Cerulein-stimulated AR42J Cells Research Articles

Lutein inhibits IL‑6 expression by inducing PPAR‑γ activation and SOCS3 expression in cerulein‑stimulated pancreatic acinar cells.

Acute pancreatitis is a severe inflammatory disease of the pancreas. In experimental acute pancreatitis, cerulein induces the expression of interleukin-6 (IL-6) by activating Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 in pancreatic acinar cells. Ligands of peroxisome proliferator activated receptor-γ (PPAR-γ) and suppressor of cytokine signaling (SOCS) 3 inhibit IL-6 expression by suppressing JAK2/STAT3 in cerulein-stimulated pancreatic acinar AR42J cells. Lutein, an oxygenated carotenoid, upregulates and activates PPAR-γ to regulate inflammation in a renal injury model. The present study aimed to determine whether lutein activated PPAR-γ and induced SOCS3 expression in unstimulated AR42J cells, and whether lutein inhibited activation of JAK2/STAT3 and IL-6 expression via activation of PPAR-γ and SOCS3 expression in cerulein-stimulated AR42J cells. The anti-inflammatory mechanism of lutein was determined using reverse transcription-quantitative PCR, western blot analysis and enzyme-linked immunosorbent assay in AR42J cells stimulated with or without cerulein. In another experiment, cells were treated with lutein and the PPAR-γ antagonist GW9662 or the PPAR-γ agonist troglitazone prior to cerulein stimulation to determine the involvement of PPAR-γ activation. The results indicated that lutein increased PPAR-γ and SOCS3 levels in unstimulated cells. Cerulein increased phospho-specific forms of JAK2 and STAT3, and mRNA and protein expression of IL-6, but decreased SOCS3 levels in AR42J cells. Cerulein-induced alterations were suppressed by lutein or troglitazone. GW9662 alleviated the inhibitory effect of lutein on JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated cells. In conclusion, lutein inhibited the activation of JAK2/STAT3 and reduced IL-6 levels via PPAR-γ-mediated SOCS3 expression in pancreatic acinar cells stimulated with cerulein.

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.

Da Cheng Qi Decoction Alleviates Cerulein-Stimulated AR42J Pancreatic Acinar Cell Injury via the JAK2/STAT3 Signaling Pathway.

Background Acute pancreatitis (AP) is a common acute abdomen inflammation, characterized by the dysregulation of digestive enzyme production and secretion. Many studies have shown that Da Cheng Qi Decoction (DCQD) is a secure, effective prescription on AP. In this study, cerulein-stimulated AR42J cells damage model was established to further explore the feasibility and underlying mechanism of DCQD as a potential inhibitor of JAK2/STAT3 pathway for the treatment of AP. Methods Cell viability of DCQD was measured using a cell counting Kit-8 assay. Pancreatic biochemical markers such as amylase, lipase, and C-reactive protein production were measured by assay kits, respectively. Cytokines (TNF-α, IL-6, IL-10, and IL-1β) were assayed by ELISA. Protein location and protein expression were detected by immunofluorescence staining and Western blotting, respectively. Gene expression was assessed by real-time PCR. For mechanistic analysis of the effect of DCQD on JAK2/STAT3 signaling pathway, selective JAK2 inhibitor (Fedratinib) and STAT3 inhibitor (Stattic) as well as STAT3 activator (Garcinone D) were used. Results DCQD protected cells by regulating cerulein-induced inflammation and reducing the secretion of pancreatic biochemical markers. Moreover, DCQD could not only inhibit the nuclear translocation of p-STAT3, but also decrease the mRNA expression of JAK2 and STAT3 as well as the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 in protein level. Additionally, DCQD could regulate the mRNA and protein expression of JAK2/STAT3 downstream effectors, Bax and Bcl-XL. The activated effect of cerulein on JAK2/STAT3 pathway was also reversed by JAK2 inhibitor Fedratinib or STAT3 inhibitor Stattic. And the overexpression of JAK2/STAT3 pathway, via STAT3 activator Garcinone D, did exert damage on cells, which bore a resemblance to cerulein. Conclusion The activation of JAK2/STAT3 pathway may play a key role in the pathogenesis of cerulein-stimulated AR42J pancreatic acinar cell injury. DCQD could improve inflammatory cytokines and cell injury, which might be mediated by suppressing the activation of JAK2/STAT3 signaling pathway.

