Anti-Inflammatory Effects and Mechanisms of Dandelion in RAW264.7 Macrophages and Zebrafish Larvae

Abstract

Dandelions (Taraxacum spp.) play an important role in the treatment of inflammatory diseases. In this study, we investigated the anti-inflammatory effects of Dandelion Extract (DE) in LPS-induced RAW264.7 macrophages and copper sulfate (CuSO₄)-induced zebrafish larvae. DE was not toxic to RAW264.7 cells at 75 μg/ml as measured by cell viability, and DE inhibited LPS-induced cell morphological changes as measured by inverted microscopy. In survival experiments, DE at 25 μg/ml had no toxicity to zebrafish larvae. By using an enzymatic standard assay, DE reduced the production of nitric oxide (NO) in LPS-induced RAW264.7 cells. Fluorescence microscopy results show that DE reduced LPS-induced ROS production and apoptosis in RAW264.7 cells. DE also inhibited CuSO4-induced ROS production and neutrophil aggregation in zebrafish larvae. The results of flow cytometry show that DE alleviated the LPS-induced cell cycle arrest. In LPS-induced RAW264.7 cells, RT-PCR revealed that DE decreased the expression of M1 phenotypic genes iNOS, IL-6, and IL-1β while increasing the expression of M2 phenotypic genes IL-10 and CD206. Furthermore, in CuSO4-induced zebrafish larvae, DE reduced the expression of iNOS, TNF-α, IL-6, and IL-10. The findings suggest that DE reduces the LPS-induced inflammatory response in RAW264.7 cells by regulating polarization and apoptosis. DE also reduces the CuSO4-induced inflammatory response in zebrafish larvae.

Keywords: M1/M2 subtype; RAW264.7 cells; apoptosis; dandelion extract (DE); inflammation; zebrafish larvae.

FIGURE 1

Effect of DE on the activity of RAW264.7 cells. Cells were treated with DE (0–300 μg/ml) for 36 h, and cell viability was measured by the cell viability assay. Compared with the control group,^### p < 0.001.

FIGURE 2

Effect of DE on the morphology of RAW264.7 cells after LPS induction (×200). Cells were pretreated with DE (18.75, 37.5, and 75 μg/ml) for 2 h and then stimulated with LPS (500 ng/ml) for another 24 h. Changes in cell morphology were observed with an inverted microscope.

FIGURE 3

NO production after LPS stimulation and after treatment with DE. Cells were pre-stimulated with different concentrations of LPS for 24 h. Cell supernatant NO production was measured using an enzyme marker (A). Cells were pretreated with DE for 2 h and then stimulated with LPS (500 ng/ml) for another 24 h. Cell supernatant NO production was measured using an enzyme marker (B). Compared with the control group, ^### p < 0.001; compared with the LPS group,***p < 0.001.

FIGURE 4

DE reduced ROS production after LPS stimulation (×40). Cells were pretreated with DE for 2 h and then stimulated with LPS (500 ng/ml) for another 3 h or 24 h. Fluorescence microscopy was used to take pictures (A) and the fluorescence intensity of ROS for 3 h (B) or 24 h (C) was obtained by analysis with Image J software. Compared with the control group, ^### p < 0.001; compared with the LPS group, ***p < 0.001.

FIGURE 5

Effect of DE on the LPS-induced RAW264.7 cell cycle. Cells were treated with DE with or without LPS (500 ng/ml) co-treatment for 6 h, 12 h, 24 h, 48 h, 72 h, and the cell viability was measured by the cell viability assay (A). The cell cycle was detected by flow cytometry at 6 h and 24 h (B), and the cycle changes were analyzed at 6 h (C) and 24 h (D). Compared with the control group, ^### p < 0.001; compared with the LPS group,***p < 0.001.

FIGURE 6

DE reduction of apoptosis in RAW264.7 cells after LPS stimulation (×100). Cells were pretreated with DE (18.75, 37.5, and 75 μg/ml) for 2 h and then stimulated with LPS (500 ng/ml) for another 3 h or 24 h. Cells were observed using a fluorescent microscope (A); fluorescence intensity recorded by flow cytometry (B); analysis of fluorescence intensity using Image J software(C). Analysis of the proportion of fluorescence intensity (D) and the average fluorescence intensity (E). Compared with the control group, ^### p < 0.001; compared with the LPS group, ***p < 0.001, **p < 0.01.

FIGURE 7

Modulation of LPS-stimulated polarization of RAW264.7 cells by DE. Cells were pretreated with DE (4.69, 9.38, 18.75, 37.5, and 75 μg/ml) for 2 h and then stimulated with LPS (500 ng/ml) for another 36 h. The expression levels of M1 phenotype genes (IL-1β, IL-6, and iNOS) (A–C), and M2 phenotype genes (CD206, IL-10) (D,E) were obtained by RT-PCR. Compared with the control group, ^### p < 0.001, ^# p < 0.05; compared with the LPS group, ***p < 0.001, **p < 0.01.

FIGURE 8

DE reduces neutrophil aggregation and ROS production in zebrafish larvae. Zebrafish larvae from 3 dpf were pretreated with DE for 1 h and then stimulated with CuSO₄ (10 μM) for a further 40 min. Fluorescence intensity of neutrophils and ROS were recorded by fluorescence microscopy (A) and analyzed using Image J software for neutrophil aggregation (B) and ROS production (C). Compared with the control group, ^### p < 0.001; compared with the CuSO₄ group, ***p < 0.001, **p < 0.01.

FIGURE 9

DE reduces the expression of inflammatory factors in zebrafish larvae. Zebrafish larvae from 3 dpf were pretreated with DE in culture for 1 h and then stimulated with CuSO₄ (10 μM) for a further 40 min. The total mRNA was extracted and RT-PCR was performed to analyse iNOS, TNF-α, IL-6, and IL-10 gene expression. β-Actin was used as an internal control (A–D). Compared with the control group, ^### p < 0.001; compared with the CuSO₄ group, ***p < 0.001.