Galantamine ameliorates experimental pancreatitis.

BACKGROUND: Acute pancreatitis is a common and serious inflammatory condition currently lacking disease modifying therapy. The cholinergic anti-inflammatory pathway (CAP) is a potent protective anti-inflammatory response activated by vagus nerve-dependent α7 nicotinic acetylcholine receptor (α7nAChR) signaling using splenic CD4+ T cells as an intermediate. Activating the CAP ameliorates experimental acute pancreatitis. Galantamine is an acetylcholinesterase inhibitor (AChEI) which amplifies the CAP via modulation of central muscarinic ACh receptors (mAChRs). However, as mAChRs also activate pancreatitis, it is currently unknown whether galantamine would be beneficial in acute pancreatitis. METHODS: The effect of galantamine (1-6 mg/kg-body weight) on caerulein-induced acute pancreatitis was evaluated in mice. Two hours following 6 hourly doses of caerulein (50 µg/kg-body weight), organ and serum analyses were performed with accompanying pancreatic histology. Experiments utilizing vagotomy, gene knock out (KO) technology and the use of nAChR antagonists were also performed. RESULTS: Galantamine attenuated pancreatic histologic injury which was mirrored by a reduction in serum amylase and pancreatic inflammatory cytokines and an increase the anti-inflammatory cytokine IL-10 in the serum. These beneficial effects were not altered by bilateral subdiaphragmatic vagotomy, KO of either choline acetyltransferase+ T cells or α7nAChR, or administration of the nAChR ganglionic blocker mecamylamine or the more selective α7nAChR antagonist methyllycaconitine. CONCLUSION: Galantamine improves acute pancreatitis via a mechanism which does not involve previously established physiological and molecular components of the CAP. As galantamine is an approved drug in widespread clinical use with an excellent safety record, our findings are of interest for further evaluating the potential benefits of this drug in patients with acute pancreatitis.

Kidney morphology and renal expression of aquaporins 2, 3 and 4 during cerulein - Induced chronic pancreatitis in pigs.

PURPOSE: Chronic pancreatitis (CP) is associated with serious complications and reduced quality of life. Kidney failure is a frequent complication of acute pancreatitis (AP), however limited information is available regarding the impact of CP on this condition. In the kidney, 9 aquaporins (AQPs) are expressed to maintain body water homeostasis and concentrate urine. The purpose of this study was to morphologically assess and analyze the location and expression of AQP2, AQP3 and AQP4 and determine whether CP affects renal structure and expression of AQPs in collecting duct (CD) principal cells. MATERIALS/METHODS: CP was induced in domestic pigs through intramuscular injections of cerulein (1 â€‹µg/kg â€‹bw/day for 6 days; n â€‹= â€‹5); pigs without CP (n â€‹= â€‹5) were used as a control group. Kidney samples were collected 6 weeks after the last injection and subjected to histological examination. Expression of AQPs was determined by immunohistochemistry and Western blot. RESULTS: The kidneys of animals with CP exhibited moderate changes, including glomerular enlargement, increased collagen percentage, numerous stromal erythrorrhages and inflammatory infiltrations compared to control group. Although the total abundance of AQP2 in the CD decreased in pigs after cerulein administration, the difference was not statistically significant. Expression of AQP3 and AQP4 was limited to the basolateral membrane of the CD cells. AQP4 abundance remained relatively stable in both groups, while AQP3 expression increased nearly three-fold in pigs with CP. CONCLUSION: This study identified morphological alterations and a statistically significant increase in the expression of renal AQP3 when pigs developed CP.

PIAS1 upregulation confers protection against Cerulein-induced acute pancreatitis via FTO downregulation by enhancing sumoylation of Foxa2.

