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Objective: Activation of the transcription factor κB (NF-κB) and secretion of pro-inflammatory mediators are major events in acute pancreatitis (AP). Recently, O-linked-N-acetylglucosamine (O-GlcNAc) modification, one type of posttranslational modifications, reportedly attunes NF-κB function. However, the expression of O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation of proteins, in AP, and the possible contribution of OGT-mediated O-GlcNAcylation to the NF-κB inflammatory activation in pancreatic acinar cells and to the AP progression have not been understood. This study focused on the effects and mechanisms of OGT-mediated O-GlcNAcylation during AP., Methods: An AP cell model was established with the caerulein-stimulated AR42 J rat pancreatic acinar cells. The secretion of pro-inflammatory cytokines TNF-α was detected by ELISA kits, and the production of NO was determined using the colorimetric Griess reaction. Expression of OGT was measured by RT-PCR and Western blot. Expression levels of RL2, phosphorylation of p65, total p65, IKKα were detected by Western blot. The NF-κB activity was evaluated by luciferase reporter gene assay. To determine the biological functions of OGT in caerulein-induced inflammatory response, RNA interference and PUGNAc, the inhibitor of O-GlcNAcase (OGA) was employed to regulate OGT expression in AR42 J cells., Results: Caerulein significantly up-regulated the expression of OGT, and increased the global protein O-GlcNAcylation level in AR42 J cells. Reduction of OGT by small interfering RNA (siRNA) inhibited caerulein-triggered inflammation, assessed by the production of pro-inflammatory mediators (TNF-α and NO). We also demonstrated that O-GlcNAcylation directly modified the NF-κB p65 subunit and its upstream activating kinases IKKα in AR42 J cells. Lowering O-GlcNAcylation by OGT knockdown attenuated p65 activating phosphorylation, nuclear translocation, NF-κB transcriptional activity and levels of NF-κB transcriptional targets TNF-α and NO; on the contrary, elevating O-GlcNAc through PUGNAc increased IKKα and p65 O-GlcNAcylation accompanied by increased p65 phosphorylation, activity and levels of TNF-α and NO in caerulein-treated cells., Conclusions: Our results demonstrate for the first time that OGT-mediated O-GlcNAcylation promotes NF-κB signaling activation and inflammation in pancreatic acinar cells, which might promote the progression of AP.