Saikosaponin D Attenuates Pancreatic Injury Through Suppressing the Apoptosis of Acinar Cell via Modulation of the MAPK Signaling Pathway

Chronic pancreatitis (CP) is a progressive fibro-inflammatory syndrome. The damage of acinar cells is the main cause of inflammation and the activation of pancreatic stellate cells (PSCs), which can thereby possibly further aggravate the apoptosis of more acinar cells. Saikosaponind (SSd), a major active ingredient derived from Chinese medicinal herb bupleurum falcatum, which exerted multiple pharmacological effects. However, it is not clear whether SSd protects pancreatic injury of CP via regulating the apoptosis of pancreatic acinar cells. This study systematically investigated the effect of SSd on pancreatic injury of CP in vivo and in vitro. The results revealed that SSd attenuate pancreatic damage, decrease the apoptosis and suppress the phosphorylation level of MAPK family proteins (JNK1/2, ERK1/2, and p38 MAPK) significantly in the pancreas of CP rats. In addition, SSd markedly reduced the apoptosis and inflammation of pancreatic acinar AR42J cells induced by cerulein, a drug induced CP, or Conditioned Medium from PSCs (PSCs-CM) or the combination of PSCs-CM and cerulein. Moreover, SSd significantly inhibited the activated phosphorylation of JNK1/2, ERK1/2, and p38 MAPK induced by cerulein or the combination of PSCs-CM and cerulein in AR42J cells. Furthermore, SSd treatment markedly decreased the protein levels of p-JNK and p-p38 MAPK caused by PSCs-CM alone. In conclusion, SSd ameliorated pancreatic injury, suppressed AR42J inflammation and apoptosis induced by cerulein, interrupted the effect of PSCs-CM on AR42J cells inflammation and apoptosis, possibly through MAPK pathway.

Glyoxalase-I Is Upregulated in Acute Cerulein-Induced Pancreatitis: A New Mechanism in Pancreatic Inflammation?

Inflammation caused by oxidative stress (ROS) demonstrates an essential mechanism in the pathogenesis of acute pancreatitis (AP). Important sources for ROS comprise the reactive compound methylglyoxal (MGO) itself and the MGO-derived formation of advanced glycation end-products (AGEs). AGEs bind to the transmembrane receptor RAGE and activate NF-κB, and lead to the production of pro-inflammatory cytokines. MGO is detoxified by glyoxalase-I (Glo-I). The importance of Glo-I was shown in different models of inflammation and carcinogenesis. Nevertheless, the role of Glo-I and MGO in AP has not been evaluated so far. This study analyzed Glo-I in cerulein-(CN)-induced AP and determined the effects of Glo-I knockdown, overexpression and pharmacological modulation. Methods: AP was induced in C57BL6/J mice by i.p. injection of CN. Glo-I was analyzed in explanted pancreata by Western Blot, qRT-PCR and immunohistochemistry. AR42J cells were differentiated by dexamethasone and stimulated with 100 nM of CN. Cells were simultaneously treated with ethyl pyruvate (EP) or S-p-bromobenzylglutathione-cyclopentyl-diester (BrBz), two Glo-I modulators. Knockdown and overexpression of Glo-I was achieved by transient transfection with Glo-I siRNA and pEGFP-N1-Glo-I-Vector. Amylase secretion, TNF-α production (ELISA) and expression of Glo-I, RAGE and NF-κB were measured. Results: Glo-I was significantly upregulated on protein and mRNA levels in CN-treated mice and AR42J cells. Dexamethasone-induced differentiation of AR42J cells increased the expression of Glo-I and RAGE. Treatment of AR42J cells with CN and EP or BrBz resulted in a significant reduction of CN-induced amylase secretion, NF-κB, RAGE and TNF-α. Overexpression of Glo-I led to a significant reduction of CN-induced amylase levels, NF-κB expression and TNF-α, whereas Glo-I knockdown revealed only slight alterations. Measurements of specific Glo-I activity and MGO levels indicated a complex regulation in the model of CN-induced AP. Conclusion: Glo-I is overexpressed in a model of CN-induced AP. Pharmacological modulation and overexpression of Glo-I reduced amylase secretion and the release of pro-inflammatory cytokines in AP in vitro. Targeting Glo-I in AP seems to be an interesting approach for future in vivo studies of AP.

