Inhibition of TRAF6 improves hyperlipidemic acute pancreatitis by alleviating pyroptosis in vitro and in vivo rat models.

OBJECTIVE: Hypertriglyceridemia (HTG) is one of the common causes of acute pancreatitis (AP). Hyperlipidemic acute pancreatitis (HTG-AP) is associated with higher mortality owing to its tendency for greater severity and rapid progression. The purpose of this study was to explore the mechanism of involvement of tumor necrosis factor receptor-related factor 6 (TRAF6) in pyroptosis during HTG-AP. METHODS: The HTG environment was simulated with palmitic acid treatment in vitro and a high-fat diet in vivo. Cerulein was used to establish the HTG-AP model, followed by genetic and pharmacological inhibition of TRAF6. Pyroptosis activation, inflammatory reaction, and the interaction between TRAF6 and pyroptosis in HTG-AP were assessed. RESULTS: HTG was found to aggravate the development of pancreatitis, accompanied by increased pyroptosis and enhanced inflammatory response in HTG-AP models. Mechanistically, TRAF6 downregulation decreased the activation of pyroptosis in cerulein-induced HTG-AP. CONCLUSION: Collectively, inhibition of TRAF6 improved HTG-AP and the associated inflammation by alleviating pyroptosis.

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.

Effects of Lipolysis-Stimulated Lipoprotein Receptor on Tight Junctions of Pancreatic Ductal Epithelial Cells in Hypertriglyceridemic Acute Pancreatitis.

Objective: We investigated the effects of lipolysis-stimulated lipoprotein receptor (LSR) on the tight junctions (TJs) of pancreatic ductal epithelial cells (PDECs) in hypertriglyceridemic acute pancreatitis (HTGAP). Methods: Sprague-Dawley rats were fed standard rat chow or a high-fat diet and injected with sodium taurocholate to obtain normal and HTGAP rats, respectively. Serum triglyceride (TG) levels, pathological changes, TJ proteins in the pancreas, and TJ ultrastructure of PDECs were assessed. LSR overexpression (OE) and knockdown (KD) HPDE6-C7 models were designed and cultured in a high-fat environment. Protein levels were quantified by Western blotting. Cell monolayer permeability was detected using FITC-Dextran. Results: Serum TG concentration and pancreatic scores were higher in the HTGAP group than in the normal group. Among the TJ proteins, LSR protein expression was significantly lower in the HTGAP group than in the acute pancreatitis (AP) group. Tricellulin (TRIC) expression in the pancreatic ductal epithelia was higher in the HTGAP group than in the AP group. The HTGAP group had lower TJ protein levels, wider intercellular space, and widespread cellular necrosis with disappearance of cell junction structures. In the cell study, TJ proteins were downregulated and the cellular barrier was impaired by palmitic acid (PA), which was reversed by LSR-OE, whereas LSR-KD downregulated the TJ proteins and aggravated PA-induced cellular barrier impairment. Conclusions: Hypertriglyceridemia downregulates the TJ proteins in PDECs, which may impair the pancreatic ductal mucosal barrier function. LSR regulation can change the effects of HTG on cellular barrier function by upregulating the TJ proteins.

Effect of TRAF6 in acute pancreatitis-induced intestinal barrier injury via TLR4/NF-κB signal pathway.

BACKGROUND: The aim of this study was to investigate the effect of tumor necrosis factor receptor-related factor 6 (TRAF6) in acute pancreatitis (AP)-induced intestinal barrier injury via the Toll-like receptor 4/nuclear factor kappa-B (TLR4/NF-κB) signal pathway. METHODS: Rat models of acute edematous pancreatitis (AEP) and acute necrotizing pancreatitis (ANP) were established by intraperitoneal injection of caerulein and retrograde infusion of sodium taurocholate solution into the biliopancreatic duct, respectively. Separate groups of model rats were pretreated with the TRAF6 inhibitor, MG-132. Rats were sacrificed at 12 h after the last injection for inducing AP. Histopathological changes, inflammatory response, intestinal barrier function, and protein expression levels were assessed by pathological score, ELISA, TUNEL, qRT-PCR, immunohistochemistry and western blotting. RESULTS: Rat models of AEP and ANP were successfully established as evidenced by the pathological changes in the pancreas and intestine. Pre-treatment with MG-132 significantly alleviated pancreatic and intestinal pathological scores, reduced serum levels of amylase, IL-1ß, and IL-6, and ameliorated apoptosis of mucosal cells. MG-132 reduced intestinal barrier injury, including serum levels of diamine oxidase and lipopolysaccharide, and intestinal expressions of ZO-1 and occludin. Moreover, it significantly suppressed the activation of the intestinal TLR4/NF-κB signaling pathway. CONCLUSIONS: TRAF6 inhibitor alleviated pancreatic and intestinal injury in AEP and ANP. This effect may be mediated through inhibition of the TLR4/NF-κB signaling pathway, which in turn regulates the inflammatory response and intestinal barrier injury.

