Effects of black radish extract (BRE) on the cell viability and nitric oxide (NO) production of RAW 264.7 macrophages. The cells were incubated for 24 h with different concentrations of BRE (25, 50, 100, and 200 μg/mL) with or without 1 mg/mL lipopolysaccharides (LPS) for 24 h. The cell viability (A and B) and NO (C) production were measured using 3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and enzyme-linked immunosorbent assay kit as described in MATERIALS AND METHODS. Data are expressed as mean± SD (n=3) of the three independent experiments. ^##P<0.01 versus control; ^*P<0.05 and ^**P<0.01 versus the untreated control group.

The effects of black radish extract (BRE) on prostaglandin E2 (PGE₂) release and pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α production. The RAW 264.7 cells were pre-treated with different BRE concentrations for 1 h and then treated with lipopolysaccharides (LPS, 1 μg/mL) for another 24 h. Release of PGE₂ (A), IL-6 (B), IL-1β (C), and TNF-α (D) in culture media were measured using enzyme-linked immunosorbent assay. Data are expressed as mean±SD of the three independent experiments. ^##P<0.01 versus control; ^**P<0.01 versus LPS treatment alone.

The effects of black radish extract (BRE) on the lipopolysaccharides (LPS)-induced expression of inducible nitric oxide synthase (iNOS) (A) and cyclooxygenase (COX)-2 (B). The cells were pre-treated with BRE (25, 50, 100, or 200 μg/mL) for 1 h and then treated with LPS (1 μg/mL) for 24 h. Representative pictures of Western blot and quantitative analysis of blots. Each immunoreactive band was digitized and expressed as a ratio of β-actin levels. Data are expressed as mean±SD of the three independent experiments. ^##P<0.01 versus control; ^*P<0.05 and ^**P<0.01 versus LPS treatment alone.

The effects of black radish extract (BRE) on inducible nitric oxide synthase (iNOS) (A), cyclooxygenase (COX)-2 (B), tumor necrosis factor (TNF)-α (C), interleukin (IL)-1β (D), and IL-6 (E) mRNA expression in lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. The cells were pre-treated with BRE (200, 100, 50, or 25 μg/mL) for 1 h and then treated with LPS (1 μg/mL) for 4 h. Cellular mRNA expression analysis was determined using quantitative polymerase chain reaction. iNOS, COX-2, TNF-α, IL-1β, and IL-6 expression levels were calculated after normalizing the signal against the β-actin gene. Data are expressed as mean±SD of the three independent experiments. ^##P<0.01 versus control; ^*P<0.05 and ^**P<0.01 versus LPS treatment alone.

The effects of black radish extract (BRE), AG490, and phenethyl isothiocyanate (PEITC) on interleukin (IL)-6-induced Janus kinase 2 (JAK2) (A and B) and signal transducer and activator of transcription 3 (STAT3) (C, D, and E) phosphorylation. The RAW 264.7 cells were pre-treated with different BRE concentrations for 30 min and then treated with IL-6 (10 ng/ mL) for 30 min. Representative pictures of Western blot and quantitative analyses. Each immunoreactive band was digitized and expressed as a ratio of STAT3 protein levels. Data are expressed as mean±SD of the three independent experiments. ^##P<0.01 versus control; ^*P<0.05 and ^**P<0.01 versus IL-6 treatment alone (B, D, and E).

The effects of AG490, phenethyl isothiocyanate (PEITC), and black radish extract (BRE) on lipopolysaccharides (LPS)-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation. The RAW 264.7 cells were pre-treated with different concentrations of AG490, PEITC, and BRE for 30 min and then treated with LPS (1 μg/mL) for 4 h. Representative pictures of Western blot and quantitative analyses. Each immunoreactive band was digitized and expressed as a ratio of STAT3 protein levels. Data are expressed as mean±SD of the three independent experiments. ^##P<0.01 versus control; ^*P<0.05 and ^**P<0.01 versus LPS treatment alone.

The inhibitory effects of black radish extract (BRE) on extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation in RAW 264.7 macrophage cells. The cells were pre-treated with BRE for 1 h prior to lipopolysaccharides (LPS) treatment. After treatment with 1 μg/mL LPS, the phosphorylated JNK (A and C) and ERK (B and D) levels were analyzed by immunoblotting. ERK and JNK levels were estimated by the loaded protein as each control. Data are expressed as mean±SD of the three independent experiments. ^##P<0.01 versus control; ^*P<0.05 and ^**P<0.01 versus LPS treatment alone.

The effects of black radish extract (BRE) on heme oxygenase-1 (HO-1) expression without (A and C) or with (B and D) lipopolysaccharide (LPS). The RAW 264.7 cells were pre-treated with different BRE concentrations. Representative pictures of Western blot and quantitative analyses. Each immunoreactive band was digitized and expressed as a ratio of β-actin levels. Data are expressed as mean±SD of the three independent experiments. ^##P<0.01 versus control; ^**P<0.01 versus LPS treatment alone.

Schematic illustration of the inhibitory effect of black radish extract (BRE) in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells. BRE may be beneficial in treating inflammation through its selective immunomodulatory effects, which may be mediated by Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) inhibition and nuclear factor erythroid-2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) signal transduction pathway activation, respectively. The arrows represent the activation, whiles the T-shaped arrows indicate inhibition. ATF4, activating transcription factor 4; IL, interleukin; NO, nitric oxide; iNOS, inducible NO synthase; KEAP1, Kelch-like epichlorohydrin-associated protein 1; MAPK, mitogen-activated protein kinase; P, phosphorylation; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor alpha.