Chlorella 11-peptide inhibits the production of macrophage-induced adhesion molecules and reduces endothelin-1 expression and endothelial permeability

Abstract

The inflammation process in large vessels involves the up-regulation of vascular adhesion molecules such as endothelial cell selectin (E-selectin), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) which are also known as the markers of atherosclerosis. We have reported that Chlorella 11-peptide exhibited effective anti-inflammatory effects. This peptide with an amino sequence Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe was further examined for its potential in preventing atherosclerosis in this study. In particular, the roles of Chlorella 11-peptide in lowering the production of vascular adhesion molecules, monocyte chemoattractant protein (MCP-1) and expression of endothelin-1 (ET-1) from endothelia (SVEC4-10 cells) were studied. The production of E-selectin, ICAM-1, VCAM-1 and MCP-1 in SVEC4-10 cells was measured with ELISA. The mRNA expression of ET-1 was analyzed by RT-PCR and agarose gel. Results showed that Chlorella 11-peptide significantly suppressed the levels of E-selectin, ICAM, VCAM, MCP-1 as well as ET-1 gene expression. The inhibition of ICAM-1 and VCAM-1 production by Chlorella 11-peptide was reversed in the presence of protein kinase A inhibitor (H89) which suggests that the cAMP pathway was involved in the inhibitory cause of the peptide. In addition, this peptide was shown to reduce the extent of increased intercellular permeability induced by combination of 50% of lipopolysaccharide (LPS)-activated RAW 264.7 cells medium and 50% normal SEVC cell culture medium (referred to as 50% RAW-conditioned medium). These data demonstrate that Chlorella 11-peptide is a promising biomolecule in preventing chronic inflammatory-related vascular diseases.

Figure 1

Effects of Chlorella 11-peptide on lipopolysaccharide (LPS)-induced monocyte chemoattractant protein (MCP-1) production. RAW264.7 cells (n = 8) were treated with LPS (1 µg/mL) with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 6 h prior to MCP-1 concentration being measured. Statistics are shown for LPS-treated cells ### p < 0.005, compared to the basal; 38 µM of Chlorella 11-peptide *** p < 0.005 and 9 M of Chlorella 11-peptide and indomethacin * p < 0.05, compared to LPS-stimulated group.

Figure 2

Effects of Chlorella 11-peptide on 50% RAW-conditioned medium-induced E-selectin production. SVEC4-10 endothelial cells (n = 8) were treated with 50% RAW-conditioned medium with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 24 h prior to E-selectin concentration being measured. Statistics are shown for 50% RAW-conditioned medium-treated cells ### p < 0.005, compared to the basal; 38 µM of Chlorella 11-peptide *** p < 0.005 and 9 µM of Chlorella 11-peptide ** p < 0.01, compared to 50% RAW-conditioned medium-treated group.

Figure 3

Effects of Chlorella 11-peptide on 50% RAW-conditioned medium-induced ICAM-1 production. SVEC4-10 endothelial cells (n = 8) were treated with 50% RAW-conditioned medium with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 24 h prior to ICAM-1 concentration being measured. Statistics are shown for 9 µM and 38 µM of Chlorella 11-peptide, and indomethacin ### p < 0.005 compared to 50% RAW-conditioned medium-treated group; 38 µM of Chlorella 11-peptide+H89 and indomethacin+H89, *** p < 0.005, compared to 38 µM of Chlorella 11-peptide and indomethacin.

Figure 4

Effects of Chlorella 11-peptide on 50% RAW-conditioned medium-induced VCAM-1 production. SVEC4-10 endothelial cells (n = 8) were treated with 50% RAW-conditioned medium with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 6 h prior to VCAM-1 concentration being measured. Statistics are shown for 38 µM of Chlorella 11-peptide, *** p < 0.005 compared to 50% RAW-conditioned medium-treated group; 38 µM of Chlorella 11-peptide+H89, ### p < 0.005, compared to 38 µM of Chlorella 11-peptide.

Figure 5

Effects of Chlorella 11-peptide on 50% RAW-conditioned medium-induced endothelin-1 mRNA expression. SVEC4-10 endothelial cells (n = 8) were treated with 50% RAW-conditioned medium with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 24 h prior to total RNA extraction and PCR were performed. Statistics are shown for 50% RAW-conditioned medium-treated cells ### p < 0.005 compared to the basal; 38 µM of Chlorella 11-peptide *** P < 0.005 and indomethacin ** p < 0.01, compared to 50% RAW-conditioned medium-treated group.

Figure 6

Effects of Chlorella 11-peptide on 50% RAW-conditioned medium-induced intercellular permeability. SVEC4-10 endothelial cells (n = 8) were treated with 50% RAW-conditioned medium with and without Chlorella 11-peptide (9 and 38 µM) or indomethacin (0.25 mM) for 24 h prior to E-selectin concentration being measured. Statistics are shown for 50% RAW-conditioned medium-treated cells ### p < 0.005, compared to the basal; 9 µM and 38 µM of Chlorella 11-peptide *** p < 0.005, compared to 50% RAW-conditioned medium-treated group.