OBJECTIVE: This research discussed the specific mechanism by which PIAS1 affects acute pancreatitis (AP). METHODS: PIAS1, Foxa2, and FTO expression was assessed in Cerulein-induced AR42J cells and mice. Loss- and gain-of-function assays and Cerulein induction were conducted in AR42J cells and mice for analysis. The relationship among PIAS1, Foxa2, and FTO was tested. Cell experiments run in triplicate, and eight mice for each animal group. RESULTS: Cerulein-induced AP cells and mice had low PIAS1 and Foxa2 and high FTO. Cerulein induced pancreatic injury in mice and inflammation and oxidative stress in pancreatic tissues, which could be reversed by PIAS1 or Foxa2 upregulation or FTO downregulation. PIAS1 elevated SUMO modification of Foxa2 to repress FTO transcription. FTO upregulation neutralized the ameliorative effects of PIAS1 or Foxa2 upregulation on Cerulein-induced AR42J cell injury, inflammation, and oxidative stress. CONCLUSION: PIAS1 upregulation diminished FTO transcription by increasing Foxa2 SUMO modification, thereby ameliorating Cerulein-induced AP.

Expression level of serum miR-374a-5p in patients with acute pancreatitis and its effect on viability, apoptosis, and inflammatory factors of pancreatic acinar cells induced by cerulein.

Acute pancreatitis (AP) is one of the life-threatening diseases of the digestive system. MicroRNA has been asserted to be a regulator of AP. This paper explored the miR-374a-5p expression in AP patients and investigated the efficacy of AR42J cells. In this study, 60 healthy people, 58 MAP patients and 58 SAP patients were included, and the serum miR-374a-5p levels of the subjects were detected by RT-qPCR technology. The pancreatitis cell model was structured by stimulating AR42J cells with cerulein. Next, cell viability and apoptosis were detected by CCK-8 assay and flow cytometry. ELISA was used to measure the concentration of cytokines, such as TNF-α, IL-6, and IL-1ß. The data showed that miR-374a-5p was downregulated in samples from AP patients, while showing discriminative power for AP populations. Attenuated miR-374a-5p were negatively bound up with patients' Ranson score and APACHE II score. Besides, miR-374a-5p was declined in cerulein-treated AR42J cells and forced elevation of miR-374a-5p was beneficial to increase cell viability, and inhibit cell apoptosis and inflammation. The present study found that miR-374a-5p was reduced in AP serum samples, and up-regulated expression level of miR-374a-5p in cell models had a protective effect on cerulein-induced inhibition of cell function and inflammatory response.

The Kruppel-like factor 4-signal transducer and activator of transcription 5A axis promotes pancreatic fibrosis in mice with caerulein-induced chronic pancreatitis.

Pancreatic fibrosis (PF) is a hallmark of chronic pancreatitis (CP), but its molecular mechanism remains unclear. This study was conducted to explore the role of Kruppel-like factor 4 (KLF4) in PF in CP mice. The CP mouse model was established using caerulein. After KLF4 interference, pathological changes in pancreatic tissues and fibrosis degree were observed by hematoxylin-eosin staining and Masson staining, and levels of Collagen I, Collagen III, and alpha-smooth muscle actin, inflammatory cytokines, KLF4, signal transducer and activator of transcription 5A (STAT5) in pancreatic tissues were measured by enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blot assay, and immunofluorescence. The enrichment of KLF4 on the STAT5 promoter and the binding of KLF4 to the STAT5 promoter were analyzed. The rescue experiments were performed by co-injection of sh-STAT5 and sh-KLF4 to confirm the regulatory mechanism of KLF4. KLF4 was upregulated in CP mice. Inhibition of KLF4 effectively attenuated pancreatic inflammation and PF in mice. KLF4 was enriched on the STAT5 promoter and enhanced the transcriptional and protein levels of STAT5. Overexpression of STAT5 reversed the inhibitory role of silencing KLF4 in PF. In summary, KLF4 promoted the transcription and expression of STAT5, which further facilitated PF in CP mice.

Downregulation of miR-146a-5p Promotes Acute Pancreatitis through Activating the TLR9/NLRP3 Signaling Pathway by Targeting TRAF6 In Vitro Rat Model.

