Anti-Inflammatory Effects of Angelica sinensis (Oliv.) Diels Water Extract on RAW 264.7 Induced with Lipopolysaccharide

Abstract

The dry root of Angelica sinensis (Oliv.) Diels, also known as &ldquo;female ginseng&rdquo;, is a popular herbal drug amongst women, used to treat a variety of health issues and cardiovascular diseases. The aim of this study is to evaluate the detailed molecular mechanism for anti-inflammatory effects of Angelica sinensis root water extract (ASW). The anti-inflammatory effect of ASW on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages was evaluated by the tetrazolium-based colorimetric assay (MTT), Griess reagent assay, multiplex cytokine assay, real time reverse transcription polymerase chain reaction (RT-PCR), and Fluo-4 calcium assay. ASW restored cell viability in RAW 264.7 at concentrations of up to 200 &micro;g/mL. ASW showed notable anti-inflammatory effects. ASW exhibited IC₅₀ = 954.3, 387.3, 191.7, 317.8, 1267.0, 347.0, 110.1, 573.6, 1171.0, 732.6, 980.8, 125.0, and 257.0 &micro;g/mL for interleukin (IL)-6, tumor necrosis factor (TNF)-&alpha;, monocyte chemotactic activating factor (MCP)-1, regulated on activation, normal T cell expressed and secreted (RANTES), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), lipopolysaccharide-induced CXC chemokine (LIX), macrophage inflammatory protein (MIP)-1&alpha;, MIP-1&beta;, MIP-2, IL-10, and intracellular calcium, respectively. Additionally, ASW inhibited the LPS-induced production of nitric oxide and the LPS-induced mRNA expression of CHOP (GADD153), Janus kinase 2 (JAK2), signal transducers and activators of transcription 1 (STAT1), first apoptosis signal receptor (FAS), and c-Fos, NOS2, and PTGS2 (COX2) in RAW 264.7 significantly (p < 0.05). Data suggest that ASW exerts an anti-inflammatory effect on LPS-induced RAW 264.7 via NO-bursting/calcium-mediated JAK-STAT pathway.

Keywords: Angelica sinensis; JAK-STAT; anti-inflammatory; calcium; cytokine; macrophage; nitric oxide.

Figure 1

Effects of Angelica sinensis root water extract (ASW) on cell viability (A) and nitric oxide (NO) production (B,C). Cells were treated for 24 h. Con means the group treated with media only. LP means the group treated with 1 µg/mL of lipopolysaccharide (LPS) alone. A25, A50, A100, A200, A400, A800, and A1000 mean 25, 50, 100, 200, 400, 800, and 1000 µg/mL of ASW respectively. Values are the mean ± standard deviation of at least three independent experiments. Statistical significance was calculated by Student t-test (A,B) and one-way ANOVA and a Tukey multiple comparison test (C). ** p < 0.01 vs. Con; *** p < 0.001 vs. Con; ^# p < 0.05 vs. LP; ^## p < 0.01 vs. LP; ^### p < 0.001 vs. LP.

Figure 1

Effects of Angelica sinensis root water extract (ASW) on cell viability (A) and nitric oxide (NO) production (B,C). Cells were treated for 24 h. Con means the group treated with media only. LP means the group treated with 1 µg/mL of lipopolysaccharide (LPS) alone. A25, A50, A100, A200, A400, A800, and A1000 mean 25, 50, 100, 200, 400, 800, and 1000 µg/mL of ASW respectively. Values are the mean ± standard deviation of at least three independent experiments. Statistical significance was calculated by Student t-test (A,B) and one-way ANOVA and a Tukey multiple comparison test (C). ** p < 0.01 vs. Con; *** p < 0.001 vs. Con; ^# p < 0.05 vs. LP; ^## p < 0.01 vs. LP; ^### p < 0.001 vs. LP.

Figure 2

Effects of Angelica sinensis root water extract (ASW) on interleukin (IL)-6, tumor necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and IL-10 production in lipopolysaccharide (LPS)-induced RAW 264.7. Cells were treated for 24 h. Con means the group treated with media only. LP means the group treated with 1 µg/mL of LPS alone. A50 and A100 mean 50 and 100 µg/mL of ASW, respectively. BA means baicalein (100 µM). Values are the mean ± standard deviation of at least three independent experiments. Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. ^### p < 0.001 vs. Con; * p < 0.05 vs. LP; ** p < 0.01 vs. LP; *** p < 0.001 vs. LP.

Figure 3

Effects of Angelica sinensis root water extract (ASW) on macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, lipopolysaccharide-induced CXC chemokine (LIX), regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic activating factor (MCP)-1, interferon gamma-induced protein 10 (IP-10), and leukemia inhibitory factor (LIF) production in lipopolysaccharide (LPS)-induced RAW 264.7. Cells were treated for 24 h. Con means the group treated with media only. LP means the group treated with 1 µg/mL of LPS alone. A50 and A100 mean 50 and 100 µg/mL of ASW respectively. BA means baicalein (100 µM). Values are the mean ± standard deviation of at least three independent experiments. Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. ^### p < 0.001 vs. Con; * p < 0.05 vs. LP; ** p < 0.01 vs. LP.

Figure 4

Effects of Angelica sinensis root water extract (ASW) on calcium release in lipopolysaccharide (LPS)-induced RAW 264.7. Cells were treated for 18 h. Con means the group treated with media only. LP means the group treated with 1 µg/mL of LPS alone. A25, A50, A100, and A200 mean 25, 50, 100, and 200 µg/mL of ASW respectively. IN means indomethacin (0.5 µM). Values are the mean ± standard deviation of at least three independent experiments. Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. ^# p < 0.05 vs. Con; * p < 0.05 vs. LP; ** p < 0.01 vs. LP.

Figure 5

Effects of Angelica sinensis root water extract (ASW) on the mRNA expression of C/EBP homologous protein (CHOP), janus kinase 2 (JAK2), signal transducers and activators of transcription 1 (STAT1), first apoptosis signal receptor (FAS), c-Fos, nitric oxide synthase 2 (NOS2), prostaglandin-endoperoxide synthase 2 (PTGS2), and STAT3 in lipopolysaccharide (LPS)-induced RAW 264.7. Cells were treated for 18 h. Each mRNA was normalized to the housekeeping gene β-Actin mRNA. Con means the group treated with media only. LP means the group treated with 1 µg/mL of LPS alone. A25, A50, A100, and A200 mean 25, 50, 100, and 200 µg/mL of ASW respectively. Values are the mean ± standard deviation of at least three independent experiments. Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. ^### p < 0.001 vs. Con; * p < 0.05 vs. LP; ** p < 0.01 vs. LP; *** p < 0.001 vs. LP.