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

AMPK-SIRT1 Pathway Modulates The Apoptosis, Proliferation and Migration of AR42J Cells By Regulating p53 and NF-κB

Background: Acute pancreatitis (AP) is an acute abdomen caused by abnormal activation of trypsin. AMPK-SIRT1 pathway has been reported to be related to various diseases, but the function in AP remains unclear. This study is designed to investigate the mechanism and effect of AMPK-SIRT1 pathway in AP.Methods: An experimental AP model of AR42J cells was stimulated with caerulein after pretreated with compound C or metformin. The mRNA and protein expressions of genes were analyzed by qRT-PCR and western blot. Cell apoptosis, proliferation and migration were measured using flow cytometry, MTT and transwell assay. Results: After pretreated with metformin, expressions of p-AMPKα, SIRT1 were elevated, ace-p53, ace-NF-κB were attenuated, cell apoptosis, proliferation, and migration were decreased. After pretreated with compound C, the reverse effects occurred. p-AMPKα and SIRT1 expressions were decreased, ace-p53 and ace-NF-κB were rasied, and cell apoptosis, proliferation, and migration were enhanced after caerulein induced in each group. Conclusion: When AP happened, expressions of p-AMPKα and SIRT1 were reduced, resulting in up-regulation of acetylation levels of p53 and NF-κB, acceleration of cell apoptosis, proliferation and migration. It hinted that AMPK-SIRT1 pathway could modulate the apoptosis, proliferation, migration and inflammation reaction of AR42J cells by regulating p53 and NF-κB.

α-Lipoic Acid Inhibits IL-6 Expression by Upregulating PPAR-γ-Mediated Expression of Antioxidants in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells

Objectives Oxidative stress is regarded as a major pathogenic factor in acute pancreatitis. Obesity is thought to be a negative prognostic factor in acute pancreatitis. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Recent study showed that resistin aggravates the expression of inflammatory cytokines such as interleukine-6 (IL-6) and production of reactive oxygen species (ROS) in pancreatic acinar cells stimulated with cerulein, a cholecystokinin analogue, as an in vitro acute pancreatitis model. Peroxisome proliferator-activated receptor (PPAR)-γ increases expression of antioxidant enzymes such as catalase and heme oxygenase-1 (HO-1). α-Lipoic acid is a powerful antioxidant and anti-inflammatory nutrient. The present study was purposed to investigate whether α-lipoic acid inhibits IL-6 expression in resistin/cerulein-stimulated pancreatic acinar cells and to determine whether it reduces ROS in AR42J cells by upregulating PPAR-γ-mediated expression of HO-1 and catalase in pancreatic acinar cells. Methods Rat pancreatic acinar cell line, AR42J cells, were stimulated with resistin (2 ng/ml) and cerulean (10−8 M), in the presence or absence of α-lipoic acid. mRNA expression of IL-6 was determined by real-time PCR analysis. ROS levels were measured using DCF-DA fluorescence. Expression of PPAR-γ, HO-1, and catalase were determined by Western blotting. Results α-Lipoic acid significantly decreased IL-6 mRNA expression and ROS production in resistin/cerulein-stimulated AR42J cells. α-Lipoic acid also increased expression of PPAR-γ, HO-1 and catalase. Inhibitory effect of α-lipoic acid on resistin/cerulein–induced IL-6 expression was suppressed by addition of a specific PPAR-γ inhibitor GW9662. GW9662 reversed the effect of α-lipoic acid on expression of HO-1 and catalase in AR42J cells. Conclusions α-Lipoic acid suppresses cerulein/resistin-induced IL-6 expression and ROS production through PPAR-γ-mediated expression of HO-1 and catalase in pancreatic acinar cells. Funding Sources This study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

Lutein Inhibits IL-6 Expression via PPARγ-Mediated SOCS3 Expression in Cerulein-Stimulated Pancreatic Acinar Cells

Objectives Cerulein pancreatitis is the best model of human edematous pancreatitis. Our previous studies showed that cerulein induced interleukin-6 (IL-6) expression through janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 signaling pathway. SOCS3 functions by inhibiting the catalytic activity of JAKs that initiate signaling within the cells. Peroxisome proliferator activated receptor-gamma (PPAR-γ) ligands, 15d-PGJ2 and troglitazone, have been known to induce suppressor of cytokine signaling (SOCS) 3 expression and inhibited JAK2/STAT3 activation in pancreatic acinar cells. Lutein is a carotenoid with reported anti-inflammatory properties. The present study was undertaken to investigate the inhibitory effect and mechanism of lutein on cerulein-induced IL-6 expression by determining expression and activation of PPAR-γ and SOCS3 in pancreatic acinar cells. Methods Rat Pancreatic acinar cells (AR42J cell line) were used. The cells were treated with lutein in the presence or absence of the PPARγ antagonist GW9662, and then stimulated with cerulein. Expression of IL-6 was assessed by real-time PCR analysis. Western blot analysis was performed to determine levels of phosphorylated JAK2/STAT3 and SOCS 3, and nuclear translocation of PPAR. Results Lutein increased expression and nuclear translocation of PPARγ, and expression of SOCS 3 in AR42J cells. Cerulein-induced IL-6 expression, and phosphorylation of JAK2 and STAT3 were inhibited by lutein. GW9662 inhibited lutein-induced expression of SOCS 3. In addition, GW9662 suppressed inhibitory effects of lutein on cerulein-induced IL-6 expression and JAK2/STAT3 activation in AR42J cells. Conclusions Lutein induces PPARγ activation and SOCS 3 expression, which inhibits JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated pancreatic acinar cells. Funding Sources This study was supported by Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

