Rubi Fructus Water Extract Alleviates LPS-Stimulated Macrophage Activation via an ER Stress-Induced Calcium/CHOP Signaling Pathway

Abstract

Despite the availability of antibiotics and vaccines, many intractable infectious diseases still threaten human health across the globe. Uncontrolled infections can lead to systemic inflammatory response syndrome and the excessive production of inflammatory cytokines, known as a cytokine storm. As cytokines also play necessary and positive roles in fighting infections, it is important to identify nontoxic and anti-inflammatory natural products that can modulate cytokine production caused by infections. Rubi Fructus, the unripe fruits of Rubus coreanus Miquel, are known to possess antioxidative properties. In this study, the effect of the water extract of Rubi Fructus (RF) on the lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 macrophages was investigated using biochemical and cell biology techniques. Our data indicated that RF inhibits p38 phosphorylation, intracellular calcium release, and the production of nitric oxide (NO), interleukin (IL)-6, monocyte chemotactic activating factor (MCP)-1, tumor necrosis factor (TNF)-α, leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine (LIX), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, and regulated on activation, normal T cell expressed and secreted (RANTES) in LPS-treated macrophages. In addition, we observed decreasing mRNA expression of Chop, Camk2a, Stat1, Stat3, Jak2, Fas, c-Jun, c-Fos, Nos2, and Ptgs2 without cytotoxic effects. We concluded that RF demonstrated immunoregulatory activity on LPS-stimulated macrophages via an endoplasmic reticulum (ER) stress-induced calcium/CCAAT-enhancer-binding protein homologous protein (CHOP) pathway and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway.

Keywords: ER stress; Rubi Fructus; Rubus coreanus; STAT; calcium; chop; cytokine; lipopolysaccharide; macrophage; nitric oxide.

Figure 1

Effects of Rubi Fructus water extract (RF) on (A) cell viability, (B) nitric oxide (NO) production, and (C) intracellular calcium release. Cells were treated with RF and LPS for 24 h (A,B) or 18 h (C). “Nor” indicates the group treated with media only. “Con” indicates the group treated with 1 µg/mL of lipopolysaccharide (LPS) alone. RF25, RF50, RF100, and RF200 indicate 25, 50, 100, and 200 µg/mL of RF, respectively. “BA” indicates treatment with baicalein (25 µM). Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. ** p < 0.01 vs. Con; *** p < 0.001 vs. Con.

Figure 2

Effects of Rubi Fructus water extract (RF) on the production of interleukin (IL)-6, monocyte chemotactic activating factor (MCP)-1, tumor necrosis factor (TNF)-α, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine (LIX), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, vascular endothelial growth factor (VEGF), and regulated on activation, normal T cell expressed and secreted (RANTES), and interferon gamma-induced protein (IP)-10. Cells were treated for 24 h with LPS and RF. “Nor” indicates the group treated with media only. “Con” indicates the group treated with 1 µg/mL of LPS alone. RF25, RF50, RF100, and RF200 indicate 25, 50, 100, and 200 µg/mL of RF, respectively. “BA” indicates treatment with baicalein (25 µM). Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. * p < 0.05 vs. Con; ** p < 0.01 vs. Con; *** p < 0.001 vs. Con.

Figure 3

Effects of Rubi Fructus water extract (RF) on mRNA expression of Chop, Camk2a, Stat1, Stat3, Jak2, Fas, c-Jun, c-Fos, Nos2, and Ptgs2. Cells were treated for 18 h. “Nor” indicates the group treated with media only. “Con” indicates the group treated with 1 µg/mL of LPS alone. RF25, RF50, RF100, and RF200 indicate treatment with 25, 50, 100, and 200 µg/mL of RF, respectively. “BA” indicates treatment with baicalein (25 µM). Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. * p < 0.05 vs. Con; ** p < 0.01 vs. Con; *** p < 0.001 vs. Con.

Figure 4

Effects of Rubi Fructus water extract (RF) on phosphorylation of p38 MAPK in RAW 264.7 cells. Cells were treated with LPS and RF for 30 min. “Nor” indicates the group treated with media only. “Con” indicates the group treated with 0.1 µg/mL of LPS alone. RF25, RF50, RF100, and RF200 indicate treatment with 25, 50, 100, and 200 µg/mL of RF, respectively. “BA” indicates treatment with baicalein (25 µM). Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. * p < 0.05 vs. Con; ** p < 0.01 vs. Con.

Figure 5

A schematic diagram of the immunoregulatory activity of Rubi Fructus water extract (RF) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. RF alleviates LPS-stimulated macrophage activation via an ER stress-induced calcium/CHOP signaling pathway and the JAK/STAT pathway.