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. 2018 Sep 4;9:911.
doi: 10.3389/fphar.2018.00911. eCollection 2018.

Activation of Nrf2/HO-1 Pathway by Nardochinoid C Inhibits Inflammation and Oxidative Stress in Lipopolysaccharide-Stimulated Macrophages

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Free PMC article

Activation of Nrf2/HO-1 Pathway by Nardochinoid C Inhibits Inflammation and Oxidative Stress in Lipopolysaccharide-Stimulated Macrophages

Jin-Fang Luo et al. Front Pharmacol. .
Free PMC article

Abstract

The roots and rhizomes of Nardostachys chinensis have neuroprotection and cardiovascular protection effects. However, the specific mechanism of N. chinensis is not yet clear. Nardochinoid C (DC) is a new compound with new skeleton isolated from N. chinensis and this study for the first time explored the anti-inflammatory and anti-oxidant effect of DC. The results showed that DC significantly reduced the release of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-activated RAW264.7 cells. The expression of pro-inflammatory proteins including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were also obviously inhibited by DC in LPS-activated RAW264.7 cells. Besides, the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also remarkably inhibited by DC in LPS-activated RAW264.7 cells. DC also suppressed inflammation indicators including COX-2, PGE2, TNF-α, and IL-6 in LPS-stimulated THP-1 macrophages. Furthermore, DC inhibited the macrophage M1 phenotype and the production of reactive oxygen species (ROS) in LPS-activated RAW264.7 cells. Mechanism studies showed that DC mainly activated nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, increased the level of anti-oxidant protein heme oxygenase-1 (HO-1) and thus produced the anti-inflammatory and anti-oxidant effects, which were abolished by Nrf2 siRNA and HO-1 inhibitor. These findings suggested that DC could be a new Nrf2 activator for the treatment and prevention of diseases related to inflammation and oxidative stress.

Keywords: HO-1; Nardochinoid C; Nardostachys chinensis; Nrf2; ROS.