Corrigendum] SiRNA‑mediated PIAS1 silencing promotes inflammatory response and leads to injury of cerulein‑stimulated pancreatic acinar cells via regulation of the P38MAPK signaling pathway.

Following the publication of the above article, an interested reader drew to the authors' attention that the data shown in Fig.2D representing the P53 and Bax data were strikingly similar. After having re‑examined their raw data, the authors have realized that this error arose inadvertently; the data shown for Bax in the original figure were selected incorrectly. In the article, the expression levels of the apoptosis‑regulatory factors P53 and Bax were investigated by western blot analysis and reverse transcription‑quantitative PCR analysis. The authors were also able to confirm that this error regarding the image placement did not influence the statistical analysis shown for the effect of PIAS1 gene silencing on pancreatic acinar cell apoptosis. The corrected version of Fig. 2, containing the correct data for Bax protein expression in Fig.2D, is shown below. The authors are grateful to the Editor of International Journal of Molecular Medicine for granting them the opportunity to publish this Corrigendum, and stress that this error did not significantly influence either the results or the conclusions of the paper. Furthermore, the authors apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 26: 919-926, 2010; DOI: 10.3892/ijmm_00000507].

Docosahexaenoic Acid Induces Expression of NAD(P)H: Quinone Oxidoreductase and Heme Oxygenase-1 through Activation of Nrf2 in Cerulein-Stimulated Pancreatic Acinar Cells.

Oxidative stress is a major risk factor for acute pancreatitis. Reactive oxygen species (ROS) mediate expression of inflammatory cytokines such as interleukin-6 (IL-6) which reflects the severity of acute pancreatitis. The nuclear factor erythroid-2-related factor 2 (Nrf2) pathway is activated to induce the expression of antioxidant enzymes such as NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) as a cytoprotective response to oxidative stress. In addition, binding of Kelch-like ECH-associated protein 1 (Keap1) to Nrf2 promotes degradation of Nrf2. Docosahexaenoic acid (DHA)—an omega-3 fatty acid—exerts anti-inflammatory and antioxidant effects. Oxidized omega-3 fatty acids react with Keap1 to induce Nrf2-regulated gene expression. In this study, we investigated whether DHA reduces ROS levels and inhibits IL-6 expression via Nrf2 signaling in pancreatic acinar (AR42J) cells stimulated with cerulein, as an in vitro model of acute pancreatitis. The cells were pretreated with or without DHA for 1 h and treated with cerulein (10−8 M) for 1 (ROS levels, protein levels of NQO1, HO-1, pNrf2, Nrf2, and Keap1), 6 (IL-6 mRNA expression), and 24 h (IL-6 protein level in the medium). Our results showed that DHA upregulates the expression of NQO1 and HO-1 in cerulein-stimulated AR42J cells by promoting phosphorylation and nuclear translocation of Nrf2. DHA increased interaction between Keap1 and Nrf2 in AR42J cells, which may increase Nrf2 activity by inhibiting Keap1-mediated sequestration of Nrf2. In addition, DHA-induced expression of NQO1 and HO-1 is related to reduction of ROS and IL-6 levels in cerulein-stimulated AR42J cells. In conclusion, DHA inhibits ROS-mediated IL-6 expression by upregulating Nrf2-mediated expression of NQO1 and HO-1 in cerulein-stimulated pancreatic acinar cells. DHA may exert positive modulatory effects on acute pancreatitis by inhibiting oxidative stress and inflammatory cytokine production by activating Nrf2 signaling in pancreatic acinar cells.

P-168 - Inhibitory effect of docosahexaenoic acid on expression of inflammatory cytokine through activating Nrf2 signaling in cerulein-stimulated pancreatic acinar cells

