical basis that CM could be a drug or agent to prevent AP.Key words : Commiphora myrrha, Acute pancreatitis, Cerulein, Amylase, Lipase

Changes of zymophagy during cell model of caerulein-induced experimental acute pancreatitis

Objective To observe the changes of zymophagy during experimental acute pancreatitis (AP) induced by caerulein.Methods Pancreatic acinar cell line AR42J cells were cultured in 6-well plates till 90％ confluent and then divided into AP group and control group.Caerulein (1 × 10-8 mol/L) was added into AP group to establish AP cell model,and 1640 cell culture medium was added into control group.After caerulein treated for one,four,six,eight,12 and 24 hours,cells and cell culture supernatant were collected.The levels of cytokine interleukin (IL)-1,tumor necrosis factor (TNF)α,trypsinogen activation (TAP) and amylase were measured with enzyme-linked immunosorbent assay (ELISA) method.The expression of LC3 and Beclin1 at mRNA of each group were detected by reverse transcription-polymerase chain reaction (RT-PCR).The LC3B protein level of each group were detected by Western blotting.The changes of autophagosome and zymophagosome were observed by transmission electron microscopy.The difference between AP group and control group was analyzed by analysis of variance.Results The level of IL-1,TNFα,amylase and TAP in cell culture supernatant of control group was (18.83±7.10) pg/mL,(14.20±3.79) pg/mL,(10.03±2.85) U/L and (39.48±8.62) pg/mL,respectively.Those of AP group significantly increased at first hour ((62.13±11.25) pg/mL,F=3.32,P＜0.01 ; (30.98±7.11) pg/mL,F=3.05,P＜0.05; (25.06±6.82) U/L,F=2.90,P＜0.05 and (128.51± 18.30) pg/mL),F=2.62,P＜0.01,at fourth or sixth hour reached peak (IL-1 at fourth hour:(71.96± 15.82) pg/mL,F=7.25,P＜0.01;TNFα at sixth hour:(39.92±8.94) pg/mL,F=4.93,P＜0.05; amylase at fourth hour:(28.83 ± 8.31) U/L,F=2.06,P＜0.05; TAP at fourth hour:(146.29± 29.36) pg/mL,F=0.14,P＜0.01) and then gradually decreased.At fourth and sixth hour,the expression of LC3 at mRNA level in AP group was 3.18±0.82,1.71±0.14,respectively,while the expression of Beclin-1 rnRNA at first,fourth hour was 2.44±0.34 and 4.13±0.30,all of them were significantly increased compared with those of control group (0.21±0.04 and 0.30±0.08,LC3 mRNA F=0.79、0.06; Beclin mRNA F=2.31、0.36,all P＜ 0.05).There were no significant differences at other time points.The numbers of autophagosome and zymophagosome of AP group were significantly higher than those of control group under transmission electron microscopy.Conclusion Zymophagy occurred during AP cell model induced by caerulein,which suggested that zymophagy might involve in the mechanism of AP. Key words: Acute pancreatitis; Autophagy; Trypsinogen

Caerulein induces β-cells to regenerate within pancreas

Caerulein induces β-cells to regenerate within pancreas

Oxidative stress and apoptosis in gut barrier dysfunction of severe acute pancreatitis

Objective By means of animal study,investigated the gut barrier function in severe acute pancreatitis ( SAP),and role of inflammatory factors releasing,gut mucosa oxidative stress,cell apoptosis in it.Methods The animal experiment was done in the animal center of first people' s hospital,shanghai jiaotong university.Twenty four BALB/c mice were randomized ( random number) divided into two groups with twelve mice each group.The SAP group,mice received six intraperitoneal injections of cerulein at 1-hour intervals, the dose was 50μg/kg, then given one intraperitoneal injection of 10 mg/kg lipopolysaccharide ( LPS from E.Coli) for the induction of severe acute pancreatitis.The control ( sham operation) group,the mice received intraperitoneal injection of 2 ml normal saline for six times at 1-hour intervals.All the animals of each group were averaged to two batches,4 h and 8h after being operated respectively,to be anesthetized and adopted blood and tissue specimen.Then we observed the pathological change of pancreas and gut,scored it.We measured the blood value of diamine oxidase ( DAO),amylase and tumor necrosis factor-α (TNF-α).We detected content of malondialdehyde (MDA),superoxide dismutase (SOD),glutathione (GSH) and activity of xanthine oxidase (XO) in gut mucosa.We detected the casepase-3 activity and cell apopotosis by means of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in gut mucosa,and conculated the apopotosis index (AI).Then using the PASW 18.0 software,we analyzed the data by anova and t-test,to make sure if the values were statistically different between the two groups and the mechanism of gut barrier dysfunction in panreatitis.Results At 4 h and 8 h after operation,the SAP-group-mice had significantly higher pancreas pathological score (P ＜0.01 ),blood amylase value ( P ＜ 0.05 ),gut pathological score and blood DAO and TNF-α value ( P ＜0.01 ),compared with the contral-group-mice.The gut mucosa MDA content and XO activity of mice in SAP group were significantly higher than which in control group ( P ＜ 0.01 ). The SAP-group-mice had significantly lower gut mucosa SOD content ( P ＜ 0.01 ) and GSH content ( P ＜ 0.05 ),compared with the contral-group-mice.The gut mucosa cells of mice in SAP group had significantly higher caspase-3 activity and apoptosis index than which in control group ( P ＜ 0.01 ).Conclusions In severe acute pancreatitis,inflammatory factors such as TNF-αwere waterfall-style released,induced gut mucosa suffer from ischemia-reperfusion injury,then serious oxidative stress developed in mucosa and activated caspase-3 pathway,inducing gut mucosa cells apoptose seriously,which was an important mechanism of gut barrier dysfunction. Key words: Systemic inflammatory response syndrome (SIRS); Inflammatory factors ; Ischemia-reperfusion injury ; Oxygen radicals; Oxidative stress; Apoptosis; Severe acute pancreatitis; Gut barrier dysfunction

Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis.

Anti-apoptosis effects of NICD gene transfer to pancreatic acinar cells

Objective To investigate effect of the recombinant adenovirus Ad-NICD on apoptosis of pancreatic acinar cells in vitro.Methods The recombinant adenovirus Ad-NICD was propagated in 293 cells and the titer of adenovirus was determined by plaque bacteriophage formation test.The adenoviruses were infected into acinar cells AR4-2J,and the expression of NICD was detected by using real-time polymerase chain reaction (PCR).Control group,Ad-CMV-transfected group and Ad-NICD-transfected group were set up.Apoptosis of the infected acinar cells was induced by cerulein and the effect of Ad-NICD was detected by Annexin V/PI double labelling cytometry.Results The titer of adenovirus was 2.2 × 1010 pfu/L and the expression of NICD was increased effectively after the acinar cells were infected by the virus.When MOI was 100,the expression of NICD was highest.After treatment with cerulein,the apoptosis index of acinar cells in Ad-NICD-transferred group was ( 7.32 ± 1.67 ) ％,which was significantly lower than in control group ( 18.55 ± 2.61 )％ and Ad-CMV-transferred group ( 19.76 ± 4.28 )％.Conclusion Recombinant adenovirus-mediated gene transfer of NICD could inhibit the apoptosis of pancreatic acinar cells induced by cerulien,which may be related to the severity of acute pancreatitis. Key words: Pancreatic acinar cells; Gene transfer; Apoptosis

The potential value of 18F-FDG-PET in the early diagnosis and evaluating the effects of interventions for severe acute pancreatitis

Objective To investigate the potential value of (F-18)-fluoro-deoxy-D-glucose positron emission tomography (18F-FDG-PET) in the early diagnosis and evaluating the effects of interventions for severe acute pancreatitis in mice.Methods Fifty-seven female ICR mice weighing (20 ± 1 ) g were used in this study.There were 19 mice in each group.The model severe acute pancreatitis was induced by the mothod reported previously.Thirty min before the administration of lipopolysaccharide,mice in the PS341 treatment group were treated intraperitoneally with 0.5 mg/kg PS-341,and those in the control groups were treated with an equal volume of vehicle.Eight h after the first cerulein injection,mice were anesthetized with 50 mg/kg pentobarbital.Three mice were used for the 18F-FDG-PET examination,8 mice for the detection of pancreatic myeloperoxidase (MPO) activity,and the rest 8 mice for pancreatic morphological changes in histological sections.Results The pancreatic appearance could not be seen in the PET image in normal group; while in those mice with severe acute pancreatitis,the 18F-FDG uptake was significantly increased (5.27 ±0.35).PS-341 treatment significantly reduced the uptake of 18F-FDG within the pancreas (3.48 ±0.49).MPO assay also demonstrated that induction of severe acute pancreatitis elevated the activity of MPO (3.46 ± 0.28 ),and PS-341 treatment significantly reduced MPO activity ( 1.82 ±0.54).This result was in accordance with that of the positron emission tomography. Conclusion 18F-FDG-Positron emission tomography could be a sensitive and promising means in evaluating the therapeutic effect and adjusting medical interventions in pancreatitis. Key words: Acute pancreatitis; Proteasome inhibitor; PS-341 ; Positron emission tomography