A Dalbergia odorifera extract improves the survival of endotoxemia model mice by inhibiting HMGB1 release

Abstract

Background: Dalbergia odorifera T. Chen (Leguminosae) is an indigenous medicinal herb that is widely used as a popular remedy in northern and eastern Asia. However, the cellular mechanisms underlying the biological activity of D. odorifera are not fully elucidated.

Methods: Anti-inflammatory effect of D. odorifera extract (DOE) was determined through intraperitoneal injection in a mouse model of endotoxemia induced by lipopolysaccharide (LPS). RAW 264.7 cells, a murine macrophage, were also treated with LPS to generate a cellular model of inflammation, and investigated the anti-inflammatory activity and underlying mechanisms of DOE and its constituent isoliquiritigenin.

Results: DOE dose-dependently inhibited LPS-induced release of high mobility group box 1 (HMGB1), a late proinflammatory cytokine, and decreased cytosolic translocation of HMGB1 in RAW264.7 cells. This inhibitory effect of DOE on HMGB1 release was observed in cells treated with DOE before or after LPS treatment, suggesting that DOE is effective for both treatment and prevention. In addition, DOE significantly inhibited LPS-induced formation of nitric oxide (NO) and expression of inducible NO synthase (iNOS) in a dose-dependent manner. These effects of DOE were accompanied by suppression of HMGB1 release triggered by LPS, suggesting a possible mechanism by which DOE modulates HMGB1 release through NO signaling. Isoriquiritigenin, a constituent of DOE, also attenuated LPS-triggered NO formation and HMGB1 release in RAW264.7 cells, indicating that isoriquiritigenin is an indexing molecule for the anti-inflammatory properties of DOE. Furthermore, c-Jun N-terminal kinase, but not extracellular signal-regulated kinase and p38, mediated DOE-dependent inhibition of HMGB1 release and NO/iNOS induction in RAW 264.7 cells exposed to LPS. Notably, administration of DOE ameliorated survival rates in a mouse model of endotoxemia induced by LPS, where decreased level of circulating HMGB1 was observed.

Conclusion: These results suggest that DOE confers resistance to LPS-triggered inflammation through NO-mediated inhibitory effects on HMGB1 release.

Keywords: Dalbergia Odorifera; Endotoxemia; HMGB1; Inflammation; Nitric oxide.

Fig. 1

Cell viability was not affected by the concentrations of DOE tested. RAW 264.7 cells were treated with the indicated concentrations of DOE. After incubation for 24 h, cell viability was assessed by the MTT assay (a) or the trypan blue exclusion method (b). The results are plotted as the means ± S.E. (n = 4)

Fig. 2

DOE inhibits LPS-triggered release of HMGB1 in RAW 264.7 cells. a Cells cultured for 24 h in serum-free medium were triggered by vehicle (DMSO) or LPS in the presence or absence of DOE. Following incubation for 24 h, aliquots of conditioned media or whole-cell lysates were analyzed by immunoblot analysis. Ponceau S staining and β-actin were used as the loading controls. b Cells were treated with vehicle (DMSO) or LPS in the presence or absence of DOE for 24 h, and then whole-cell lysates were fractionated into cytosolic (C) and nuclear (N) fractions. The localization of HMGB1 was analyzed by immunoblotting with the indicated antibodies. Representative blots from three independent experiments are shown

Fig. 3

Treatment with DOE after LPS exposure attenuates LPS-triggered release of HMGB1 in RAW 264.7 cells. Cells cultured for 24 h in serum-free medium were triggered by vehicle (DMSO) or LPS. DOE was added at the indicated time points after LPS treatment for 24 h. Equal volumes of conditioned medium were subjected to immunoblot analysis to determine the levels of HMGB1. As a loading control, Ponceau S staining was used. The results are representative of three or four independent experiments