Acute pancreatitis is a severe inflammatory condition of the pancreas. The development of acute pancreatitis involves increased oxidative stress. Increased reactive oxygen species (ROS) production induces expression of pro-inflammatory cytokines such as IL-6 in cerulein-stimulated pancreatic acinar cells. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a key regulator for expression of antioxidant enzymes such as NAD(P) H quinone dehydrogenase 1 (NQO1) and heme oxygenase-1 (HO-1). Docosahexaenoic acid (DHA), an essential omega-3 fatty acid, shows antioxidant and anti-inflammatory effects. DHA may inhibit oxidative stress in experimental acute pancreatitis using cerulean which is a cholecystokinin analog. The aim of this study was to investigate whether DHA inhibits cerulein-induced expression of pro-inflammatory cytokines such as interleukine-6 (IL-6) via Nrf2 signaling pathway in pancreatic acinar cells. As a result, DHA suppressed ROS levels and IL-6 expression in cerulein-stimulated AR42J cells. In addition, DHA increased phosphorylation of Nrf2, and expression of NQO1 and HO-1. These results suggest that DHA decreases ROS levels and IL-6 expression by upregulation of Nrf2-mediated NQO1 and HO-1 expression in cerulein-stimulated pancreatic acinar cells.

Protective Role of TNIP2 in Myocardial Injury Induced by Acute Pancreatitis and Its Mechanism.

BackgroundAberrant regulation of nuclear factor-κB (NF-κB) and the signaling pathways that regulate its activity have been found to be involved in various pathologies, particularly cancers, as well as inflammatory and autoimmune diseases. Acute pancreatitis (AP) is a complex pathological process, depending on autodigestion caused by premature activation of zymogens. This study aimed to investigate the effect of high expression of TNIP2 gene on AP and AP-induced myocardial injury.Material/MethodsTo investigate the effect of TNIP2 on AP and AP-induced myocardial injury, we established an AP cell model and rat model. HE staining was applied for histological examination. ELISA was used to determine the level of pro-inflammatory cytokines (TNF-α and IL-6) and myocardial injury markers (LDH and CK-MB). QRT-PCR and Western blot analysis were performed to determine the mRNA and protein level of related genes, respectively.ResultsWe found that the protein level of TNIP2 was relatively higher in the normal AR42J cells. At 4 h after stimulating with cerulein, the protein level of TNIP2 decreased, reached a minimum at 8 h, and then gradually increased. We also found that TNIP2 was correlated with the activation of NF-κB in cerulein-stimulated AR42J cells, and TNIP2 over-expression inhibited the inflammatory response caused by cerulein. Moreover, our results suggest that TNIP2 over-expression relieved the cerulein-triggered inflammatory response and AP-induced myocardial injury in mice.ConclusionsTNIP2 was shown to exert a protective effect on AP and AP-induced myocardial injury.

Peroxisome Proliferator-activated Receptor-gamma Inhibits the Activation of STAT3 in Cerulein-stimulated Pancreatic Acinar Cells.

Cerulein-induced pancreatitis is similar to human edematous pancreatitis, characterized by the dysregulation of digestive enzyme production, edema formation, and an infiltration of inflammatory cells into the pancreas. We previously showed that the Janus kinase 2 (JAK2)/STAT3 pathway mediates inflammatory signaling in cerulein-stimulated pancreatic acinar cells. PPAR-γ has been implicated in the regulation of inflammatory responses in several cells. In the present study, we investigated the role of PPAR-γ in cerulein-induced activation of JAK2/STAT3 in pancreatic acinar cells. Treatment with cerulein induced the activation of JAK2/STAT3 and PPAR-γ expression in AR42J cells. Cerulein-induced PPAR-γ expression was inhibited by AG490, a JAK2/STAT3 inhibitor, in AR42J cells. An immunoprecipitation analysis showed that PPAR-γ binds to STAT3 in cerulein-stimulated AR42J cells. Down-regulation of PPAR-γ by siRNA increased STAT3 phosphorylation in AR42J cells stimulated with cerulein. These results show that PPAR-γ inactivates STAT3 by directly interacting with STAT3 in cerulein-stimulated pancreatic acinar cells. Overexpression of PPAR-γ may be beneficial for preventing pancreatitis by suppressing the activation of STAT3 in pancreatic acinar cells.

Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in cerulein-stimulated pancreatic acinar cells

Cerulein pancreatitis mirrors human acute pancreatitis. In pancreatic acinar cells exposed to cerulein, reactive oxygen species (ROS) mediate inflammatory signaling by Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, and cytokine induction. Docosahexaenoic acid (DHA) acts as an agonist of peroxisome proliferator activated receptor γ (PPARγ), which mediates the expression of some antioxidant enzymes. We hypothesized that DHA may induce PPARγ-target catalase expression and reduce ROS levels, leading to the inhibition of JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated acinar cells. Pancreatic acinar AR42J cells were treated with DHA in the presence or absence of the PPARγ antagonist GW9662, or treated with the PPARγ agonist troglitazone, and then stimulated with cerulein. Expression of IL-6 and catalase, ROS levels, JAK2/STAT3 activation, and nuclear translocation of PPARγ were assessed. DHA suppressed the increase in ROS, JAK2/STAT3 activation, and IL-6 expression induced nuclear translocation of PPARγ and catalase expression in cerulein-stimulated AR42J cells. Troglitazone inhibited the cerulein-induced increase in ROS and IL-6 expression, but induced catalase expression similar to DHA in AR42J cells. GW9662 abolished the inhibitory effect of DHA on cerulein-induced increase in ROS and IL-6 expression in AR42J cells. DHA-induced expression of catalase was suppressed by GW9662 in cerulein-stimulated AR42J cells. Thus, DHA induces PPARγ activation and catalase expression, which inhibits ROS-mediated activation of JAK2/STAT3 and IL-6 expression in cerulein-stimulated pancreatic acinar cells.

Expression of the Transcription Factor Egr-1 in Pancreatic Acinar Cells Following Stimulation of Cholecystokinin or Gαq-Coupled Designer Receptors

Backgound/Aims: The injection of cerulein, an analogue of the pancreatic secretagogue cholecystokinin (CCK), induces acute pancreatitis in mice that is accompanied by the synthesis of the transcription factor Egr-1. The signaling cascade that connects cerulein stimulation with enhanced Egr-1 biosynthesis was analyzed. Methods: AR42J rat pancreatic acinar cells were used as a model system to measure cerulein-induced Egr-1 biosynthesis. For comparison, the signaling cascade induced by activation of Gαq-coupled designer receptors with the designer drug clozapine-N-oxide (CNO) was investigated. Results: Stimulation of AR42J cells with cerulein induced a robust and transient biosynthesis of Egr-1. The signaling cascade connecting cerulein stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca²⁺ and the activation of the protein kinases PKC, Raf and ERK, while expression of MKP-1 prevented Egr-1 biosynthesis in cerulein-stimulated AR42J cells. In addition, ternary complex factors are required to connect cerulein stimulation with enhanced transcription of the Egr-1 gene. Egr-1 biosynthesis induced in CNO-stimulated AR42J pancreatic acinar cells expressing Gαq-coupled designer receptors required identical signaling molecules, although subtle differences were observed in comparison to cerulein/CCK receptor signaling. Conclusion: We propose that overstimulation of the canonical Gαq-induced signaling pathway may be crucial for inducing acute pancreatitis.

SiRNA-based targeting of fractalkine overexpression suppresses inflammation development in a severe acute pancreatitis rat model

Fractalkine (FKN), a chemokine that acts as both an adhesion molecule and a chemoattractant, is expressed in many inflammatory diseases. Chemokines play a crucial role in severe acute pancreatitis (SAP). This study used adenovirus-mediated siRNA to target FKN overexpression and assessed its ability to suppress inflammation development in a SAP rat model. Adenovirus-mediated FKN siRNA was transfected into cerulein-stimulated AR42J cells. The growth of cerulein-stimulated AR42J cells was determined by colony formation and MTT assays. The inhibitory effect of the FKN siRNA was studied in a SAP rat model in vivo and detected by ELISA, RT-PCR, western blot analysis and immunohistochemistry. FKN, IL-8 and TNF-α were found to be overexpressed in cerulein-stimulated AR42J cells by ELISA and western blot analysis (P<0.05). The animal experiments confirmed that FKN siRNA could inhibit inflammation development in SAP. The values of serum FKN, TNF-α and IL-8 levels were decreased after FKN siRNA treatment (P<0.05). Furthermore, western blotting and RT-PCR analysis showed that FKN protein and mRNA levels were decreased after injection with FKN siRNA (P<0.05). Immunohistochemistry also showed that inflammation was decreased after injection with FKN-siRNA in the SAP rat model. Treatment with siRNA can inhibit FKN overexpression and also suppresses inflammation development in a SAP rat model. More importantly, this study indicated that FKN, which is overexpressed in the SAP rat model, may serve as a novel and effective therapeutic target for SAP.

