N-Acetylcysteine Inhibits NLRP3 Inflammasome Activation by Suppressing the Noncanonical Pyroptosis Pathway, Thereby Promoting Uterine Quiescence

Abstract

Background: The NOD-like receptor protein 3 (NLRP3) inflammasome plays a crucial role in the immune microenvironment. Therefore, we investigated the involvement of NLRP3 in the inflammatory response associated with term and preterm birth, as well as the potential of N-acetylcysteine (NAC) to mitigate this inflammatory response. Methods: First, we induced infectious preterm birth in mice using Lipopolysaccharide (LPS) and treated them with NAC. Next, LPS was used to trigger inflammation in uterine smooth muscle cells (USMC), which were then treated with NAC. The levels of cleaved caspase-4/5/11, NLRP3, and Apoptosis-associated speck-like protein containing CARD (ASC) proteins in both the myometrium and USMC were determined by western blot. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of caspase-1, interleukin-1β (IL-1β), and IL-18 in the myometrium and USMC. Subsequently, siRNA targeting NLRP3 was transfected into LPS-treated USMC, and the expression levels of NLRP3, oxytocin receptor (OTR), ASC, connexin 43 (CX43), and inflammatory cytokines were evaluated using western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: The NLRP3 inflammasome, cleaved caspase-4/5/11, and ASC were found to be upregulated in USMC and myometrium tissues treated with LPS (p < 0.01). Administration of NAC not only prolonged the duration of pregnancy but also suppressed the activation of NLRP3 and the noncanonical pyroptosis pathway (p < 0.01 and p < 0.001). Deletion of NLRP3 under LPS conditions led to a decrease in the expressions of OTR and CX43 (p < 0.05), along with a reduction in the levels of associated inflammatory factors (p < 0.05). Conclusions: NAC inhibits immune inflammation and promotes uterine quiescence by suppressing the noncanonical pyroptosis pathway and NLRP3 inflammasome activation.