Quercetin prevents LPS-induced high-mobility group box 1 release and proinflammatory function

Abstract

The pathogenesis of sepsis is mediated in part by the pathogen-associated molecular pattern molecule bacterial endotoxin, which stimulates macrophages to sequentially release early (e.g., TNF-alpha, IL-1beta) and late (e.g., high-mobility group box [HMGB] 1 protein) proinflammatory mediators. The recent discovery of HMGB1 as a late mediator of lethal sepsis has prompted investigation into development of several new experimental therapeutics that limit release, either blocking HMGB1 itself or its nominal receptors. Quercetin was recently identified as an experimental therapeutic that significantly protects against oxidative injury. Here, we report that quercetin attenuates lethal systemic inflammation caused by endotoxemia, even if treatment is started after the early TNF response. Quercetin treatment reduced circulating levels of HMGB1 in animals with established endotoxemia. In macrophage cultures, quercetin inhibited release as well as the cytokine activities of HMGB1, including limiting the activation of mitogen-activated protein kinase and NF-kappaB, two signaling pathways that are critical for HMGB1-induced subsequent cytokine release. Quercetin and autophagic inhibitor, wortmannin, inhibited LPS-induced type-II microtubule-associated protein 1A/1B-light chain 3 production and aggregation, as well as HMGB1 translocation and release, suggesting a potential association between autophagy and HMGB1 release. Quercetin delivery, a strategy to pharmacologically inhibit HMGB1 release that is effective at clinically achievable concentrations, now warrants further evaluation in sepsis and other systemic inflammatory disorders.

Figure 1.

Quercetin pretreatment prevents endotoxin lethality, attenuating TNF-α and high-mobility group box (HMGB) 1 release in vivo. (A) Mice (n = 20 per group) were injected with a single dose of quercetin (Q) as indicated, followed 30 minutes later by a lethal infusion of endotoxin (LPS, 10 mg/kg, intraperitoneally). Quercetin conferred significant protection against lethality (^#different from vehicle control group, P < 0.05). The Kaplan-Meyer method was used to compare the differences in mortality rates between groups. (B) In a parallel group of quercetin-treated animals, circulating TNF levels were measured by ELISA of sera prepared at 1 hour after LPS injection. Quercetin pretreatment (50 and 100 mg/kg, intraperitoneally) significantly attenuated the release of serum TNF in response to LPS (*P < 0.05). (C) Circulating levels of HMGB1 20 hours after LPS infusion (LPS, 10 mg/kg, intraperitoneally) were measured in a parallel experimental group of endotoxemic animals by Western blot analysis. Pretreatment with quercetin (100 mg/kg, intraperitoneally) attenuated serum HMGB1 levels at 20 hours after LPS.

Figure 2.

Effects of delayed administration of quercetin on the lethality of endotoxemia and serum HMGB1 level. (A) Mice (n = 20 per group) received a lethal infusion of endotoxin (LPS, 10 mg/kg, intraperitoneally) and were treated with quercetin (as indicated) 4, 8, 12, 24, and 36 hours later. Quercetin conferred significant protection against lethality (^#different from vehicle control group, P < 0.05). The Kaplan-Meyer method was used to compare the differences in mortality rates between groups. (B) In a parallel set of endotoxemic mice, HMGB1 release was analyzed by Western blot of serum collected at 20 hours. Slight delayed treatment with quercetin (100 mg/kg, intraperitoneally) inhibited the release of serum HMGB1. (C) Mice (n = 20 per group) received a lethal infusion of endotoxin (LPS, 10 mg/kg, intraperitoneally) and were treated with quercetin (as indicated) 12, 24, and 36 hours later. Quercetin conferred no protection against lethality (P > 0.05). The Kaplan-Meyer method was used to compare the differences in mortality rates between groups. (D) In a parallel set of endotoxemic mice, HMGB1 release was analyzed by Western blot of serum collected at 20 hours.

Figure 3.