Acute pancreatitis (AP) is mainly caused by acinar cells releasing various inflammatory factors, causing inflammatory storms and leading to severe pancreatitis. Detection methods and treatment targets for pancreatitis are lacking, raising the urgency of identifying diagnostic markers and therapeutic targets for AP. MicroRNAs (miRNAs) have recently been identified as molecular markers for various biological processes such as tumors, immunity, and metabolism, and the involvement of miRNAs in inflammatory responses has been increasingly studied. To explore the role of miRNAs in AP is the primary objective of this study. By using qPCR on our cerulein-induced pancreatitis cell model, it is worth noting that the change of miR-146a-5p expression in inflammation-related miRNAs in AP was predominant. Next, ELISA, CCK8, and flow cytometry were used to inspect the impact of miR-146a-5p on pancreatitis. BiBiServ bioinformatics anticipated binding ability of miR-146a-5p and 3'-untranslated region (3'UTR) of TNF receptor-associated factor 6 (TRAF6), and the dual-luciferase assay verified the combination of the two. TRAF6 knockdown verified the effect of TRAF6 on the progression of pancreatitis. Finally, rescue experiments verified the capability of miR-146a-5p and TRAF6 interaction on the Toll-like receptor 9 (TLR9)/NOD-like receptor protein 3 (NLRP3) signaling pathway and cell function. The expression of miR-146a-5p decreased in cerulein-induced AR42J pancreatic acinar cells. Functional experiments verified that miR-146a-5p facilitated the proliferation of AR42J pancreatic acinar cells and inhibited their apoptosis. Bioinformatic predictions and dual-luciferase experiments verified the actual binding efficiency between miR-146a-5p and 3'UTR of TRAF6. Our study confirmed that knockdown of TRAF6 restrained the progression of pancreatitis, and knockdown of TRAF6 rescued pancreatitis caused by miR-146a-5p downregulation by the TLR9/NLRP3 signaling pathway. Therefore, downregulation of miR-146a-5p in the induced pancreatitis cell model promotes the progression of pancreatitis via the TLR9/TRAF6/NLRP3 signaling pathway. There is potential for miR-146a-5p to serve as a diagnostic marker and therapeutic nucleic acid drug for AP.

Inhibition of ANXA2 regulated by SRF attenuates the development of severe acute pancreatitis by inhibiting the NF-κB signaling pathway.

BACKGROUND: Acute pancreatitis (AP) is an inflammatory process of the pancreas resulting from biliary obstruction or alcohol consumption. Approximately, 10-20% of AP can evolve into severe AP (SAP). In this study, we sought to explore the physiological roles of the transcription factor serum response factor (SRF), annexin A2 (ANXA2), and nuclear factor-kappaB (NF-κB) in SAP. METHODS: C57BL/6 mice and rat pancreatic acinar cells (AR42J) were used to establish an AP model in vivo and in vitro by cerulein with or without lipopolysaccharide (LPS). Production of pro-inflammatory cytokines (IL-1ß and TNF-α) were examined by ELISA and immunoblotting analysis. Hematoxylin and eosin (HE) staining and TUNEL staining were performed to evaluate pathological changes in the course of AP. Apoptosis was examined by flow cytometric and immunoblotting analysis. Molecular interactions were tested by dual luciferase reporter, ChIP, and Co-IP assays. RESULTS: ANXA2 was overexpressed in AP and correlated to the severity of AP. ANXA2 knockdown rescued pancreatic acinar cells against inflammation and apoptosis induced by cerulein with or without LPS. Mechanistic investigations revealed that SRF bound with the ANXA2 promoter region and repressed its expression. ANXA2 could activate the NF-κB signaling pathway by inducing the nuclear translocation of p50. SRF-mediated transcriptional repression of ANXA2-protected pancreatic acinar cells against AP-like injury through repressing the NF-κB signaling pathway. CONCLUSION: Our study highlighted a regulatory network consisting of SRF, ANXA2, and NF-κB that was involved in AP progression, possibly providing some novel targets for treating SAP.

Activity of acute pancreatitis is modified by secreted protein acidic and rich in cysteine ablation.

