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-ß (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.
Subject(s)
Membrane Microdomains/drug effects , NADPH Oxidases/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Saponins/pharmacology , Toll-Like Receptor 4/metabolism , ATPases Associated with Diverse Cellular Activities/antagonists & inhibitors , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Acetylcysteine/antagonists & inhibitors , Acetylcysteine/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , DNA Helicases/antagonists & inhibitors , DNA Helicases/genetics , DNA Helicases/metabolism , Inflammation/chemically induced , Inflammation/drug therapy , Lipopolysaccharides/toxicity , Macrophages/drug effects , Macrophages/metabolism , Mice , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Onium Compounds/pharmacology , RAW 264.7 Cells , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Signal Transduction , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/geneticsABSTRACT
It appears to be more practical and effective to prevent carcinogenesis by targeting the tumor promotion stage. Gap junctional intercellular communication (GJIC) is strongly involved in carcinogenesis, especially the tumor promotion stage. Considerable interest has been focused on the chemoprevention activities of soyasaponin (SS), which are major phytochemicals found in soybeans and soy products. However, less is known about the preventive effects of SS (especially SS with different chemical structures) against tumor promoter-induced inhibition of GJIC. We investigated the protective effects of SS-A1, SS-A2, and SS-I against hydrogen peroxide (H2O2)-induced GJIC inhibition and reactive oxygen species (ROS) production in Buffalo rat liver (BRL) cells. The present results clearly show for the first time that SS-A1, SS-A2, and SS-I prevent the H2O2-induced GJIC inhibition by scavenging ROS in BRL cells in a dose-dependent manner at the concentration range of from 25 to 100 µg/mL. Soyasaponins attenuated the H2O2-induced ROS through potentiating the activities of superoxide dismutase and glutathione peroxidase. This may be an important mechanism by which SS protects against tumor promotion. In addition, various chemical structures of SS appear to exhibit different protective abilities against GJIC inhibition. This may partly attribute to their differences in ROS-scavenging activities.
Subject(s)
Cell Communication/drug effects , Free Radical Scavengers/pharmacology , Gap Junctions/drug effects , Hepatocytes/drug effects , Reactive Oxygen Species/metabolism , Saponins/pharmacology , Animals , Catalase/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Comet Assay , DNA Damage/drug effects , Gap Junctions/metabolism , Glutathione Peroxidase/metabolism , Hepatocytes/metabolism , Hydrogen Peroxide/adverse effects , Lipid Peroxidation/drug effects , Liver/cytology , Liver/drug effects , Malondialdehyde/metabolism , Rats , Glycine max/chemistry , Superoxide Dismutase/metabolismABSTRACT
We and others have recently shown that soyasaponins abundant in soybeans can decrease inflammation by suppressing the nuclear factor kappa B (NF-kB)-mediated inflammation. However, the exact molecular mechanisms by which soyasaponins inhibit the NF-kB pathway have not been established. In this study in macrophages, soyasaponins (A1, A2 and I) inhibited the lipopolysaccharide (LPS)-induced release of inflammatory marker prostaglandin E2 (PGE2) to a similar extent as the NF-kB inhibitor (BAY117082). Soyasaponins (A1, A2 and I) also suppressed the LPS-induced expression of cyclooxygenase 2 (COX-2), another inflammatory marker, in a dose-dependent manner by inhibiting NF-kB activation. In defining the associated mechanisms, we found that soyasaponins (A1, A2 and I) blunted the LPS-induced IKKα/ß phosphorylation, IkB phosphorylation and degradation, and NF-kB p65 phosphorylation and nuclear translocation. In studying the upstream targets of soyasaponins on the NF-kB pathway, we found that soyasaponins (A1, A2 and I) suppressed the LPS-induced activation of PI3K/Akt similarly as the PI3K inhibitor LY294002, which alone blocked the LPS-induced activation of NF-kB. Additionally, soyasaponins (A1, A2 and I) reduced the LPS-induced production of reactive oxygen species (ROS) to the same extent as the anti-oxidant N-acetyl-L-cysteine, which alone inhibited the LPS-induced phosphorylation of Akt, IKKα/ß, IkBα, and p65, transactivity of NF-kB, PGE2 production, and malondialdehyde production. Finally, our results show that soyasaponins (A1, A2 and I) elevated SOD activity and the GSH/GSSG ratio. Together, these results show that soyasaponins (A1, A2 and I) can blunt inflammation by inhibiting the ROS-mediated activation of the PI3K/Akt/NF-kB pathway.
