Abstract
BackgroundThis study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms.MethodsLipopolysaccharide (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.ResultsImipramine, 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.ConclusionThis 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.
Highlights
Inflammation is a local protective mechanism evoked by the exposure of cells to harmful stimuli, such as irritants, pathogens, or damaged cells
C2-Cer, and verapamil on cell viability The effects of imipramine (0, 25, 50, 75, 100 μmol/L), C2-Cer (0, 15, 30, 45, 60 μmol/L), and verapamil (0, 25, 50, 75, 100 μmol/L) on the viability of J774A.1 cells and THP-1 macrophages were measured by the MTT method
Imipramine pretreatment inhibits LPS/adenosine 5′-triphosphate (ATP)-induced Acid sphingomyelinase (ASM) activity and Cer production To unravel the role of the ASM/Cer pathway in thioredoxin interacting protein (TXNIP)/ NOD-Like Receptor Protein 3 (NLRP3) inflammasome activation, the effect of imipramine, an ASM activity inhibitor, on LPS/ATPinduced ASM activation and Cer generation was examined in J774A.1 cells and THP-1 macrophages
Summary
Inflammation is a local protective mechanism evoked by the exposure of cells to harmful stimuli, such as irritants, pathogens, or damaged cells. Excessive or chronic inflammation gives rise to multiple pathological complications, such as tissue damage and dysfunction, diabetes, sepsis, arteriosclerosis, Alzheimer’s disease, liver disorders, and various malignancies [1]. Prescribed anti-inflammatory drugs fall under two categories: non-steroidal and steroidal. These drugs produce a range of side-effects, which restricts their clinical utility [2, 3]. Chronic activation of the NLRP3 inflammasome culminates in the accumulation of pro-inflammatory cytokines, i.e., interleukin-1β (IL-1β) and interleukin-18 (IL-18), contributing to excessive inflammation and inflammatory disorders [4]. Unraveling the precise regulatory mechanism leading to the activation of NLRP3 inflammasome could help determine molecular drug targets to treat inflammatory diseases. This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
