Inhibiting NLRP3/Caspase-1/GSDMD-Mediated Pyroptosis: A New Role of GADD45B's Role in Hypertriglyceridemia-Induced Acute Pancreatitis

Abstract

Background: Hypertriglyceridemia-induced acute pancreatitis (HTG-AP) is a severe form of pancreatitis, rapidly progressing to multiorgan failure. Growth arrest and DNA damage-inducible beta (GADD45B) play a crucial role in stress responses; however, its precise role in HTG-AP pathogenesis remains unknown. Therefore, this study aimed to elucidate the role of GADD45B in HTG-AP using both in vitro cellular and in vivo animal models.

Methods: AR42J cells were transfected with GADD45B si-RNA or overexpressed plasmids and induced with palmitic acid (PA) and caerulein (Cae) to establish an HTG-AP cellular model. The HTG-AP animal model was successfully developed by treating mice with P-407 and Cae, alongside adeno-associated virus (AAV)-shRNA interference. The transfection efficiency was assessed using quantitative PCR (qPCR) and Western blot analyses. Furthermore, cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay, and cell death rate, inflammation levels, and pyroptosis were examined using Hoechst 33342/propidium iodide (PI) staining, enzyme-linked immunosorbent assay (ELISA), and transmission electron microscope (TEM). Moreover, protein expression levels of the pyroptotic pathway, nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain containing 3/Cysteine-dependent aspartate-specific protease 1/Gasdermin D (NLRP3/Caspase-1/GSDMD), were evaluated using Western blot analysis.

Results: The Co-IP assay confirmed the interaction between GADD45B and NLR family pyrin domain containing 3 (NLRP3). In the AR42J cell model of HTG-AP, GADD45B interference promoted cell viability, attenuated cell death, pro-inflammation, pyroptotic cytokines interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-1β, IL-18, amylase, and intracellular vesicle counts (p < 0.05). Furthermore, AAV-shGADD45B treatment improved pancreatic injury, cell death, and pyroptosis in HTG-AP model mice (p < 0.05). Moreover, GADD45B knockdown suppressed the pyroptosis-related pathway NLRP3/Caspase-1/GSDMD protein levels (p < 0.05). However, GADD45B overexpression exhibited opposite effects, which was reserved by NLRP3 inhibitor MCC950 (p < 0.05).

Conclusions: This study revealed that GADD45B downregulation reduces pyroptosis by suppressing the NLRP3/Caspase-1/GSDMD axis in HTG-AP, underscoring GADD45B as a promising therapeutic target and enhancing its clinical application.

Keywords: GADD45B; HTG-AP; NLRP3/Caspase-1/GSDMD; inflammatory; pyroptosis.