BACKGROUND: Acute pancreatitis (AP) is a frequent cause for hospitalization. However, molecular determinants that modulate severity of experimental pancreatitis are only partially understood. OBJECTIVE: To investigate the role of secreted protein acidic and rich in cysteine (SPARC) during cerulein-induced AP in mice. METHODS: AP was induced by repeated cerulein injections in SPARC knock-out mice (SPARC-/- ) and control littermates (SPARC+/+ ). Secreted protein acidic and rich in cysteine expression and severity of AP were determined by histopathological scoring, immunohistochemistry, and biochemical assays. For functional analysis, primary murine acinar cell cultures with subsequent amylase release assays were employed. Proteome profiler assay and ELISA were conducted from pancreatic tissue lysates, and co-immunofluorescence was performed. RESULTS: Upon cerulein induction, SPARC expression was robustly induced in pancreatic stellate cells (PSCs) but not in acinar cells. Genetic SPARC ablation resulted in attenuated severity of AP with significantly reduced levels of pancreatic necrosis, apoptosis, immune cell infiltration, and reduced fibrosis upon chronic stimulation. However, the release of amylase upon cerulein stimulation in primary acinar cell culture from SPARC+/+ and SPARC-/- was indistinguishable. Notably, immune cell derived C-C Motif Chemokine Ligand 2 (CCL2) was highly elevated in SPARC+/+ pancreatic tissue potentially linking PSC derived SPARC with CCL2 induction in AP. CONCLUSION: SPARC mediates the severity of AP. The potential link between SPARC and the CCL2 axis could open new avenues for tailored therapeutic interventions in AP patients and warrants further investigations.

MicroRNA profiling predicts positive nodal status in papillary thyroid carcinoma in the preoperative setting.

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.

Stem bark of Fraxinus rhynchophylla ameliorates the severity of pancreatic fibrosis by regulating the TGF-ß/Smad signaling pathway.

Chronic pancreatitis (CP) is a pathological fibroinflammatory syndrome of the pancreas. Currently, there are no therapeutic agents available for treating CP-associated pancreatic fibrosis. Fraxinus rhynchophylla (FR) reportedly exhibits anti-inflammatory, antioxidative and antitumor activities. Although FR possesses numerous properties associated with the regulation of diverse diseases, the effects of FR on CP remain unknown. Herein, we examined the effects of FR on CP. For CP induction, mice were intraperitoneally administered cerulein (50 µg/kg) 6 times a day, 4 days per week for 3 weeks. FR extract (100 or 400 mg/kg) or saline (control group) was intraperitoneally injected 1 hour before the first cerulein injection. After 3 weeks, the pancreas was harvested for histological analysis. In addition, pancreatic stellate cells (PSCs) were isolated to examine the antifibrogenic effects and regulatory mechanisms of FR. Administration of FR significantly inhibited histological damage in the pancreas, increased pancreatic acinar cell survival, decreased PSC activation and collagen deposition, and decreased pro-inflammatory cytokines. Moreover, FR treatment inhibited the expression of fibrotic mediators, such as α-smooth muscle actin (α-SMA), collagen, fibronectin 1, and decreased pro-inflammatory cytokines in isolated PSCs stimulated with transforming growth factor (TGF)-ß. Furthermore, FR treatment suppressed the phosphorylation of Smad 2/3 but not of Smad 1/5 in TGF-ß-stimulated PSCs. Collectively, these results suggest that FR ameliorates pancreatic fibrosis by inhibiting PSC activation during CP.

TSPAN1 silencing protects against cerulein-induced pancreatic acinar cell injury via targeting AGR2.

