Ginsenoside Rb2 enhances the anti-inflammatory effect of ω-3 fatty acid in LPS-stimulated RAW264.7 macrophages by upregulating GPR120 expression

Abstract

Recent studies confirm that chronic low-grade inflammation is closely associated with metabolic syndromes, and anti-inflammatory therapy is a potential approach for treating cardiovascular diseases and type 2 diabetes. Accumulating evidence suggests that GPR120 activation is a feasible solution to ameliorating chronic inflammation and improving glucose metabolism. In this study we investigated whether ginsenoside Rb2 (Rb2), which exhibited regulatory activities in glucose and lipid metabolism, affected GPR120 expression in lipopolysaccharide (LPS)-activated mouse macrophage RAW264.7 cells, and examined the contribution of GPR120 activation to reducing the LPS-induced inflammatory response. LPS (100 ng/mL) activated the macrophages, resulting in dramatic increases in TNF-α, IL-6, IL-1β and NO production. Treatment with a ω-3 fatty acid α-linolenic acid (ALA, 50 μmol/L) produced moderate reduction in LPS-stimulated inflammatory cytokines and NO production (TNF-α and IL-6 were decreased by 46% and 42%, respectively). Pre-incubation with Rb2 (1 or 10 μmol/L) for 12 h before ALA treatment dramatically amplified the inhibitory effects of ALA (TNF-α and IL-6 were decreased by 74% and 86%, respectively). Compared to the treatment with ALA alone, pre-incubation with Rb2 resulted in a more prominent reduction in LPS-stimulated expression of iNOS and COX-2 and LPS-stimulated IKK/NF-κB phosphorylation and MAPK pathway activation. Rb2 (0.1-100 μmol/L) dose- and time-dependently increased both mRNA and protein expression of GPR120 in RAW264.7 cells, but treatment with Rb2 alone did not exert anti-inflammatory effect in LPS-activated RAW264.7 cells. In RAW264.7 cells transfected with GPR120 shRNA, the ameliorating effects of Rb2 on LPS-induced inflammation were abolished. In conclusion, Rb2 exerts anti-inflammatory effect in LPS-stimulated mouse macrophage RAW264.7 cells in vitro by increasing GPR120 expression and subsequently enhancing ω-3 fatty acid-induced GPR120 activation.

Figure 1

Effects of Rb2 on the viability of RAW 264.7 macrophages. RAW264.7 cells were incubated with various concentrations of Rb2 in the absence or presence of ALA (50 μmol/L) for 48 h. Cell viability was determined by MTT assay. The data are presented as mean±SD of three independent experiments. 'ns' (no significant difference) means P>=0.05 vs vehicle (DMSO) group which acts as control.

Figure 2

Effect of Rb2 on GPR120 expression in RAW264.7 macrophages. RAW264.7 cells were incubated with Rb2 (0.1–100 μmol/L) or vehicle (DMSO) for 24 h. The expression levels of GPR120 protein (A) in cell lysates were detected by Western blot analysis and mRNA (B) in cells were detected by quantitative RT-PCR. The expression levels of GPR120 protein (C) and mRNA (D) in cells transfected with GPR120 shRNA or control shRNA were detected by Western blot analysis and quantitative RT-PCR, respectively. (E) The induction of GPR120 by Rb2 behaved in a time-dependent manner as determined by Western blot analysis. The data are presented as mean±SD of three independent experiments. ^*P<0.05 vs vehicle (DMSO) group, control shRNA or '0 h' group which acts as control.

Figure 3

Rb2 enhanced reduction effect of ALA on inflammatory cytokines and NO production induced by LPS in RAW264.7 macrophages. RAW264.7 cells were transfected with either GPR120 shRNA or negative control shRNA for 12 h, then pretreated with Rb2 (1 and 10 μmol/L) or DMSO for 12 h prior to incubation of ALA (50 μmol/L) or DMSO for 1 h and finally stimulated with LPS (100 ng/mL) or DMSO for 24 h. The production of cytokines TNF-α (A) and IL-6 (B) in culture supernatants were measured by ELISA, NO (F) in supernatant was measured by Total NO Assay Kit. The mRNA expression levels of TNF-α (C), IL-6 (D) and IL-1β (E) in cells were detected by quantitative RT-PCR. The data are presented as mean±SD of three independent experiments. ^*P<0.05 vs 'LPS alone' group. ^#P<0.05 vs 'LPS plus ALA' group.

Figure 4

Rb2 enhanced the reduction effect of ALA on expression of iNOS and COX-2 induced by LPS. RAW264.7 cells were transfected with either GPR120-specific shRNA or negative control shRNA for 12 h, then pretreated with Rb2 (1 and 10 μmol/L) or DMSO for 12 h prior to incubation of ALA (50 μmol/L) or DMSO and finally stimulated with LPS (100 ng/mL) or DMSO for 6 h (for RT-PCR) or 12 h (for Western blot). The expression levels of iNOS and COX-2 protein (A) were detected by Western blot and relative density was normalized by Actin. Relative mRNA expression levels (B) of iNOS and COX-2 were detected by quantitative RT-PCR and were normalized by 18s. The data are presented as mean±SD of three independent experiments. ^*P<0.05 vs 'LPS alone' group. ^#P<0.05 vs 'LPS plus ALA' group.

Figure 5

The Inhibitory effect of Rb2 on LPS-induced IKKβ/NF-κB signal pathway activation is GPR120 activation-dependent. RAW264.7 cells were transfected with either GPR120 shRNA or negative control shRNA for 12 h, then pretreated with Rb2 (1 and 10 μmol/L) or DMSO for 12 h prior to incubation of ALA (50 μmol/L) or DMSO for 1 h and finally stimulated with LPS (100 ng/mL) or DMSO for 15 min. (A, B) Protein levels of phosphorylated IKKβ (p-IKKβ) and phosphorylated NF-κB (p-NF-κB) were detected by Western blotting and relative density was normalized by total IKKβ (t-IKKβ) and total NF-κB (t-NF-κB), respectively. The data are presented as mean±SD of three independent experiments. ^*P<0.05 vs 'LPS alone' group. ^#P<0.05 vs 'LPS plus ALA' group.

Figure 6

Different role of MAPKs in the anti-inflammatory process of Rb2. RAW264.7 cells were transfected with either GPR120 shRNA or negative control shRNA for 12 h, then pretreated with Rb2 (1 and 10 μmol/L) or DMSO for 12 h prior to incubation of ALA (50 μmol/L) or DMSO for 1 h and finally stimulated with LPS (100 ng/mL) or DMSO for 15 min. Protein levels of phosphorylated ERK (p-ERK), phosphorylated JNK (p-JNK) and phosphorylated p38 (p-p38) were detected by Western blot (A) and relative density was normalized by total ERK (t-ERK), total JNK (t-JNK) and total p38 (t-p38) (B), respectively. The data are presented as mean±SD of three independent experiments. ^*P<0.05 vs LPS alone group. ^#P<0.05 vs 'LPS plus ALA' group.