Abstract
Previously, we have shown that N,N-dimethylacetamide (DMA) prevents inflammation-induced preterm birth in a murine model, inhibits LPS-induced increases in placental pro-inflammatory cytokines and up-regulates the anti-inflammatory cytokine Interleukin-10 (IL-10). However, DMA's mechanism of action remains to be elucidated. In the current study we investigate how DMA produces its anti-inflammatory effect. Using in vitro and ex vivo models, we show that DMA suppresses secretion of pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 cells, TNFα-challenged JEG-3 cells and LPS-stimulated human placental explants. DMA significantly attenuated the secretion of TNFα, IL-6, IL-10, and granulocyte macrophage colony stimulating factor (GM-CSF) from LPS-stimulated RAW 264.7 cells, IL-6 secretion from TNFα-stimulated JEG-3 cells and TNFα, IL-6, IL-10, GM-CSF and Interleukin-8 (IL-8) from LPS-stimulated human placental explants. We further investigated if DMA's effect on cytokine expression involves the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. DMA (10 mM) significantly inhibited nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) degradation in LPS-stimulated RAW 264.7 cells, but there was no significant change in the expression of phosphorylated or native forms of downstream proteins in the MAPK pathway. In addition, DMA significantly attenuated luciferase activity in cells co-transfected with NF-κB-Luc reporter plasmid, but not with AP-1-Luc or CEBP-Luc reporters. Overall, our findings suggest that the anti-inflammatory activity of DMA is mediated by inhibition of the NF-κB pathway via decreased IκBα degradation.
Highlights
Inflammation has been implicated in a broad spectrum of disorders, ranging from cardiovascular to neurodegenerative disease [1,2]
Cytokines such as IL-1β, IL-6 and TNFα in turn increase the transcription of genes that mediate tissue remodeling, such as matrix metalloproteinases (MMPs) [22], and increase synthesis of prostaglandins and other inflammatory mediators [23,24,25,26,27]
At 20 mM, viability was significantly decreased to approximately 75%, 80% and 85% in RAW 264.7, JEG-3 and HEK 293/TLR4 cells, respectively
Summary
Inflammation has been implicated in a broad spectrum of disorders, ranging from cardiovascular to neurodegenerative disease [1,2]. Ascending infection and chorioamnionitis trigger the innate immune system, resulting in an inflammatory response that initiates the process of parturition [3,4,5,6,7] This paradigm has been the basis of recently developed in vivo models of inflammation-driven preterm birth, where preterm labor is induced by injecting lipopolysac-. Nuclear translocation of NF-κB triggers the production of proinflammatory cytokines such as tumor necrosis factor (TNF)α and interleukin (IL)-1, which regulate the innate immune response and inflammation [13,14,15,18,19,20,21] Cytokines such as IL-1β, IL-6 and TNFα in turn increase the transcription of genes that mediate tissue remodeling, such as matrix metalloproteinases (MMPs) [22], and increase synthesis of prostaglandins and other inflammatory mediators [23,24,25,26,27]. NF-κB plays a central role in inflammation-associated PTB and represents one potential target for pharmacotherapeutic approaches aimed at controlling preterm labor and delivery [32,33,34]
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