Acute pancreatitis (AP) is an inflammatory gastrointestinal disorder affecting the pancreas. Previous study reported that tetraspanin 1 (TSPAN1) expression was significantly upregulated in the pancreas of AP patients. However, the underlying molecular mechanism of TSPAN1 in the pathogenesis of AP remains unclear. Thus, the aim of the present study was to investigate the potential role of TSPAN1 in development of AP. RT-qPCR was carried out to quantify the relative mRNA levels of TSPAN1 and anterior gradient-2 (AGR2). The CCK-8 assay was used to detect the cell viability. The TUNEL assay was performed to visualize the apoptotic cells. Western blot was performed to determine the expressions of proteins related to endoplasmic reticulum (ER) stress and apoptosis. ELISA kits were adopted to detect the concentration of inflammatory cytokines including TNF-α and IL-6. Finally, immunoprecipitation (IP) was used to verify the interaction between TSPAN1 and AGR2. TSPAN1 was upregulated in serum of AP patients and AP cell models. TSPAN1 silencing promoted the cell proliferation and inhibited inflammatory response in cerulein-induced AR42J cells. Moreover, TSPAN1 induced endoplasmic reticulum stress by binding AGR2. Interestingly, the overexpression of AGR2 abolished the effects of TSPAN1 silencing on cell proliferation and inflammatory response in cerulein-induced AR42J cells. In summary, TSPAN1 silencing protects against cerulein-induced pancreatic acinar cell injury through inhibiting ER stress-mediated by AGR2. Hence, TSPAN1 may serve as a promising therapeutic target for AP treatment.

Interfering hsa_circ_0073748 alleviates caerulein-induced ductal cell injury in acute pancreatitis by inhibiting miR-132-3p/TRAF3/NF-κB pathway.

Circular RNA hsa_circ_0073748 (circ_0073748) is upregulated in patients with acute pancreatitis (AP), a clinically common sudden inflammatory response. MicroRNA (miR)-132-3p is a stress-induced factor with high conservation between species. Herein, expression and role of circ_0073748 and miR-132-3p in caerulein-induced pancreatitis were studied. Expression levels of circ_0073748, miR-132-3p, TNF receptor associated factor 3 (TRAF3), Bcl-2 and Bcl-2-associated X protein (Bax) were examined by reverse transcription-quantitative PCR and Western blotting. Cell proliferation was measured by MTS and EdU assays. Flow cytometry and assay kits detected apoptosis, inflammatory, and oxidative responses. Western blotting detected nuclear factor (NF)-κB signaling pathway. Circ_0073748 was upregulated and miR-132-3p was downregulated in AP patients' plasma and human pancreatic ductal HPDE6-C7 cells with caerulein induction. Interfering circ_0073748 and reinforcing miR-132-3p improved cell viability, EdU incorporation, and superoxide dismutase (SOD) activity of caerulein-treated HPDE6-C7 cells but suppressed malonaldehyde (MDA), IL-6 and TNF-α levels and apoptosis rate. Moreover, TRAF3 downregulation was allied with circ_0073748 silencing and miR-132-3p overexpression in caerulein-induced HPDE6-C7 cells. Mechanically, circ_0073748 was identified as a sponge for miR-132-3p to modulate TRAF3 expression, thus establishing a competitive endogenous RNA (ceRNA) regulation model. Notably, circ_0073748 blockage could suppress expressions of phosphorylated P65 (p-P65) and p-IκB in caerulein-induced HPDE6-C7 cells by promoting miR-132-3p and inhibiting TRAF3. Silencing circ_0073748 and upregulating miR-132-3p could alleviate caerulein-induced HPDE6-C7 injury and inactivate canonical NF-κB signal by inhibiting TRAF3. Circ_0073748/miR-132-3p/TRAF3 ceRNA pathway might be one underlying mechanism and therapeutic target of caerulein-induced AP.

Role of tumor necrosis factor receptor­associated factor 6 in pyroptosis during acute pancreatitis.