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.

Bioinformatics analysis identified MMP14 and COL12A1 as immune-related biomarkers associated with pancreatic adenocarcinoma prognosis.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is one of the most common malignant tumors with high mortality rates and a poor prognosis. There is an urgent need to determine the molecular mechanism of PAAD tumorigenesis and identify promising biomarkers for the diagnosis and targeted therapy of the disease. METHODS: Three GEO datasets (GSE62165, GSE15471 and GSE62452) were analyzed to obtain differentially expressed genes (DEGs). The PPI networks and hub genes were identified through the STRING database and MCODE plugin in Cytoscape software. GO and KEGG enrichment pathways were analyzed by the DAVID database. The GEPIA database was utilized to estimate the prognostic value of hub genes. Furthermore, the roles of MMP14 and COL12A1 in immune infiltration and tumor-immune interaction and their biological functions in PAAD were explored by TIMER, TISIDB, GeneMANIA, Metascape and GSEA. RESULTS: A total of 209 common DEGs in the three datasets were obtained. GO function analysis showed that the 209 DEGs were significantly enriched in calcium ion binding, serine-type endopeptidase activity, integrin binding, extracellular matrix structural constituent and collagen binding. KEGG pathway analysis showed that DEGs were mainly enriched in focal adhesion, protein digestion and absorption and ECM-receptor interaction. The 14 genes with the highest degree of connectivity were defined as the hub genes of PAAD development. GEPIA revealed that PAAD patients with upregulated MMP14 and COL12A1 expression had poor prognoses. In addition, TIMER analysis revealed that MMP14 and COL12A1 were closely associated with the infiltration levels of macrophages, neutrophils and dendritic cells in PAAD. TISIDB revealed that MMP14 was strongly positively correlated with CD276, TNFSF4, CD70 and TNFSF9, while COL12A1 was strongly positively correlated with TNFSF4, CD276, ENTPD1 and CD70. GSEA revealed that MMP14 and COL12A1 were significantly enriched in epithelial mesenchymal transition, extracellular matrix receptor interaction, apical junction, and focal adhesion in PAAD development. CONCLUSIONS: Our study revealed that overexpression of MMP14 and COL12A1 is significantly correlated with PAAD patient poor prognosis. MMP14 and COL12A1 participate in regulating tumor immune interactions and might become promising biomarkers for PAAD.

KRT7 Overexpression is Associated with Poor Prognosis and Immune Cell Infiltration in Patients with Pancreatic Adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is a deadly tumor with a high recurrence rate and poor prognosis. Keratin 7 (KRT7) is a member of the keratin gene family that is involved in the regulation of cell growth, migration and apoptosis in many cancers. However, the role of KRT7 and its biological functions in PAAD remain unclear. We systemically analyzed the expression and clinical values of KRT7 in PAAD. METHODS: The Gene Expression Profiling Interactive Analysis (GEPIA), Oncomine and Human Protein Atlas (HPA) databases were used to analyze the mRNA and protein expression of KRT7 in PAAD. The prognosis and subgroup analysis of KRT7 in PAAD patients was performed using the GEPIA, PROGgeneV2 and UALCAN databases. Later, the correlation between KRT7 expression and tumor immune molecules in PAAD was evaluated using the Immune Cell Abundance Identifier (ImmuCellAI) and TISIDB databases. Finally, the functional enrichment pathway of KRT7 and its coexpressed genes were analyzed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Metascape databases and Gene Set Enrichment Analysis (GSEA). RESULTS: The mRNA and protein expression of KRT7 was increased in PAAD tissues compared with normal tissues. High KRT7 expression was closely associated with tumor grade, TP53 mutations and poor prognosis in PAAD patients. Cox regression analysis proved that overexpressed KRT7 was an important and independent risk factor for poor overall survival (P = 0.006, HR =1.87) and disease-free survival (P = 0.019, HR =1.793) in PAAD. Additionally, KRT7 expression was significantly associated with immune infiltration of tumor immune cells and immunomodulators. Functional enrichment analyses and GSEA indicated that KRT7 might be involved in the regulation of the p53 pathway in PAAD. CONCLUSION: Overexpressed KRT7 could be a promising prognostic and therapeutic target biomarker for PAAD by bioinformatics analysis.