Fig. 4

DOE inhibits LPS-triggered iNOS expression and NO formation in RAW 264.7 cells. a Cells cultured for 24 h in serum-free medium were triggered by vehicle (DMSO) or LPS. DOE was added at the indicated time points after LPS treatment for 24 h. The levels of nitrite were determined by incubation with Griess reagents. b Cells cultured for 24 h in serum-free medium were triggered by vehicle (DMSO) or LPS with or without indicated concentrations of DOE. After incubation for 24 h, aliquots of whole-cell lysates or conditioned media were subjected to immunoblot or nitrite analysis, respectively. The results are plotted as the means ± S.E. (n = 6). ^*, p < 0.01 vs untreated group; ^#, p < 0.01 vs LPS-treated group

Fig. 5

DOE inhibits LPS-triggered release of HMGB1 through inhibition of NO formation in RAW 264.7 cells. a and b Cells cultured for 24 h in serum-free medium were pretreated with or without L-NMMA and then stimulated with vehicle (DMSO) or LPS in the presence or absence of DOE. After incubation for 24 h, aliquots of whole-cell lysates or conditioned media were subjected to immunoblot or nitrite analysis, respectively (s). The levels of HMGB1 were analyzed by immunoblot using equal volumes of conditioned media (b). Ponceau S staining and β-actin were used as the loading controls. The blots are representative of three independent experiments. The results are plotted as the means ± S.E. (n = 6). ^*, p < 0.01 vs untreated group; ^#, p < 0.01 vs LPS-treated group

Fig. 6

LC-MS/MS analysis. Chromatographic separation was performed on a C18 analytical column eluted with an acetonitrile gradient at 0.2 ml/min. Isoliquiritigenin, daidzein, formononetin, dalbergin, piperidine, and sativanone were identified using authentic standards

Fig. 7

Isoliquiritigenin inhibits LPS-induced NO formation and HMGB1 release in RAW264.7 cells. a Cells cultured for 24 h in serum-free medium were stimulated with vehicle (DMSO) or LPS in the presence of DOE, isoliquiritigenin, dalbergin, or sativanone. After incubation for 24 h, aliquots of conditioned media were subjected to nitrite analysis. b Cells cultured for 24 h in serum-free medium were triggered by vehicle (DMSO) or LPS in the absence or presence of isoliquiritigenin. After incubation for 24 h, equal volumes of conditioned medium were subjected to nitrite analysis or immunoblot analysis to determine the levels of HMGB1. Ponceau S staining was used as a loading control. The results are plotted as the means ± S.E. (n = 6). ^*, p < 0.01 vs untreated group; ^#, p < 0.01 vs LPS-treated group

Fig. 8

DOE inhibits the LPS-triggered release of HMGB1 by suppressing phosphorylation of JNK via NO in RAW 264.7 cells. a Cells were treated with LPS for the indicated amounts of time. b Cells were stimulated with vehicle (DMSO) or LPS in the presence or absence of DOE for 30 min. Aliquots of whole-cell lysates were immunoblotted with activation-specific antibodies, and parallel immunoblots were analyzed for total kinase levels. c and d Cells were pretreated with SP600125 for 1 h and then exposed to vehicle (DMSO) or LPS with or without DOE. Following incubation for 24 h, aliquots of conditioned media or whole-cell lysates were subjected to immunoblot or nitrite analysis to detect HMGB1 (c) or iNOS and nitrite (d), respectively. The blots are representative of three independent experiments. The results are plotted as the means ± S.E. (n = 5). ^*, p < 0.01 vs untreated group; ^#, p < 0.01 vs LPS-treated group

Fig. 9

DOE improves the survival rates of mice treated with endotoxin through blockade of HMGB1 release. a BALB/c mice (n = 8 per group) were treated with LPS (10 mg/kg, i.p.) and then injected with a single dose of DOE (20 mg/kg, i.p.) 0, 3, 6, and 9 h later. Motality was monitored for up to 2 weeks every day. b BALB/c mice (n = 4 per group) were treated with a single dose of DOE (20 mg/kg, i.p.) with or without a lethal injection of endotoxin (LPS, 10 mg/kg, i.p.). Circulating levels of HMGB1 were detected by immunoblot analysis of sera collected at 20 h post-LPS injection. As a loading control, Ponceau S staining was used. c Schematic representation of DOE-mediated inhibition of HMGB1 release. ^#, p < 0.01 vs LPS-treated group