Figures

FIGURE 1
FIGURE 1
The effect of Nardochinoid C (DC) on the release of NO and PGE2 in LPS-stimulated RAW264.7 cells. (A) The chemical structure of DC. (B) Cytotoxicity of DC on LPS-unstimulated RAW264.7 cells. (C) Cytotoxicity of DC on LPS-stimulated RAW264.7 cells. The cells were treated with DC at various concentrations (1.25–40 μM) for 1 h, and then stimulated with or without LPS for 18 h, the cell viability was analyzed with MTT method. Effects of DC on the productions of NO (D) and PGE2 (E) in LPS-stimulated RAW264.7 cells. The cells were incubated with indicated concentrations of DC or DEX for 1 h, and then stimulated with LPS for 18 h. The concentration of NO (expressed as nitrite) and PGE2 in the culture medium were quantified by ELISA kits. Statistical analysis was carried out by using one-way ANOVA with Tukey’s multiple comparison tests in GraphPad Prism7 (P < 0.05, ANOVA). Results are expressed as mean ± SEM of three independent experiments (N = 3), #p < 0.05, ##p < 0.01, vs. LPS-unstimulated cells (B,C) or p < 0.05, ∗∗p < 0.01, vs. LPS-stimulated cells (D,E).
FIGURE 2
FIGURE 2
Effects of DC on the expressions of iNOS, COX-2, TNF-α, and IL-6 in LPS-stimulated RAW264.7 cells. The cells were plated in 24-well plates and incubated for 24 h, next the cells were pretreated with indicated concentrations of DC for 1 h and stimulated with LPS for 18 h. The total proteins of the cells were prepared and the expressions of iNOS (A) and COX-2 (B) were analyzed by Western blot. Total mRNA was prepared, the mRNA expressions of iNOS (C) and COX-2 (D) were analyzed by real time PCR. The protein expression levels of TNF-α (E) and IL-6 (F) in cell supernatant were analyzed by ELISA. The mRNA expression of TNF-α (G) and IL-6 (H) were analyzed by real time PCR. Statistical analysis was carried out by using one-way ANOVA with Tukey’s multiple comparison tests in GraphPad Prism7 (P < 0.05, ANOVA). Results are expressed as mean ± SEM of three independent experiments (N = 3). p < 0.05, ∗∗p < 0.01, vs. LPS-stimulated cells.
FIGURE 3
FIGURE 3
Effects of DC on the levels of COX-2, PGE2, TNF-α, and IL-6 in LPS-stimulated THP-1 cells. (A) Cytotoxicity of DC on LPS-unstimulated THP-1 cells. (B) Cytotoxicity of DC on LPS-stimulated THP-1 cells. (C) Effects of DC on the expression of COX-2 in LPS-stimulated THP-1 cells. (D) Effects of DC on the productions of PGE2 in LPS-stimulated THP-1 cells. The mRNA expression of TNF-α (E) and IL-6 (F) were analyzed by real time PCR. THP-1 cells (1 × 106 cells/well) were seeded in six well plates. After being incubated for 24 h, the cells were treated with 50 nM PMA for 48 h, then incubated with various concentration (2.5, 5, and 10 μM) of DC or DEX (0.5 μM) for 1 h, finally, exposed to LPS (1 μg/mL) for 24 h. The cell supernatant, total proteins and mRNA of the cells were prepared as described before for Western blot and real time PCR. Statistical analysis was carried out by using one-way ANOVA with Tukey’s multiple comparison tests in GraphPad Prism7 (P < 0.05, ANOVA). Results are expressed as mean ± SEM of three independent experiments (N = 3). #p < 0.05, ##p < 0.01, vs. LPS-unstimulated cells (A,B) or p < 0.05, ∗∗p < 0.01, vs. LPS-stimulated cells (C–F).
FIGURE 4
FIGURE 4
The effect of DC on the activation of NF-κB and MAPK pathways in LPS-stimulated RAW264.7 cells. The cells were plated in 24-well plates and incubated for 24 h, next the cells were pretreated with indicated concentration of DC for 1 h and stimulated with LPS for 15 min. The total proteins of the cells were prepared and the expression of phosphorylation level of IKKα/β and p65 (A) were detected by Western blot. The cells were treated with DC (10 μM) for 1 h and stimulated with LPS for 15 min. The subcellular localization of p65 was detected with immunofluorescence assay (B) and the images were acquired using the Leica DM2500 fluorescent microscopy. The total proteins of the cells were prepared and the expressions of phosphorylated JNK, ERK and p38 (C) were detected by Western blot.
FIGURE 5
FIGURE 5
The effect of DC on the expressions of M1, ROS, HO-1, and NQO1 in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pretreated with indicated concentrations of DC for 1 h, then cells were incubated for 6 h in the absence of LPS, then collected all the cells and cellular staining for F/480 and CD11c, the M1 markers (A) according to the manufacturer’s directions. The intracellular level of ROS (B) was determined using the fluorescent probe DCFH-DA. The cells were plated in 24-well plates and incubated for 24 h, next the cells were pretreated with indicated concentrations of DC for 1 h and stimulated with LPS for 18 h. The total proteins of the cells were prepared and the expression of HO-1 (C) and NQO1 (D) were analyzed by Western blot. The mRNA expressions of HO-1 (E) and NQO1 (F) were analyzed by real time PCR. Statistical analysis was carried out by using one-way ANOVA with Tukey’s multiple comparison tests in GraphPad Prism7 (P < 0.05, ANOVA). Results are expressed as mean ± SEM of three independent experiments (N = 3). p < 0.05, ∗∗p < 0.01, vs. LPS-stimulated cells.
FIGURE 6
FIGURE 6
The effect of DC on the nuclear translocation of Nrf2 and the expressions of HO-1, NQO1, Keap1, and p62 in LPS-unstimulated RAW264.7 cells. The cells were treated with SFN or DC (10 μM) for 6 h. The subcellular localization of Nrf2 was detected with immunofluorescence assay (A) and the images were acquired using the Leica DM2500 fluorescent microscopy. The nuclear protein of the cells was prepared and the expression of Nrf2 in nuclear protein (B) of the cells were analyzed by Western blot. The cells were plated in 24-well plates and incubated for 24 h, next the cells were pretreated with indicated concentrations of DC for 1 h and stimulated with or without LPS for 18 h. The total proteins of the cells were prepared and the expressions of HO-1 (C), NQO1 (D), Keap1 (G), and p62 (H) were analyzed by Western blot. The mRNA levels of HO-1 (E) and NQO1 (F) were analyzed by real time PCR. Statistical analysis was carried out by using one-way ANOVA with Tukey’s multiple comparison tests in GraphPad Prism7 (P < 0.05, ANOVA). Results are expressed as mean ± SEM of three independent experiments (N = 3). p < 0.05, ∗∗p < 0.01, vs. LPS-unstimulated cells.
FIGURE 7
FIGURE 7
The effect of Nrf2 siRNA and HO-1 inhibitor on the anti-inflammatory effect of DC. For transfection, the cells were seeded in 24-well culture plates and incubated with the control siRNA or Nrf2 siRNA at 300 nM for 24–48 h in serum-free OPTI-MEM media. The total proteins of Nrf2 (A) were analyzed by Western blot. The mRNA level of Nrf2 (B) were analyzed by real time PCR. Statistical analysis was carried out by using unpaired t-test in GraphPad Prism7 (P < 0.05, unpaired t-test). Results are expressed as mean ± SEM of three independent experiments (N = 3). p < 0.05, ∗∗p < 0.01, vs. NS siRNA treated cells (B). The transfected cells were also pretreated with indicated concentration of DC for 1 h and stimulated with or without LPS for 18 h. The total proteins of the cells were prepared and the expressions of Nrf2 (C,E) and HO-1 (D,F) were analyzed by Western blot. The concentration of NO (G) in the culture medium were quantified. Statistical analysis was carried out by using one-way ANOVA with Tukey’s multiple comparison tests in GraphPad Prism7 (P < 0.05, ANOVA). Results are expressed as mean ± SEM of three independent experiments (N = 3). p < 0.05, ∗∗p < 0.01, vs. DC, LPS, and NS siRNA treated cells (E–G).
FIGURE 8
FIGURE 8
The effect of HO-1 inhibitor on the anti-inflammatory effect of DC. The cells were pretreated with indicated concentration of ZnPP for 1 h, and then stimulated with DC for 1 h, at last, treated with LPS for 18 h. The total proteins of iNOS (A), and HO-1 (B) were analyzed by Western blot. The concentration of NO (C) in the culture medium were quantified. The levels of Bilirubin (D) in cell microsome fraction were detected. Statistical analysis was carried out by using one-way ANOVA with Tukey’s multiple comparison tests in GraphPad Prism7 (P < 0.05, ANOVA). Results are expressed as mean ± SEM of three independent experiments (N = 3). p < 0.05, ∗∗p < 0.01, vs. LPS treated cells, #p < 0.05 vs. ZnPP treated cells.
FIGURE 9
FIGURE 9
Proposed molecular mechanisms underlying the inhibitory effect of DC on the activation of macrophage induced by LPS. DC activates Nrf2 pathway to increase anti-oxidant genes and protein, which in turn reduce inflammation and oxidative stress.

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