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The NF-κB Inhibitor Pyrrolidinedithiocarbamate Ammonium (PDTC) Exerts an Anti-Pyroptotic Effect in Monocytes
Xianxian Mao1,2, Yihan Wang3, Hongru Lin3,4
1Department of Obstetrics and Gynecology, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, 330006, PR China.
Objectives:
Pyroptosis is a type of inflammatory programmed cell death that is triggered by inflammasomes. Pyroptosis exacerbates inflammatory responses and promotes the progression of various inflammation-related diseases, yet effective intervention strategies remain inadequately defined. This study aimed to screen small-molecule inhibitors for their anti-pyroptotic activity in monocytes and clarify the mechanism of the most potent candidate.
Materials And Methods:
Human monocytic leukemia THP-1 cells were used as the in vitro model via stimulation with 1 μg/mL lipopolysaccharide (LPS) for 3 hours, followed by 8 μg/mL nigericin for 1 hour. Fourteen small molecules were initially screened. Cell viability was assessed via a CCK-8 assay, and pyroptosis-related cytotoxicity was evaluated by measuring lactate dehydrogenase (LDH) release. Calcein AM/PI staining was performed to visualize live/dead cells and observe pyroptotic morphological changes. Western blotting was used to detect the expression and activation of key proteins in the canonical pyroptosis pathway and the Nuclear Factor-κB (NF-κB) signaling pathway. RNA isolation and quantitative PCR (qPCR) were conducted to analyze NOD-like Receptor Family Pyrin Domain Containing 3 (NLRP3) mRNA expression. Additionally, proteasomal inhibitors (MG132) and autophagy/lysosomal inhibitors were used to explore the mechanism of NLRP3 protein regulation.
Results:
The NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) exerted a remarkable anti-pyroptotic effect (p < 0.05), as evidenced by reduced LDH release and improved cell viability following LPS + nigericin challenge. PDTC suppressed activation of the canonical pyroptosis pathway by downregulating NLRP3, consequently reducing downstream caspase-1 and Gasdermin D (GSDMD) cleavage, as well as interleukin-1β (IL-1β) secretion (p < 0.01). Mechanistically, PDTC suppressed activation of the NF-κB signaling pathway and dual-regulated NLRP3 expression by inducing proteasomal degradation and suppressing transcription (p < 0.05).
Discussion:
Pyroptosis drives uncontrolled inflammation, and pharmacological inhibition of it is a potential strategy for inflammatory diseases. Non-NF-κB inhibitors failed to protect THP-1 cells from pyroptosis. PDTC's dual regulation of NLRP3 goes beyond NF-κB suppression, with its optimal NF-κB inhibitory and anti-pyroptotic concentrations not fully overlapping.
Conclusion:
These findings suggest that PDTC is a potent inhibitor of pyroptosis in monocytes, exerting effects by suppressing the NF-κB pathway and regulating NLRP3 at transcriptional and post-transcriptional levels, representing therapeutic potential for inflammatory disorders.
Insights
Pyrrolidinedithiocarbamate ammonium (PDTC) effectively inhibits pyroptosis, a form of inflammatory cell death, in monocytes. PDTC works by suppressing the NF-κB pathway and regulating NLRP3 expression, offering potential for treating inflammatory diseases.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Pyroptosis is inflammatory programmed cell death triggered by inflammasomes.
- It exacerbates inflammation and disease progression, but effective interventions are lacking.
Purpose of the Study:
- Screen small-molecule inhibitors for anti-pyroptotic activity in monocytes.
- Elucidate the mechanism of the most potent inhibitor.
Main Methods:
- Used human monocytic leukemia THP-1 cells stimulated with LPS and nigericin.
- Screened 14 small molecules, assessing cell viability (CCK-8) and cytotoxicity (LDH release).
- Analyzed pyroptosis pathway proteins (Western blotting) and NLRP3 mRNA (qPCR), exploring regulatory mechanisms with proteasomal and lysosomal inhibitors.
Main Results:
- Pyrrolidinedithiocarbamate ammonium (PDTC), an NF-κB inhibitor, showed significant anti-pyroptotic effects.
- PDTC reduced LDH release, improved cell viability, and suppressed canonical pyroptosis pathway activation by downregulating NLRP3, caspase-1, GSDMD cleavage, and IL-1β secretion.
- PDTC inhibited NF-κB signaling and regulated NLRP3 via proteasomal degradation and transcriptional suppression.
Conclusions:
- PDTC is a potent monocyte pyroptosis inhibitor, acting via NF-κB suppression and dual NLRP3 regulation.
- Its mechanism extends beyond NF-κB inhibition, with distinct optimal concentrations for NF-κB inhibition and anti-pyroptosis.
- PDTC demonstrates therapeutic potential for inflammatory disorders.
