Anti-inflammatory activities of Sigesbeckia glabrescens Makino: combined in vitro and in silico investigations

Abstract

Background: Sigesbeckia glabrescens Makino (SG) is one of the important plant origins of Sigesbeckiae herba and has been widely used for the treatment of chronic inflammatory diseases in China. However, the underlying anti-inflammatory mechanism of SG is rarely investigated and reported. There are more than 40 kinds of chemical constituents in SG, but the action of the bioactive compounds of SG is still unclear. Therefore, we aimed to systemically investigate the mechanisms behind the anti-inflammatory properties of SG by combining in vitro and in silico investigations.

Methods: Cytotoxicity was measured using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Nitric oxide (NO) release was detected using the Griess assay. The secretion of pro-inflammatory cytokines and the expression of relevant proteins were assessed using ELISA kits and Western blots, respectively. Molecular docking was performed and scored using AutoDock via a comparison with the molecular docking of N-acetyl-d-glucosamine (NAG).

Results: In lipopolysaccharides (LPS)-stimulated macrophages, SG significantly inhibited NO, MCP-1, and IL-6 secretion; iNOS expression; and NF-κB activation but did not significantly affect MAPK signalling (p38, ERK, and JNK). Moreover, the results from the molecular docking prediction suggested that over 10 compounds in SG could likely target TLR4, p105, and p65.

Conclusions: These findings suggest that the anti-inflammatory effects of SG are highly related to the inactivation of NF-κB. Moreover, this study provides a novel approach to investigate the effects of herbal medicine using combined in vitro and in silico investigations.

Keywords: Docking; Inflammation; Lipopolysaccharide; NF-κB; Sigesbeckia glabrescens Makino; TLR4.

Fig. 1

Quantitative analysis of kirenol and darutoside in Sigesbeckia glabrescens Makino (SG). HPLC chromatograms of a SG extract (1 mg/mL) and b mixed standards (7.5 μg/mL of kirenol and darutoside). 1: kirenol; 2: darutoside. c Chemical structure of kirenol. d Chemical structure of darutoside. RAW 264.7 cells were pretreated with e kirenol (0, 25, 50, and 100 μg/mL) or f darutoside (0, 25, 50, and 100 μg/mL) for 4 h followed by the addition of lipopolysaccharides (LPS, 1 μg/mL) for 12 h. Nitric oxide (NO) release was detected using the Griess reagent

Fig. 2

Effects of Sigesbeckia glabrescens Makino (SG) on a cell density, b cell viability, and c cell cytotoxicity. RAW 264.7 cells were treated with SG (0, 50, 100, 200, 400, and 800 μg/mL) for 24 h. *P < 0.05 vs. control, ns stands for not statistically significant

Fig. 3

Effects of Sigesbeckia glabrescens Makino (SG) on cell proliferation and cell cytotoxicity. a RAW 264.7 cells were treated with SG (0, 25, 50, 100, 200, and 400 μg/mL) for 6 days. Cell proliferation was determined with the CFDA-SE probe using flow cytometry. b Statistical results of a. c RAW 264.7 cells were pretreated with SG (0, 25, 50, and 100 μg/mL) for 4 h followed by the addition of lipopolysaccharides (LPS, 1 μg/mL) for 12 h. Indomethacin (IND) was selected as a positive drug. *P < 0.05 vs. control

Fig. 4

Effects of Sigesbeckia glabrescens Makino (SG) on the secretions of a nitric oxide (NO), b IL-6, c MCP-1, and d TNF-α. RAW 264.7 cells were pretreated with SG (0, 25, 50, 100 or 200 μg/mL) for 4 h followed by the addition of lipopolysaccharides (LPS, 1 μg/mL) for 12 h. Indomethacin (IND) was selected as a positive drug. *P < 0.05 vs. LPS-induced cells, **P < 0.01 vs. LPS-induced cells, ***P < 0.001 vs. LPS-induced cells, and ns stands for not statistically significant

Fig. 5

Effects of Sigesbeckia glabrescens Makino (SG) on the relevant pathways. RAW 264.7 cells were pretreated with SG (0, 25, 50, and 100 μg/mL) for 4 h followed by the addition of lipopolysaccharides (LPS, 1 μg/mL) for 12 h. iNOS pathway (a) and the timing assay (b) for the MAPK pathway (c) were evaluated by Western blotting

Fig. 6

Effects of Sigesbeckia glabrescens Makino (SG) on the relevant pathways. RAW 264.7 cells were pretreated with SG (0, 25, 50, 100 or 200 μg/mL) for 4 h followed by the addition of lipopolysaccharides (LPS, 1 μg/mL) for 12 h. The NF-κB pathway was investigated by a, b Western blotting (p-p65, Ser536) and c a dual-luciferase reporter assay. Indomethacin (IND) was selected as a positive drug. *P < 0.05 vs. LPS-induced cells, **P < 0.01 vs. LPS-induced cells, and ***P < 0.001 vs. LPS-induced cells