Pekson Ryan, Poltoratsky Vladimir, Gorasiya Samir, Sundaram Sruthi, Ashby Charles R, Vancurova Ivana, Reznik Sandra E
Dept of Pharmaceutical Sciences, St. John's University.
Toxicon, Bedford, MA.
Mol Med. 2016 Dec;22:747-758. doi: 10.2119/molmed.2016.00017. Epub 2016 Oct 24.
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 and 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.
此前，我们已经证明，N，N-二甲基乙酰胺（DMA）可在小鼠模型中预防炎症诱导的早产，抑制脂多糖（LPS）诱导的胎盘促炎细胞因子增加，并上调抗炎细胞因子白细胞介素-10（IL-10）。然而，DMA的作用机制仍有待阐明。在当前研究中，我们探究了DMA如何产生其抗炎作用。使用RAW 264.7细胞和JEG-3细胞模型，我们发现DMA可抑制脂多糖（LPS）诱导的RAW 264.7细胞、TNFα刺激的JEG-3细胞以及LPS刺激的人胎盘外植体中促炎细胞因子的分泌。DMA显著减弱了LPS刺激的RAW 264.7细胞中TNFα、IL-6、IL-10和粒细胞巨噬细胞集落刺激因子（GM-CSF）的分泌，TNFα刺激的JEG-3细胞中IL-6的分泌，以及LPS刺激的人胎盘外植体中TNFα、IL-6、IL-10、GM-CSF和白细胞介素-8（IL-8）的分泌。我们进一步研究了DMA对细胞因子表达的影响是否涉及活化B细胞核因子κB（NF-κB）和丝裂原活化蛋白激酶（MAPK）途径。DMA（10 mM）显著抑制了LPS刺激的RAW 264.7细胞中B细胞κ轻链多肽基因增强子抑制因子α（IκBα）的降解，但MAPK途径中磷酸化或天然形式的下游蛋白表达没有显著变化。此外，DMA显著减弱了与NF-κB-Luc报告质粒共转染的细胞中的荧光素酶活性，但与AP-1-Luc或CEBP-Luc报告质粒共转染的细胞中则没有。总体而言，我们的研究结果表明，DMA的抗炎活性是通过抑制NF-κB途径介导的，其机制是IκBα降解减少。
