USP33 promotes caerulein-induced apoptotic, oxidative and inflammatory injuries in acute pancreatitis by deubiquitinating TRAF3.

Abstract: Background: Tumor necrosis factor receptor associated factor 3 (TRAF3) and deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) have been identified to play important roles in inflammatory diseases, including acute pancreatitis (AP). Here, we aimed to explore whether USP33 affected AP progression by affecting TRAF3 expression through deubiquitination.Methods: Caerulein-treated HPDE6-C7 cells were used to mimic AP conditions in vitro. Levels of mRNAs and proteins were examined by qRT-PCR and Western blot. Cell proliferation and apoptosis were evaluated using CCK-8 assay, EdU assay and flow cytometry. Cell oxidative stress was assessed by detecting the production of superoxide dismutase and malonaldehyde. ELISA analysis detected IL-6 and TNF-α levels. Macrophage M1 polarization was evaluated by flow cytometry. Cellular ubiquitination analyzed the ubiquitination effect on TRAF3. Protein interaction between USP33 and TRAF3 was identified by immunofluorescence staining.Results: Caerulein dose-dependently induced apoptosis, oxidative stress, and inflammatory response in HPDE6-C7 cells and promoted macrophage M1 polarization to enhance inflammation (P < 0.05). TRAF3 was highly expressed in AP patients (3.5±1.10 vs. 1.0 ±0.74, P < 0.05) and caerulein-induced HPDE6-C7 cells (3.3 ±0.34 vs. 1.0 ±0.10, P < 0.05). Knockdown of TRAF3 protected HPDE6-C7 cells from caerulein-induced apoptotic, oxidative and inflammatory injuries. Mechanistically, USP33 interacted with TRAF3 and induced TRAF3 deubiquitination to up-regulate its expression (P < 0.05). Further analyses showed that USP33 knockdown reversed caerulein-induced apoptosis, oxidative stress and inflammation in HPDE6-C7 cells by TRAF3 (P < 0.05). Moreover, USP33-TRAF3 activated the NF-κB pathway (P < 0.05).Conclusion: USP33 promoted caerulein-induced apoptosis, oxidative stress and inflammation in pancreatic ductal cells by deubiquitinating TRAF3, indicating a novel insight into the pathogenesis of AP.