Background: This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms.Methods: 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.Results: 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.Conclusion: 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.

Background: The NLRP3 inflammasome serves as a crucial component in an array of inflammatory conditions by boosting the secretion of pro-inflammatory cytokines: IL-1β and IL-18. Hence, a thorough investigation of the underlying mechanism of NLRP3 activation could ascertain the requisite directionality to the ongoing studies, along with the identification of the novel drug targets for the management of inflammatory diseases. Previous studies have established the vital role of the Acid sphingomyelinase (ASM)/Ceramide (Cer) pathway in the functional outcome of cells, with a particular emphasis on the inflammatory processes. ASM mediates the ceramide production by sphingomyelin hydrolysis. Furthermore, the participation of the ASM/Cer in NLRP3 activation remains ambiguous. Methods: We employed lipopoysaccharide (LPS)/Adenosine Triphosphate (ATP)-induced activation of NLRP3 inflammasome in J774A.1 cells as an in vitro inflammatory model. Results: We observed that imipramine, a well-known inhibitor of ASM, significantly inhibited ASM activity & increased ceramide accumulation, which indicates ASM activation. Besides, it also suppressed the LPS/ATP-induced expression of proteins and mRNA: Thioredoxin interacting protein (TXNIP), NLRP3, Caspase-1, IL-1β and IL-18. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced TXNIP/NLRP3 inflammasome activation; however, it did not affect LPS/ATP-induced ASM activity and ceramide production. Further examination showed that the exogenous C2-ceramide-treated J774A.1 cells induce the overexpression of TXNIP, NLRP3, Caspase-1, IL-1β, and IL-18. Furthermore, verapamil inhibited C2-Ceramide mediated TXNIP overexpression and NLRP3 inflammasome activation. These findings infer that TXNIP overexpression leads to Cer mediated NLRP3 inflammasome activation. Conclusion: Our study validated the crucial role of the ASM/Cer/TXNIP signaling pathway in NLRP3 inflammasome activation.

Background: The NOD-Like Receptor Protein 3 (NLRP3) inflammasome is a crucial component of an array of inflammatory conditions. It functions by boosting the secretion of pro-inflammatory cytokines: interleukin-1β (IL-1β) and interleukin-18 (IL-18). Previous studies have established the vital role of the acid sphingomyelinase (ASM)/ceramide (Cer) pathway in the functional outcome of cells, with a particular emphasis on the inflammatory processes. This study aimed to explore the effects and associated underlying mechanism of Cer-induced NLRP3 inflammasome activation.Methods: Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells was used as an in vitro inflammatory model. 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 evaluated using ELISA kits. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content.Results: Imipramine, a well-known inhibitor of ASM, significantly inhibited ASM activity and inhibited Cer accumulation, which indicated ASM activation. Besides, it also suppressed the LPS/ATP-induced expression of proteins and mRNA: thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1β and IL-18. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced TXNIP/NLRP3 inflammasome activation; however, it did not affect LPS/ATP-induced ASM activation and ceramide production. Further analysis showed that the exogenous C2-Cer treated J774A.1 cells induced the overexpression of TXNIP, NLRP3, caspase-1, IL-1β and IL-18. Besides, TXNIP siRNA or verapamil inhibited C2-Cer-induced TXNIP overexpression and NLRP3 inflammasome activation.Conclusion: This study demonstrated the involvement of the ASM/Cer/TXNIP signaling pathway in NLRP3 inflammasome activation.