Anti-Inflammatory Activity and ROS Regulation Effect of Sinapaldehyde in LPS-Stimulated RAW 264.7 Macrophages

Abstract

We evaluated the anti-inflammatory effects of SNAH in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages by performing nitric oxide (NO) assays, cytokine enzyme-linked immunosorbent assays, Western blotting, and real-time reverse transcription-polymerase chain reaction analysis. SNAH inhibited the production of NO (nitric oxide), reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, and interleukin (IL)-6. Additionally, 100 μM SNAH significantly inhibited total NO and ROS inhibitory activity by 93% (p < 0.001) and 34% (p < 0.05), respectively. Protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) stimulated by LPS were also decreased by SNAH. Moreover, SNAH significantly (p < 0.001) downregulated the TNF-α, IL-6, and iNOS mRNA expression upon LPS stimulation. In addition, 3-100 µM SNAH was not cytotoxic. Docking simulations and enzyme inhibitory assays with COX-2 revealed binding scores of -6.4 kcal/mol (IC₅₀ = 47.8 μM) with SNAH compared to -11.1 kcal/mol (IC₅₀ = 0.45 μM) with celecoxib, a known selective COX-2 inhibitor. Our results demonstrate that SNAH exerts anti-inflammatory effects via suppression of ROS and NO by COX-2 inhibition. Thus, SNAH may be useful as a pharmacological agent for treating inflammation-related diseases.

Keywords: anti-inflammatory effect; cytokine; docking simulation; nitric oxide; reactive oxygen species; sinapaldehyde.

Figure 1

Effects of SNAH (sinapaldehyde) on cell viability and LPS (lipopolysaccharide)-induced NO (nitric oxide) production in RAW 264.7 cells. (a) The chemical structure of SNAH. (b) The viability of Raw264.7 cells was determined by FACS (fluorescence activated cell sorter) analysis. (c) Cells were pretreated with 3, 10, 30, and 100 μM SNAH for 1 h before treatment with 1 μg/mL LPS. After incubation for 18 h, NO production was detected by Griess test. (d) The cell viability was detected by XTT assay. The values are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, compared to the LPS-treated group. # p < 0.05, ## p < 0.01, compared to the LPS non-treated group.

Figure 2

Effects of SNAH on LPS-induced TNF-α (a) and IL-6 (b) production. RAW 264.7 cells were pretreated with 3, 10, 30, and 100 μM SNAH for 1 h before treatment with 1 μg/mL LPS. After incubation for 18 h, IL-6 and TNF-α production was detected by ELISA. The values are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the LPS-treated group.

Figure 3

Effects of SNAH on LPS-induced iNOS and COX-2 protein expression. RAW 264.7 Cells were pretreated with 3-100 μM SNAH for 1 h before treatment with 1 μg/mL LPS. After incubation for 24 h, iNOS (a) and COX-2 (b) protein levels were detected by Western blot. The values are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the LPS-treated group.

Figure 4

Effect of SNAH on TNF-α, iNOS, and IL-6 gene expression in RAW 264.7 cells. RAW264.7 cells were pretreated with 10-100 μM SNAH for 1 h prior to LPS stimulation. After 6h LPS stimulation, total RNA was isolated using Trizol reagent. TNF-α (a), iNOS (b), and IL-6 (c) mRNA levels were analyzed by real-time RT-PCR. Relative mRNA levels were determined using the Ct-value method and normalized to GAPDH expression. The values are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the LPS-treated group.

Figure 5

Effects of SNAH on LPS-induced ROS (reactive oxygen species) production. (a) RAW 264.7 cells were pretreated with 10–100 μM SNAH for 1 h before treatment with 1 μg/mL LPS for 18 h. Cells were incubated with 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 30 min at 37 °C. Then, cells were harvested, and dichlorofluorescein (DCF) fluorescence was immediately analyzed by flow cytometry. DCF fluorescence intensity was determined from the same numbers of cells in a randomly selected area. (b) The mean relative ROS level is presented in a bar graph. The values are presented as means ± SD of three independent experiments ** p < 0.01, compared to the LPS-treated group.

Figure 6

Effect of SNAH on MAPK and p65 signaling pathways. (a) The time course of ERK (extracellular-signal-regulated kinase) and SAPK/JNK (Stress-activated protein kinase/Jun-amino-terminal kinase) phosphorylation stimulated by LPS in RAW264.7 cells. (b) RAW 264.7 cells were treated for 15 min with LPS (1 μg/mL) alone or with LPS (1 μg/mL) coupled with 3–100 μM SNAH. Cell lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, SAPK/JNK, and NF-κB (p65) were probed as quantitative controls.

Figure 7

Docking simulation between SNAH and COX-2 (6COX). (a) The pink-colored chemical structure represents SNAH, and the COX-2 protein structure complexed with celecoxib is indicated by blue colored. The magnified rectangles indicate the active sites of COX-2 proteins. In the box image, hydrogen bond interactions between COX-2 and SNAH are depicted by green-colored lines. The binding energies of SNAH and celecoxib for COX-2 were −6.4 kcal/mol and −11.1 kcal/mol, respectively. (b) COX-2 enzyme inhibitory activities of SNAH. The commercial standard, celecoxib was used as a positive control at identical concentrations.