This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms. Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1β and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content. Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1β, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression. This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

中文翻译：

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ASM/Cer/TXNIP 信号模块在 NLRP3 炎症小体激活中的作用

本研究旨在探讨神经酰胺（Cer）对 NLRP3 炎症小体激活的影响及其潜在机制。J774A.1 细胞和 THP-1 巨噬细胞中脂多糖 (LPS)/三磷酸腺苷 (ATP) 诱导的 NLRP3 炎症小体激活被用作体外炎症模型。分别使用蛋白质印迹和实时 PCR (RT-PCR) 检测蛋白质和 mRNA 水平。通过ELISA测量IL-1β和IL-18水平。ASM检测试剂盒和免疫荧光用于检测ASM活性和Cer含量。丙咪嗪是一种众所周知的 ASM 抑制剂，可显着抑制 LPS/ATP 诱导的 ASM 活性和随之而来的 Cer 积累。此外，丙咪嗪在蛋白质和 mRNA 水平上抑制了 LPS/ATP 诱导的硫氧还蛋白相互作用蛋白 (TXNIP)、NLRP3、caspase-1、IL-1β 和 IL-18 的表达。有趣的是，维拉帕米（一种 TXNIP 抑制剂）抑制了 LPS/ATP 诱导的 TXNIP/NLRP3 炎症小体激活，但不影响 LPS/ATP 诱导的 ASM 激活和 Cer 形成。TXNIP siRNA 和维拉帕米抑制 C2-Cer 诱导的 TXNIP 上调和 NLRP3 炎症小体的激活。此外，用不可逆的清道夫受体 CD36 抑制剂磺基-N-琥珀酰亚胺油酸酯 (SSO) 预处理细胞，可阻断 Cer 诱导的核因子-κB (NF-κB) 活性、TXNIP 表达和 NLRP3 炎症小体的上调激活。SN50 对 NF-κB 活化的抑制阻止了 Cer 诱导的 TXNIP 上调和 NLRP3 炎性体的激活，但不影响 CD36 表达。该研究表明 ASM/Cer/TXNIP 信号通路参与了 NLRP3 炎症小体的激活。