Dauricine Attenuates Vascular Endothelial Inflammation Through Inhibiting NF-κB Pathway

Abstract

Endothelial cells are the fundamental components of blood vessels that regulate several physiological processes including immune responses, angiogenesis, and vascular tone. Endothelial dysfunction contributes to the development of various diseases such as acute lung injury, and endothelial inflammation is a vital part of endothelial dysfunction. Dauricine is an extract isolated from Menispermum dauricum DC, a traditional Chinese medical plant that can be used for pharyngitis. In this work, we found that IL-1β-induced overexpression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin was inhibited by dauricine in primary human umbilical vein endothelial cells (HUVECs). Correspondingly, adhesion of human acute monocytic leukemia cell line (THP-1) to HUVECs was decreased by dauricine. Further studies showed that dauricine inhibited the activation of nuclear factor-κB (NF-κB) pathway in HUVECs stimulated with IL-1β. In vivo, dauricine protected mice from lipopolysaccharide (LPS)-induced acute lung injury. In lung tissues, the activation of NF-κB pathway and the expression of its downstream genes (ICAM-1, VCAM-1, and E-selectin) were decreased by dauricine, consistent with what was found in vitro. In summary, we concluded that dauricine could alleviate endothelial inflammation by suppressing NF-κB pathway, which might serve as an effective candidate for diseases related with endothelial inflammation.

Keywords: NF-κB pathway; acute lung injury; dauricine; endothelial dysfunction; inflammation.

FIGURE 1

Chemical structure and cytotoxicity induced by dauricine. (A) Chemical structure of dauricine. (B) Dauricine was safe to use for HUVECs at concentrations of 0–40 μM, as determined by MTT assay (n = 5). HUVECs, human umbilical vein endothelial cells.

FIGURE 2

Dauricine inhibited monocyte–endothelial cell interaction. (A) Representative images of THP-1 cells (green) that adhered to HUVECs (scale bar: 100 μm). (B) Quantitative analysis of THP-1 cells that adhered to HUVECs. Data are expressed as mean ± SD. ###p < 0.001 versus the vehicle group; ***p < 0.001 versus the vehicle + IL-1β group. DMSO, dimethyl sulfoxide; IL-1β, interleukin-1β; HUVECs, human umbilical vein endothelial cells.

FIGURE 3

Dauricine decreased the expression of ICAM-1, VCAM-1, and E-selectin in HUVECs. (A) Relative mRNA expression levels of ICAM-1, VCAM-1, and E-selectin in HUVECs treated with IL-1β and dauricine by quantitative PCR (qPCR). (B) The protein levels of ICAM-1, VCAM-1, and E-selectin in HUVECs by Western blotting. (C) Quantification of the relative expression level of proteins in panel B. Data are expressed as mean ± SD. ###p < 0.001 versus the vehicle group; ***p < 0.001, **p < 0.01, *p < 0.05 versus the vehicle + IL-1β group. HUVECs, human umbilical vein endothelial cells; IL-1β, interleukin-1β.

FIGURE 4

Dauricine inhibited IL-1β-induced NF-κB activation. (A) Phosphorylation of p65 and IκBα in HUVECs treated with IL-1β and dauricine at different concentrations. (B) Quantification of the phosphorylation of p65 and IκBα displayed in panel A. ##p < 0.01, #p < 0.05 versus the vehicle group; *p < 0.05 versus the vehicle + IL-1β group. (C) Nuclear translocation of p65, as indicated by the immunofluorescence in HUVECs treated with IL-1β and dauricine (scale bar: 200 μm). DAPI was used for nuclear staining (blue). P65 (green) was stained with the Alexa Fluor 488 secondary antibody. (D) Cytoplasmic and nuclear distribution of p65 in HUVECs as detected by Western blotting analysis. IL-1β, interleukin-1β; HUVECs, human umbilical vein endothelial cells.

FIGURE 5

Dauricine suppressed NF-κB activation-induced expression of ICAM-1, VCAM-1, and E-selectin. (A) Dauricine inhibited ICAM-1, VCAM-1, and E-selectin promoter-derived luciferase activity individually in 293T cells cotransfected with a p65-overexpressing vector. ###p < 0.001 versus the VCAM-1, ICAM-1, E-selectin-Luc + empty vector + TK groups; ***p < 0.001, **p < 0.05 and *p < 0.05 versus the VCAM-1, ICAM-1, E-selectin-Luc + p65-expressing vector + vehicle + TK groups. (B) Dauricine reduced NF-κB p65 binding to its downstream gene promoters, including the ICAM-1, VCAM-1, and E-selectin promoters, as detected by ChIP assay. ChIP, chromatin immunoprecipitation.

FIGURE 6

Dauricine attenuated LPS-induced acute lung injury in vivo. (A) Survival curves for mice treated with LPS and dauricine (20 mg/kg), n = 10/group. (B) Protein levels of ICAM-1, VCAM-1, and E-selectin and the phosphorylation of IκBα and p65 in lung tissues. (C) Quantification analysis of the protein levels in panel B. ###p < 0.001, ##p < 0.01, #p < 0.05 versus the vehicle group; **p < 0.01, *p < 0.05 versus the vehicle + LPS group. (D) Level of endothelial cell-expressed ICAM-1 in lung tissues, as detected by immunofluorescence assay (scale bar: 20 μm). DAPI was used for nucleus staining (blue), ICAM-1 was stained red, and CD31 was stained green. (E) Nuclear translocation of p65 in endothelial cells as detected by immunofluorescence assay in lung tissues (scale bar: 10 μm). DAPI was used for nucleus staining (blue), p65 was stained red, and CD31 was stained green. (F) Quantification analysis of ICAM-1 in panel D. ##p < 0.01 versus the vehicle group; *p < 0.05 versus the vehicle + LPS group. (G) Quantification analysis of nucleic p65 in panel E. ###p < 0.001 versus the vehicle group; *p < 0.05 versus the vehicle + LPS group. LPS, lipopolysaccharide.

FIGURE 7

Schematic illustration of the anti-inflammatory effect of dauricine. Proinflammatory stimulus such as LPS and IL-1β increased the phosphorylation and nuclear translocation of the NF-κB p65 subunit, which resulted in the upregulation of adhesion molecules and many other inflammatory cytokines that could aggravate vascular inflammation. Dauricine showed an inhibition of NF-kB pathway and downregulated the expression of its downstream genes to attenuate endothelial inflammation. IL-1β, interleukin-1β; LPS, lipopolysaccharide.