Amomum tsao-ko fruit extract suppresses lipopolysaccharide-induced inducible nitric oxide synthase by inducing heme oxygenase-1 in macrophages and in septic mice

Abstract

Amomum tsao-ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao-ko (AOM) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS-induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase-1 (HO-1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO-1) and silencing HO-1 using siRNA prevented the AOM-mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF-E2-related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N-acetyl-l-cysteine (NAC; a ROS scavenger) diminished the AOM-induced phosphorylation of ERK and JNK and AOM-induced HO-1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM-induced signalling of HO-1 expression. In LPS-induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO-1 expression and survival rates. These results indicate that AOM strongly inhibits LPS-induced NO production by activating the ROS/MAPKs/Nrf2-mediated HO-1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.

Keywords: Amomum tsao-ko; HO-1; NO; Nrf2; ROS; sepsis.

Figure 1

AOM suppressed LPS‐induced NO production and iNOS expression and induced HO‐1 expression in RAW 264.7 macrophages. (a) Following pretreatment with AOM (5, 10, or 20 μg/ml) for 1 h, cells were treated with LPS (1 μg/ml) for 24 h. Culture media were collected and subjected to a Griess assay to determine NO production levels. Controls were not treated with LPS or AOM. _L‐NIL (20 μM) was used as a positive control. (b) Lysates were prepared from cells pretreated with/without indicated concentrations of AOM for 1 h and then treated with LPS (1 μg/ml) for 24 h. Total cellular proteins were resolved by SDS‐PAGE, transferred to PVDF membranes and detected using a specific iNOS antibody. (c) Total RNAs for qRT‐PCR were prepared from cells pretreated with/without the indicated concentrations of AOM for 1 h and then treated with LPS (1 μg/ml) for 4 h. iNOS levels were normalized against β‐actin. Data are presented as the means ± SDs of three independent experiments. ^# P < 0.05 vs. the non‐treated control group; ***P < 0.001 vs. LPS‐stimulated group. (d) Lysates were prepared from cells treated with AOM (20 μg/ml) for the indicated times. Total cellular proteins were resolved by SDS‐PAGE, transferred to PVDF membranes, and detected using a specific HO‐1 antibody. (e) Total RNAs for qRT‐PCR analysis were prepared from cells treated with AOM (20 μg/ml) for the indicated times. HO‐1 levels were normalized against β‐actin. (f) Cells were treated with AOM from 6.25 to 100 μg/ml for 24 h in the presence or absence of LPS (1 μg/ml). Cell viability were determined by MTT assay. Data are presented as the means ± SDs of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. non‐treated control group.

Figure 2

The induction of HO‐1 mediated the AOM‐induced suppression of NO production and iNOS expression in RAW 264.7 macrophages. (a) Cells were pretreated with AOM (5, 10, or 20 μg/ml) in the presence or absence of SnPP (10 μM) for 1 h and then treated with LPS (1 μg/ml) for 24 h. (b) Cells were transfected with HO‐1 siRNA or control siRNA prior to AOM (20 μg/ml) treatment. HO‐1 deletion was determined by Western blotting using a specific HO‐1 siRNA. (c and d) After siRNA transfection, cells were pretreated with AOM (5, 10, or 20 μg/ml) for 1 h, and then treated with LPS (1 μg/ml) for 24 h. The culture medium and total protein extracts were then subjected to Griess reaction assay to determine NO levels (c) or to Western blotting to determine iNOS protein expression (d). Data are presented as the means ± SDs of three independent experiments. ^# P < 0.05 vs. the non‐treated control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. SnPP‐treated or HO‐1 siRNA‐transfected group.

Figure 3

AOM induced Nrf2 activation in RAW 264.7 macrophages. (a) Nuclear extracts were prepared from cells treated with AOM (20 μg/ml) for the indicated times or cells treated with LPS (1 μg/ml) for 1 h, and analysed for Nrf2 binding by EMSA. LPS was used as a positive control. The specificity of binding was examined by competition with 100‐fold unlabelled Nrf2 oligonucleotide (CP). (b and c) Cells were treated with AOM (20 μg/ml) for the indicated times (b) or with the indicated concentrations of AOM for 1 h (c). Total protein or nuclear protein extracts were resolved by SDS‐PAGE, transferred to PVDF membranes and detected with specific Nrf2 antibody. β‐Actin and PARP were used as internal controls. (d–f) Total RNAs for qRT‐PCR analysis were prepared from cells treated with indicated concentrations of AOM for 12 h (d) or 8 h (e) or 4 h (f). NQO‐1, GCLM and GCLC levels were normalized against β‐actin. Data are presented as the means ± SDs of three independent experiments. ***P < 0.001 vs. non‐treated control group.

Figure 4

AOM induced HO‐1 expression via ERK and JNK activation in RAW 264.7 macrophages. (a) Cells were pretreated with SB203580 (20 μM), PD98059 (20 μM) or SP600125 (10 μM) for 1 h and then treated with AOM (20 μM) for 8 h. (b) Lysates were prepared from cells treated with AOM (20 μg/ml) for the indicated times. Total protein extracts were resolved by SDS‐PAGE, transferred to PVDF membranes, and detected using specific HO‐1, pERK, ERK, pJNK and JNK antibodies.

Figure 5

AOM‐induced ROS production enhanced HO‐1 expression via ERK and JNK activation in RAW 264.7 macrophages. (a) Cells were incubated with H₂ DCFDA (20 μM) for 30 min in the presence of AOM (20 μg/ml) or H₂O₂ (200 μM). Fluorescence intensities were measured by flow cytometry. H₂O₂ (200 μM) was used as a positive control for ROS production. (b and c) Cells were pretreated with NAC (5 mM) for 1 h and then treated with AOM (20 μM) for 8 h (HO‐1), 30 min (pERK) or 20 min (pJNK). Total protein extracts were resolved by SDS‐PAGE, transferred to PVDF membranes, and detected with specific HO‐1, pERK, ERK, pJNK and JNK antibodies.

Figure 6

AOM reduced NO production and iNOS expression and increased HO‐1 expression and survival rates in LPS‐induced septic mice. (a) AOM (10, 20, or 50 mg/kg, p.o.) was administered to mice 1 h before injecting LPS (25 mg/kg, i.p.). Serum samples were collected 6 h after injecting LPS (25 mg/kg, i.p.). NO levels were determined using a Griess reaction assay. (b and c) Liver tissues were collected 6 h after injecting LPS (25 mg/kg, i.p.) and iNOS and HO‐1 expressions were assessed by qRT‐PCR. ^# P < 0.05 vs. non‐treated control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. LPS‐injected mice (n = 8). (d) Survival rates were observed over 48 h after LPS administration (n = 5) and percentage was shown;**P < 0.01 vs. LPS‐injected mice.