PSD-95 protects the pancreas against pathological damage through p38 MAPK signaling pathway in acute pancreatitis

Acute pancreatitis is one of the leading causes of gastrointestinal disorder-related hospitalizations, yet its pathogenesis remains to be fully elucidated. Postsynaptic density protein-95 (PSD-95) is closely associated with tissue inflammation and injury. We aimed to investigate the expression of PSD-95 in pancreatic acinar cells, and its function in regulating the inflammatory response and pancreatic pathological damage in acute pancreatitis. A mouse model of edematous acute pancreatitis was induced with caerulein and lipopolysaccharide in C57BL/6 mice. Tat-N-dimer was injected to inhibit the PSD-95 activity separately, or simultaneously with SB203580, inhibitor of p38 MAPK phosphorylation. Rat pancreatic acinar cells AR42J were cultured with 1 μM caerulein to build a cell model of acute pancreatitis. PSD-95-knockdown and negative control cell lines were constructed by lentiviral transfection of AR42J cells. Paraffin-embedded pancreatic tissue samples were processed for routine HE staining to evaluate the pathological changes of human and mouse pancreatic tissues. Serum amylase and inflammatory cytokine levels were detected with specific ELISA kits. Immunofluorescence, immunohistochemical, Western-blot, and qRT-PCR were used to detect the expression levels of PSD-95, p38, and phosphorylated p38. Our findings showed that PSD-95 is expressed in the pancreatic tissues of humans, C57BL/6 mice, and AR42J cells, primarily in the cytoplasm. PSD-95 expression increased at 2 h, reaching the peak at 6 h in mice and 12 h in AR42J cells. IL-6, IL-8, and TNF-α increased within 2 h of disease induction. The pancreatic histopathologic score was greater in the PSD-95 inhibition group compared with the control ( P < 0.05), while it was lesser when phosphorylation of p38 MAPK was inhibited compared with the PSD-95 inhibition group ( P < 0.05). Moreover, phosphorylation of p38 MAPK increased statistically after PSD-95 knocked-down. In conclusion, PSD-95 effectively influences the pathological damage of the pancreas in acute pancreatitis by affecting the phosphorylation of p38 MAPK.

Angiotensin‐(1‐7) Alleviates Autophagy Response Through the PI3K/Akt/mTOR Signaling Pathway in Severe Acute Pancreatitis

Background: Severe acute pancreatitis (SAP) is a fatal medical emergency. The autophagy response is essential for cellular homeostasis, and plays an important role in SAP. We aimed to determine if angiotensin‐(1‐7), abbreviated as Ang1‐7, regulates the autophagy response in SAP and to elucidate the underlying mechanism.Methods: We used a rat model to investigate the effects of Ang1-7 on pancreatic pathomorphological damage and the autophagy response, which were evaluated using histological scoring and the quantification of the autophagy markers microtubule-associated protein 1 light chain 3 (LC3) and p62/SQSTM (p62) by western blotting and immunohistochemistry. We treated rat pancreatic acinar AR42J cells with caerulein (CAE) to build an in vitro model. To prevent degradation of the autophagy markers, so that we could determine the increase in autophagic vacuolization, we used chloroquine to inhibit autophagosome and lysosome fusion. The PI3K inhibitor BEZ235 was used to suppress PI3K/Akt/mTOR signaling. We observed the impact of Ang1-7 on the autophagy response and evaluated the underlying mechanism by detecting protein expressions of LC3 and p62.Results: In the rat SAP model, Ang1-7 significantly relieved pancreatic pathological damage. Ang1-7 also reduced autophagy protein markers, including the LC3-Ⅱ to LC3-Ⅰ ratio and the p62 level. In AR42J cells, the autophagy markers significantly increased after treatment with CAE and chloroquine. The autophagy response was significantly alleviated after treatment of the cells with Ang1-7, while blocking the PI3K/Akt/mTOR pathway remarkably counteracted this effect.Conclusions: Our results indicated that Ang1-7 alleviated the autophagy response in SAP via the PI3K/Akt/mTOR signaling pathway.