Anti-Inflammatory Potential of Cultured Ginseng Roots Extract in Lipopolysaccharide-Stimulated Mouse Macrophages and Adipocytes

Figures

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  Figure 1. Effects of cultured ginseng roots (GR) extract on cell viability in RAW264.7, mouse bone-marrow-derived macrophages (BMDM), and 3T3-L1 pre-adipocytes. The culture of (A) RAW264.7, (B) BMDM, and (C) 3T3-L1 were treated with 25–250 μg/mL of GRE for 24 h. 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) reagent was added 3 h before measurement of OD 450nm. Data are expressed as percentages of the vehicle control (dimethyl sulfoxide (DMSO)). Data are represented as the mean ± SEM of three independent experiments (n = 8/group for each experiment); * p < 0.05; ** p < 0.01; compared with vehicle vs. GRE-treated group by one-way ANOVA with Bonferroni’s comparison test.

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  Figure 2. GR extract attenuated lipopolysaccharide (LPS)-induced inflammatory responses, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) in mouse RAW264.7 macrophage cell lines. RAW264.7 cells were pretreated with GRE (50–250 μg/mL) for 48 h in FBS-containing completed media and then starved for 12–18 h in DMEM, followed by LPS stimulation (1 μg/mL) for 2 h in the presence or absence of GRE. (A) TNFα and IL-1β gene expression by qPCR. (B) TNFα cytokine production by ELISA; cells were pretreated with GRE (100 μg/mL) for 48 h, followed by LPS stimulation (1 μg/mL) for 4, 6, and 8 h. (C) TNFα and IL-1β gene expression by qPCR. (D) TNFα cytokine production by ELISA. (E) Protein extracts from LPS-treated RAW264.7 cells were immunoblotted with antibodies targeting phosphor (p)-JNK, p-ERK, p-p38, total (t)-ERK, or β-actin (loading control). (F) Relative ROS production in RAW264.7 with or without GRE, using 2′,7′–dichlorofluorescein (DCF) fluorescence for detecting ROS production. All values are presented as the mean ± SEM (n = 3–4/group for each experiment); * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; compared with LPS-treated group vs. LPS + GRE-treated group by…

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  Figure 3. GR extract attenuated LPS-induced inflammatory responses and ER stress with the induction of antioxidant gene expression in mouse bone-marrow-derived macrophages (BMDMs). BMDMs were pretreated with GRE (50–250 μg/mL) for 48 h in FBS-containing completed media and then starved in DMEM for 12–18 h, followed by LPS stimulation (100 ng/mL) for 2 h (A,D) or 0.5, 2, and 4 h (B,C), in the presence or absence of GRE. (A) TNFα gene expression by qPCR. (B) TNFα and IL-1β gene expression by qPCR. (C) TNFα cytokine production by ELISA. (D) Protein extracts from LPS-treated BMDMs were immunoblotted with antibodies targeting p-JNK, p-ERK, p-p38, t-ERK, or β-actin (loading control); BMDMs were pretreated with GRE (100 μg/mL) for 48 h in FBS-containing completed media and then starved in DMEM, followed by LPS stimulation (100 ng/mL) for 4 h in the presence or absence of GRE. (E) GPx, SOD1, SOD2, and HO-1 gene expression by qPCR. All values are presented as the mean ± SEM (n = 3–4/group for each experiment). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; compared with LPS-treated group vs. LPS + GRE-treated group by one-way ANOVA with Bonferroni’s comparison test. +, treatment; −,…

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  Figure 4. GR extract protected from LPS-mediated impairment of energy metabolism. RAW264.7 cells were pretreated with GRE (100 μg/mL) for 48 h in FBS-containing completed media and then starved in DMEM for 12–18 h, followed by LPS stimulation (1 μg/mL) for 2–4 h in the presence or absence of GRE. (A,B) Oxygen consumption rate (OCR) in RAW264.7 cells treated with vehicle without LPS (white circle, white bar), vehicle with LPS (black circle, black bar), and LPS + GRE (grey triangle, grey bar), as determined by Seahorse extracellular analyzer. Arrow indicates the treatment of inhibitors of oligomycin (Oligo), carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and a combination of antimycin A and rotenone (Rot/AA). All values are presented as the mean ± SEM (n = 5/group for each experiment). #### p < 0.0001; compared with non-LPS-treated group vs. LPS-treated group. ** p < 0.01; *** p < 0.001; compared with LPS-treated group vs. LPS + GRE-treated group by one-way ANOVA or two-way ANOVA with Bonferroni’s comparison test.

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  Figure 5. GR extracts attenuated the LPS-induced inflammatory responses in 3T3-L1 adipocytes and adipocytes exposed to macrophage-conditioned media (CM). The 3T3-L1 cells were seeded and induced to differentiation in the presence of dimethyl sulfoxide (DMSO, vehicle control) and GRE (100 μg/mL) for 7 days and then starved in DMEM for 12–18 h, followed by LPS stimulation (100 ng/mL, 2h) in the presence or absence of GRE. (A) IL-6, IL-1β, and TNFα gene expression by qPCR. (B) Protein extracts from LPS-treated 3T3-L1 adipocytes in the presence or absence of GRE (100 μg/mL) were immunoblotted with antibodies targeting NF-kB, p-JNK, p-ERK, p-p38, t-ERK, or β-actin (loading control); 3T3-L1 adipocytes were preincubated with DMSO or GRE before receiving the CM of LPS-treated BMDMs with or without GRE. (C) Experimental scheme. (D) IL-6, MCP1, and TNFα gene expression by qPCR. All values are presented as the mean ± SEM (n = 3–4/group for each experiment). #### p < 0.0001; compared with non-macrophage (mφ) CM vs. mφ CM. * p < 0.05; *** p < 0.001; **** p < 0.0001 compared with LPS-treated group or m mφ CM vs. GRE-treated group by one-way ANOVA with Bonferroni’s comparison test. +, treatment; −,…