Inhibitory effects of chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ and their synthetic derivatives on LPS-induced NO production in microglia
Introduction
Microglia, resident immune cells in the central nervous system (CNS), are involved in the progression of neurodegeneration in many CNS disorders, such as ischemic stroke and Alzheimer’s disease.1, 2 It has been reported that cerebral ischemia stimulates activation of microglia in the injured area and elicits a robust neuroinflammatory response.1 Activated microglia produce inflammatory mediators including nitric oxide (NO). NO, which is a multifunctional effector molecule, is produced by inducible NO synthase (iNOS) in activated microglia. Increasing evidence suggests that the excess NO released from activated microglia results in neuronal cell death.3, 4
Toll-like receptor 4 (TLR4) plays a key role in innate immunity. TLR4-mediated signaling stimulates expression of various inflammatory response genes including iNOS and proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β).5 Recent studies suggest that TLR4 signaling exacerbates ischemic outcomes.5, 6 For example, it was reported that a loss of function of TLR4 in TLR4 mutant mice protects against focal cerebral ischemia-induced neurodegeneration.7 Therefore, activation of TLR4 signaling caused by ischemic stroke is thought to be responsible for neuronal cell death associated with brain ischemia. Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, is a ligand of TLR4 and triggers expression of iNOS and proinflammatory cytokines through activation of TLR4. Nuclear factor-κB (NF-κB) and signal transduction and activator of transcription 1 (STAT1) have been shown to be key transcription factors responsible for LPS-induced iNOS expression.8
NF-κB is sequestered in the cytoplasm by the inhibitory protein, inhibitor-κB (I-κB), under normal conditions. LPS promotes the degradation of I-κB, and in turn stimulates the translocation of NF-κB to the nucleus.9 The binding site of NF-κB is reported to be located in the regulatory region of iNOS gene.10 On the other hand, activation of STAT1 is mainly regulated via an autocrine loop involving interferon-β (IFN-β) produced by LPS-activated macrophages.11, 12 Secreted IFN-β stimulates phosphorylation of STAT1, and in turn promotes formation of STAT1 dimerization and its translocation to the nucleus. Since LPS-induced iNOS expression has been shown to be suppressed in macrophages prepared from STAT1−/− mice,13 SATA1 is known to be indispensable for this phenomenon.
Chalcone, a subgroup of flavonoids, has been demonstrated to possess anti-inflammatory,14 anti-allergic15 and anti-oxidant16 activities. Recently, we reported that chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ have inhibitory effects on antigen-induced degranulation in basophilic leukemia RBL-2H3 cells.17, 18 In this study, we found that ‘hidabeni’ chalcones and their synthetic derivatives suppress LPS-induced iNOS expression and NO production in rat immortalized microglia, HAPI cells. Furthermore, we also addressed the mechanism underlying the inhibitory effects of these compounds on LPS-induced iNOS expression.
Section snippets
Effects of compounds on LPS-induced iNOS expression
To confirm whether LPS induces iNOS expression under our conditions, HAPI cells were treated with 100 ng/mL LPS. As shown in Figure 1A, the induction of iNOS mRNA reached a peak at 6 h. LPS induced iNOS mRNA in a dose-dependent manner (Fig. 1B). Next, to test the effects of six compounds isolated from Brassica rapa L. ‘hidabeni’ (A1–A6, Fig. 2) on LPS-induced iNOS expression, we performed RT-PCR. HAPI cells were pretreated with each compound (50 μM) for 30 min, followed by the treatment with LPS
Discussion
NO is thought to be an important mediator of progression of neurodegeneration in many CNS diseases. Excessive NO release under pathological conditions results in neuronal cell death. We have demonstrated here that A2 from the aerial parts of Brassica rapa L. ‘hidabeni’ prevents LPS-induced iNOS expression and NO production in HAPI cells. In addition, we addressed the mechanism by which this compound suppresses LPS-induced iNOS expression, and found that it inhibits the activation of STAT1
Materials
LPS was purchased from Sigma (St. Louis, MO). Anti-STAT1, anti-phospho JNK (Thr183/Thr185), anti-JNK, anti-phospho ERK, anti-ERK, anti-phospho p38 and anti-p38 antibodies were purchased from Cell Signaling Technology (Denvers, MA). Anti-phospho-STAT1 (Thr701) antibody was purchased from Signalway Antibody (Pearland, TX). Anti-NF-κB p65 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin antibody was purchased from Millipore (Billerica, MA). l-NAME was purchased
Acknowledgment
This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (20590543 to H.H.).
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