Acute pancreatitis (AP) is hypothesized to be related to the activation of an inflammatory response induced by pyroptosis. The aim of the present study was to investigate the potential role of tumor necrosis factor receptor­associated factor 6 (TRAF6) in pyroptosis in an AP rat model and the human pancreatic ductal epithelial HPDE6C7 cell line. In vivo, AP was induced by intraperitoneal injection of caerulein (CAE) in rats. The rats were sacrificed at 24 or 48 h after the final CAE injection. In vitro, HPDE6C7 cells were treated with CAE for 12, 24 and 48 h. Moreover, TRAF6 was overexpressed and treated with CAE for 48 h. Histopathological changes of pancreatic, serum and supernatant inflammatory cytokines and pyroptosis­related mRNA and protein expression levels were determined by histopathological scores, ELISA, reverse transcription­quantitative PCR and western blotting. In addition, pyroptosis morphological changes were also determined by Hoechst/PI staining in HPDE6C7 cells. Results showed that AP was observed in the CAE­induced rat model, and that serum IL­1ß and IL­18 levels, and TRAF6, NLR pyrin domain containing 3 (NLRP3), caspase­1 and caspase­3 mRNA and protein expression levels were increased. Similar in HPDE6C7 cells, CAE treatment caused supernatant IL­1ß level, NLRP3 and caspase­1 mRNA expression levels to significantly increase. After TRAF6 overexpression and CAE treatment, supernatant IL­1ß level, caspase­1 protein expression level, and NLRP3 and caspase­3 mRNA and protein expression levels were also significantly increased. Furthermore, cells exhibited red fluorescence in Hoechst/PI staining, which can be used as a method of detecting pyroptosis activation. The results also showed that the red fluorescence was stronger after CAE treatment or TRAF6 overexpression plus CAE treatment. In conclusion, TRAF6 and caspase­1/3 signaling pathways were involved in the pathogenesis of CAE­induced AP in rats. Pyroptosis was activated by CAE and TRAF6 overexpression via the caspase­1/3 signaling pathways in HPDE6C7 cells.

Distinct Murine Pancreatic Transcriptomic Signatures during Chronic Pancreatitis Recovery.

We have previously demonstrated that the pancreas can recover from chronic pancreatitis (CP) lesions in the cerulein-induced mouse model. To explore how pancreatic recovery is achieved at the molecular level, we used RNA-sequencing (seq) and profiled transcriptomes during CP transition to recovery. CP was induced by intraperitoneally injecting cerulein in C57BL/6 mice. Time-matched controls (CON) were given normal saline. Pancreata were harvested from mice 4 days after the final injections (designated as CP and CON) or 4 weeks after the final injections (designated as CP recovery (CPR) and control recovery (CONR)). Pancreatic RNAs were extracted for RNA-seq and quantitative (q) PCR validation. Using RNA-seq, we identified a total of 3,600 differentially expressed genes (DEGs) in CP versus CON and 166 DEGs in CPR versus CONR. There are 132 DEGs overlapped between CP and CPR and 34 DEGs unique to CPR. A number of selected pancreatic fibrosis-relevant DEGs were validated by qPCR. The top 20 gene sets enriched from DEGs shared between CP and CPR are relevant to extracellular matrix and cancer biology, whereas the top 10 gene sets enriched from DEGs specific to CPR are pertinent to DNA methylation and specific signaling pathways. In conclusion, we identified a distinct set of DEGs in association with extracellular matrix and cancer cell activities to contrast CP and CPR. Once during ongoing CP recovery, DEGs relevant to DNA methylation and specific signaling pathways were induced to express. The DEGs shared between CP and CPR and the DEGs specific to CPR may serve as the unique transcriptomic signatures and biomarkers for determining CP recovery and monitoring potential therapeutic responses at the molecular level to reflect pancreatic histological resolution.

Protective effects of baicalin on caerulein-induced AR42J pancreatic acinar cells by attenuating oxidative stress through miR-136-5p downregulation.

Baicalin, the main active component of Scutellaria baicalensis, has antioxidant and anti-apoptotic effects and is used to treat acute pancreatitis; however, its specific mechanism is unclear. This study aims to determine the protective effect and underlying mechanism of baicalin on AR42J pancreatic acinar cell injury. AR42J acinar cells (caerulein, 10 nmol/L) were induced in vitro to establish a cell model for acute pancreatitis. Cell relative survival was measured by thiazolyl blue tetrazolium bromide, and cell apoptosis and death were examined by flow cytometry. The expression levels of superoxide dismutase1 (SOD1), Bax, survivin, Bcl-2, caspase-3, and caspase-7 proteins were analyzed by Western blot, and those of SOD1 mRNA and miR-136-5p were determined by RT-PCR. The activities of GSH, SOD1, ROS, and MDA were also investigated. Compared with those of the caerulein group, the relative survival rate and activity of AR42J pancreatic acinar cells with different baicalin concentrations were significantly increased (p < 0.05), and the supernatant amylase level was markedly decreased (p < 0.05). In addition, the ROS and MDA activities and mir-136-5p expression were significantly decreased, and the GSH activities and SOD1 gene and protein expression levels were markedly increased (p < 0.05). These results suggest that baicalin reduced the caerulein-induced death of AR42J acinar cells and alleviated the caerulein-induced injury in pancreatic acinar cells by inhibiting oxidative stress. The mechanism may be related to the decreased expression of Mir-136-5p and the increased expression of SOD1 gene and protein.