Effects of quercetin on LPS- and TNF-α-induced HMGB1 expression, release and translocation in macrophage. (A–D) RAW 264.7 macrophages were pretreated for 1 hour with quercetin at the indicated dose before stimulation with LPS (A and C) or TNF-α (B and D) at the indicated doses for 24 hours, cell viability was determined by MTT assay, and expressed as the mean (±SEM) of four experiments in duplicate. In parallel experiments, HMGB1 levels in the whole-cell lysate (A and B) or culture medium (C and D) were determined by the relative optical intensity (in arbitrary units [AU]) of the immunoreactive bands on Western blots, and expressed as the mean (±SEM) of three experiments in duplicate. *P < 0.05. (E) RAW 264.7 macrophages were pretreated for 1 hour with quercetin (100 μM) before stimulation with LPS (500 ng/ml) or TNF-α (5 ng/ml) for 12 hours, and monitored for HMGB1 cytoplasmic translocation by immunocytochemistry (E). Red, HMGB1; blue, nuclei; original magnification, ×400. (F) RAW 264.7 cells were stimulated with LPS, and quercetin (100 μM) was added at 0.5, 4, and 8 hours after LPS stimulation. Levels of HMGB1 in the culture medium were determined at 20 hours after LPS stimulation, and expressed (AU) as mean (±SEM) of three experiments in duplicate.

Figure 4.

Effects of quercetin on LPS- and HMGB1-induced TNF-α and IL-1β release and expression in macrophages. (A) RAW 264.7 macrophages were pretreated for 1 hour with quercetin at the indicated dose before stimulation with LPS (500 ng/ml) for 1 hour or HMGB1 (1 μg/ml) for 4 hours, and TNF-α and IL-1β levels in the culture medium were detected by ELISA analysis. Values are means (±SEM) of three experiments in duplicate. *P < 0.05. (B) In parallel experiments, the mRNA expression level of TNF-α and IL-1β were detected by RT-PCR analysis. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as a loading control. Values are means (±SEM) of three experiments in duplicate. *P < 0.05.

Figure 5.

Effects of quercetin on LPS- and HMGB1-induced MAPKs phosphoration in macrophage cultures. RAW 264.7 macrophages were pretreated with quercetin (100 μM) for 1 hour before stimulation with LPS (500 ng/ml) or HMGB1 (1 μg/ml) for 30–60 minutes, and phosphorylated p38 (P-p38), c-Jun NH₂-terminal kinase (JNK) 1/2 (P-JNK1/2), or extracellular signal-regulated kinase (ERK) 1/2 (P-ERK1/2) protein levels in the whole-cell lysate were detected by Western blotting analysis. GAPDH was used as a loading control. Values are means (±SEM) of three experiments in duplicate. *P < 0.05 versus LPS group or HMGB1 group.

Figure 6.

Quercetin inhibits LPS- and HMGB1-induced inhibitor of NF-κB (IκBα) degradation, NF-κB p65 nuclear translocation, and NF-κB DNA binding activation in macrophage cultures. (A and B) Cell pretreatment with quercetin (100 μM) for 1 hour before stimulation with LPS (500 ng/ml) or HMGB1 (1 μg/ml) for indicated times, and IκBα level (A) in cytosolic fractions, and p65 level (B) in cytosolic (C) or nuclear (N) fractions were detected by Western blotting analysis. GAPDH, actin, or PCNA was used as a loading control. (C) Activities of transcriptional factor NF-κB in cells pretreated with quercetin (100 μM) for 1 hour in the absence or presence of LPS (500 ng/ml) or HMGB1 (1 μg/ml). At 1 hour after LPS or HMGB1 stimulation, NF-κB activities were determined by electrophoretic mobility shift assay with biotin-labeled NF-κB probe, and unlabeled (Cold) NF-κB probe. Specificity was determined by addition of p65 antibody to the nuclear extracts. All blots shown are representative of three experiments with similar results.

Figure 7.

Quercetin and wortmannin inhibited microtubule-associated protein 1A/1B–light chain 3 (LC3) expression and aggregation as well as HMGB1 translocation and release after treatment with LPS in macrophage cultures. RAW 264.7 macrophages were pretreated with quercetin (Q; 50 μM) or wortmannin (WM; 100 nM) for 1 hour before stimulation with LPS (500 ng/ml) for 24 hours, and LC3 protein levels in the whole-cell lysate (A) and HMGB1 release (C) were detected by Western blotting analysis. In parallel experiments, average LC3 spots (B) and nuclear and cytosole HMGB1 intensity (D) per cell were detected by ArrayScan assay. Values are means (±SEM). *P < 0.05. (E) HMGB1 translocation, determined by imaging analysis, was plotted against HMGB1 nuclear and cytosolic area. Each point represents a single cell. (F) RAW 264.7 cells were treated with Beclin1 shRNA, then stimulated with LPS (500 ng/ml) for 24 hours. Extracellular HMGB1 (Ex-HMGB1) was assayed by Western blotting analysis.