Fractalkine Upregulates Inflammation through CX3CR1 and the Jak–Stat Pathway in Severe Acute Pancreatitis Rat Model

Based on the function of chemokine fractalkine (FKN), acting as both adhesion and chemoattractant, FKN plays a role in acute inflammatory response. In this study, we investigated the mechanism of FKN mediated upregulation inflammation in severe acute pancreatitis (SAP) rat models. Western blot, reverse transcriptase-polymerase chain reaction, and immunofluorescence demonstrated that FKN and its receptor CX3CR1 were overexpressed in cerulein-stimulated AR42J cells. AG490 and FKN-siRNA inhibited activation of Janus kinase/signal transducers and activators of transcription (Jak/Stat) in cerulein-stimulated AR42J cells. Following exposure AG490 and FKN-siRNA inhibited tumor necrosis factor-alpha expression by enzyme-linked immunosorbent assay and immunohistochemistry in vivo the SAP rat models. These results showed FKN and CX3CR1 were involved inflammatory response in cerulein-stimulated AR42J cells. FKN upregulates inflammation through CX3CR1 and the Jak/Stat pathway in SAP rat models.

SiRNA-mediated PIAS1 silencing promotes inflammatory response and leads to injury of cerulein-stimulated pancreatic acinar cells via regulation of the P38MAPK signaling pathway

Our aim in this study was to investigate the changes of inflammatory response by protein inhibitor of activated signal transducer and activator of transcription 1 (PIAS1) gene silencing treatment in cerulein-stimulated AR42J cells, and relate them to changes in cell injury, thus providing evidence for developing clinical therapies. This study examined the effects of cerulein on the activity of P38 mitogen activated protein kinase (P38MAPK), c-jun NH2-terminal kinase/stress-activated protein kinase and the inflammatory mediators released by PIAS1 gene-silenced AR42J cells. Consequently, the markers including DNA ladder, cell apoptotic rat, cell cycles, levels of cell cycle and apoptotic related factors were used to determine the effects of PIAS1 gene silencing on the cerulein-induced cell injury. The results indicated that in the cerulein-stimulated PIASI silencing cells, the activity of P38MAPK was enhanced, while at the same time, the levels of inflammatory mediators such as the tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and matrix metallopeptidase-9, were markedly higher than those of other cerulein-stimulated cells. Thus, the cerulein-stimulated PIASI gene-silenced cells obviously increased cell arrest in the G1/M phase by increasing P21 and P27 expression, and also induced apoptosis by regulating the P53 signaling pathway. This study suggests that the down-regulation of PIAS1 is efficacious at enhancing the expression of inflammatory mediators and inducing cell injury in acute pancreatitis (AP), thus deteriorating the severity of disease. It provides evidence that PIAS1 is a potential therapeutic target for AP.

SOCS 3 and PPAR-γ ligands inhibit the expression of IL-6 and TGF-β1 by regulating JAK2/STAT3 signaling in pancreas

Induction of proinflammatory cytokines IL-6 and TGF-β1 are the hallmark of human pancreatitis. Cerulein pancreatitis is similar to human edematous pancreatitis involving dysregulation of digestive enzyme production, cytoplasmic vacuolization, and increased cytokine production. We previously showed that cerulein induced IL-1β expression through the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway in pancreatic acinar cells. Suppressor of cytokine signaling (SOCS) is a negative feedback regulator of JAK/STAT signaling. In this study, we demonstrate that SOCS 3 is induced by cerulein in pancreatic acinar AR42J cells and in the rat pancreas. In both AR42J cells and rat pancreas, cerulein induced expression of IL-6 and TGF-β1, which is enhanced by transfection or injection of SOCS 3 antisense oligonucleotide (AS ODN). Pre-treating cerulein-stimulated AR42J cells or rats with the peroxisome proliferator activated receptor-γ (PPAR-γ) ligands, 15d-PGJ2 and troglitazone, induced SOCS 3 expression and inhibited JAK2/STAT3 activation. This treatment regimen also inhibited IL-6 and TGF-β1 induction, vacuolization, and α-smooth muscle actin (α-SMA) expression. Thus, SOCS 3 expression is associated with a reduction in IL-6 and TGF-β1 expression, edema formation, vacuolization, and α-SMA expression, possibly by direct regulation of JAK2/STAT3 signaling. 15d-PGJ2 and troglitazone are potentially useful pancreatitis therapies by suppressing the JAK2/STAT3 inflammatory signaling through SOCS 3 induction.