Abstract
Scope:
We and others recently showed that soyasaponin Bb (SSBb ) inhibited lipopolysaccharide (LPS)-induced inflammation in macrophages. Since the recruitment of toll-like receptor 4 (TLR4) into lipid rafts is vital for LPS-initiated signaling, we investigated whether this process would be modulated by SSBb .
Methods and results:
By using sucrose gradient ultracentrifuge, we found that pretreatment of macrophages with SSBb inhibited LPS-induced recruitments of TLR4, myeloid differentiation primary response protein 88 (MyD88) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF) into fractions enriched with lipid rafts marker flotillin-1. We also found SSBb decreased co-localization of TLR4 and lipid rafts by utilizing confocal immunofluorescence microscopy. Additionally, we observed that SSBb suppressed LPS-induced formation of TLR4/MyD88 and TLR4/TRIF complexes, production of pro-inflammatory molecules, and activation of nuclear factor kappa B (NF-κB). Furthermore, we found that these inhibitory effects of SSBb were associated with reduced reactive oxygen species (ROS) because pretreating cells with N-acetyl-L-cysteine and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited LPS-induced TLR4 recruitment into lipid rafts and NF-κB activation. SSBb also inhibited NADPH oxidase activation by blocking interaction between gp91(phox) and p47(phox) similarly as DPI.
Conclusion:
SSBb can inhibit TLR4 recruitment into lipid rafts and its signaling by suppressing the NADPH oxidase-dependent ROS generation.
Keywords:
Lipid raft/ Nicotinamide adenine dinucleotide phosphate/ Reactive oxygen species/ Soyasaponin/ Toll-like receptor 4.
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATPases Associated with Diverse Cellular Activities / antagonists & inhibitors
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ATPases Associated with Diverse Cellular Activities / genetics
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ATPases Associated with Diverse Cellular Activities / metabolism
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Acetylcysteine / antagonists & inhibitors
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Acetylcysteine / metabolism
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Adaptor Proteins, Vesicular Transport / genetics
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Adaptor Proteins, Vesicular Transport / metabolism
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Animals
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DNA Helicases / antagonists & inhibitors
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DNA Helicases / genetics
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DNA Helicases / metabolism
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Inflammation / chemically induced
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Inflammation / drug therapy
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Lipopolysaccharides / toxicity
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Macrophages / drug effects
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Macrophages / metabolism
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Membrane Microdomains / drug effects*
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Mice
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Myeloid Differentiation Factor 88 / genetics
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Myeloid Differentiation Factor 88 / metabolism
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NADPH Oxidases / antagonists & inhibitors*
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NADPH Oxidases / genetics
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NADPH Oxidases / metabolism
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NF-kappa B / antagonists & inhibitors
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NF-kappa B / genetics
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NF-kappa B / metabolism
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Onium Compounds / pharmacology
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RAW 264.7 Cells
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Reactive Oxygen Species / metabolism*
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Receptors, Immunologic / antagonists & inhibitors
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Receptors, Immunologic / genetics
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Receptors, Immunologic / metabolism
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Saponins / pharmacology*
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Signal Transduction
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Toll-Like Receptor 4 / antagonists & inhibitors
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Toll-Like Receptor 4 / genetics
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Toll-Like Receptor 4 / metabolism*
Substances
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Adaptor Proteins, Vesicular Transport
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Lipopolysaccharides
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Myd88 protein, mouse
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Myeloid Differentiation Factor 88
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NF-kappa B
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Onium Compounds
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Pirb protein, mouse
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Reactive Oxygen Species
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Receptors, Immunologic
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Saponins
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TICAM-1 protein, mouse
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Tlr4 protein, mouse
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Toll-Like Receptor 4
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soyasaponin Bb
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diphenyleneiodonium
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NADPH Oxidases
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ATPases Associated with Diverse Cellular Activities
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DNA Helicases
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RUVBL2 protein, mouse
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Acetylcysteine