Epimedium sagittatum inhibits TLR4/MD-2 mediated NF-κB signaling pathway with anti-inflammatory activity

Abstract

Background: Epimedium sagittatum (Sieb.et Zucc.) Maxim., Ying-Yang-Huo in Chinese has been used as a traditional Chinese medicine and is deemed to "reinforce the kidney Yang". Previous studies showed that E. sagittatum could modulate the immune system and treat some chronic disease such as rheumatic arthritis, cardiovascular diseases and osteoporosis. The aim of this study is to evaluate the anti-inflammatory effects of ethyl acetate extracts (YYHs) of E. sagittatum and its mechanisms of action.

Methods: In order to explore the composition of YYHs, YYHs was analyzed using high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS/MS) and in comparison with reference standards. Anti-inflammatory model was established in LPS-induced RAW264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Production of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were measured by enzyme-linked immunosorbent assays (ELISA). In addition, expression of p-p65 protein and TLR4/MD-2 complex was detected by western blots and flow cytometric, respectively. Nuclear factor kappa B (NF-κB) nuclear translocation was observed by fluorescence microscope.

Results: A total of eight compounds were identified, of which icariside II was the most abundant compound. YYHs (12.5-50 μg/mL) had no obvious cytotoxic effect on cells, and remarkably inhibited LPS-induced production of NO, TNF-α and IL-2 with a dose-dependent manner. Additionally, YYHs up-regulated expression of p-p65 and TLR4/MD-2 complex. Further research showed that YYHs significantly suppressed NF-κB p65 nuclear translocation.

Conclusion: In brief, YYHs contributed to the inhibition of LPS-induced inflammatory response through the TLR4/MD-2-mediated NF-κB pathway and may be a potential choice to combat inflammation diseases. It includes a schema of pathways at the end of the paper.

Keywords: Anti-inflammation; E, sagittatum; NF-κB; TLR4/MD-2.

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Figures

Fig. 1

Representative HPLC chromatogram of YYHs at 270 nm

Fig. 2

Cytotoxicity of YYHs on RAW 264.7 cells. Cells were treated in the presence of YYHs or in combination with LPS (1 μg/mL) for 24 h. Cell viability was determined by MTT assay (black bar, YYHs treated; white bar, YYHs+LPS treated). Compared with the control, #P < 0.05, ##P < 0.01. Compared with LPS group, *P < 0.05, **P < 0.01

Fig. 3

Effect of YYHs on TNF-α (a), IL-2 (b) and NO (c) production in LPS-induced RAW 264.7 cells. Cells were pretreated with YYHs (12.5–50 μg/mL) and LPS for 24 h and collected culture supernatant. The culture supernatant was detected to ELISA kits and Griess reagent, respectively. Compared with the control, #P < 0.05, ##P < 0.01. Compared with LPS group, *P < 0.05, **P < 0.01

Fig. 4

Flow cytometric analysis for TLR4/MD-2 complex. Cells were treated with YYHs and LPS (1 μg/mL) for 18 h. And TLT4/MD-2 complex was detected with FCM by PE conjugated mAb TLR4/MD-2 complex antibody (a, blank group; b, LPS group; c-e, 12.5–50 μg/mL YYHs group). Compared with the control, #P < 0.05, ##P < 0.01. Compared with LPS group, *P < 0.05, **P < 0.01

Fig. 5

Effect of YYHs on NF-κB activation in RAW 264.7 cells. The cells were seeded in a 6-well culture plate, and treated with YYHs for 1 h and followed by stimulated with LPS for another 1 h. After treatment, fixed cells were incubated with p65 antibody and Cy3-conjugated secondary antibody, and nuclei were stained with DAPI. The images were obtained by fluorescence microscopy and overlay (Control, untreated cells; LPS, LPS (1 μg/ml) only; YYHs+LPS, YYHs (50 μg/mL) + LPS)

Fig. 6

Effect of YYHs on LPS-induced activation of NF-κB pathway in RAW 264.7 cells. The cells were pretreated with different concentrations of YYHs for 2 h and then stimulated with LPS(1 μg/mL) for another 1 h. The NF-κB p65 and phosphorylated p65 in the total protein was detected by western blotting. β–acting was used as the internal control for normalization. Compared with the control, #P < 0.05, ##P < 0.01. Compared with LPS group, *P < 0.05, **P < 0.01