Concerted cell and in vivo screen for pancreatic ductal adenocarcinoma (PDA) chemotherapeutics.

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.

The role of calcium-binding protein S100g (CalbindinD-9K) and annexin A10 in acute pancreatitis.

BACKGROUND: We reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis. METHOD: We induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca2+]i), and cell viability in these cells, when incubated with cholecystokinin (CCK). RESULTS: The expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca2+]i increase in AR42J-S100g cells. These effects were weak in AR42J-Anxa10 cells. Cell viability was not changed by incubation with cerulein. CONCLUSION: In cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca2+ buffer in acute pancreatitis.

Leelamine Is a Novel Lipogenesis Inhibitor in Prostate Cancer Cells In Vitro and In Vivo.

Increased de novo synthesis of fatty acids is implicated in the pathogenesis of human prostate cancer, but a safe and effective clinical inhibitor of this metabolic pathway is still lacking. We have shown previously that leelamine (LLM) suppresses transcriptional activity of androgen receptor, which is known to regulate fatty acid synthesis. Therefore, the current study was designed to investigate the effect of LLM on fatty acid synthesis. Exposure of 22Rv1, LNCaP, and PC-3 prostate cancer cells, but not RWPE-1 normal prostate epithelial cell line, to LLM resulted in a decrease in intracellular levels of neutral lipids or total free fatty acids. LLM was superior to another fatty acid synthesis inhibitor (cerulenin) for suppression of total free fatty acid levels. LLM treatment downregulated protein and/or mRNA expression of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase, and sterol regulatory element-binding protein 1 (SREBP1) in each cell line. Consistent with these in vitro findings, we also observed a significant decrease in ATP citrate lyase and SREBP1 protein expression as well as number of neutral lipid droplets in vivo in 22Rv1 tumor sections of LLM-treated mice when compared with that of controls. LLM-mediated suppression of intracellular levels of total free fatty acids and neutral lipids was partly attenuated by overexpression of SREBP1. In conclusion, these results indicate that LLM is a novel inhibitor of SREBP1-regulated fatty acid/lipid synthesis in prostate cancer cells that is not affected by androgen receptor status.

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis.

BACKGROUND: During pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence. METHODS: We examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis. RESULTS: Pancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin. CONCLUSION: Our findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.

Azithromycin does not improve disease severity in acute experimental pancreatitis.

Acute pancreatitis is a severe systemic disease triggered by a sterile inflammation and initial local tissue damage of the pancreas. Immune cells infiltrating into the pancreas are main mediators of acute pancreatitis pathogenesis. In addition to their antimicrobial potency, macrolides possess anti-inflammatory and immunomodulatory properties which are routinely used in patients with chronic airway infections and might also beneficial in the treatment of acute lung injury. We here tested the hypothesis that the macrolide antibiotic azithromycin can improve the course of acute experimental pancreatitis via ameliorating the damage imposed by sterile inflammation, and could be used as a disease specific therapy. However, our data show that azithromycin does not have influence on caerulein induced acute pancreatitis in terms of reduction of organ damage, and disease severity. Furthermore Infiltration of immune cells into the pancreas or the lungs was not attenuated by azithromycin as compared to controls or ampicillin treated animals with acute experimental pancreatitis. We conclude that in the chosen model, azithromycin does not have any beneficial effects and that its immunomodulatory properties cannot be used to decrease disease severity in the model of caerulein-induced pancreatitis in mice.