Ghrelin inhibits AngII -induced expression of TNF-α, IL-8, MCP-1 in human umbilical vein endothelial cells

Abstract

Aim: Ghrelin, a gastric peptide, is involved in several metabolic and cardiovascular processes. Emerging evidence indicates the potential involvement of ghrelin in low-grade inflammatory diseases such as atherosclerosis and hypertension. Cytokine-induced inflammation is critical in these pathological states. The growth hormone secretagogue receptor (GHSR) has been identified in blood vessels, so we predict that ghrelin might inhibit proinflammatory responses in human umbilical vein endothelial cells (HUVECs). The aim of this study is to examine the effect of ghrelin on angiotension II (AngII)-induced expression of TNF-α, MCP-1, IL-8 in HUVECs.

Method: HUVECs were pretreated with ghrelin, with or without the specific antagonist of GHSR [D-Lys(3)]-GHRP-6, the selective inhibitor of nuclear factor-kappaB (NF-κB) PDTC, and the selective inhibitor of extracellular signal-regulated kinase (ERK1/2) PD98059. The cells were finally treated with AngII. The expression of TNF-α, MCP-1, IL-8 was examined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The activity of ERK1/2 and NF-κB was analyzed by Western blot.

Result: our study showed that ghrelin inhibited AngII -induced expression of IL-8, TNF-α and MCP-1 in the HUVECs via GHSR pathway in concentration- and time-dependent manners. We also found that ghrelin inhibited AngII -induced activation of ERK1/2 and NF-κB.

Conclusions: these results suggest that Ghrelin may play novel antiinflammatory and immunoregulatory roles in HUVECs.

Keywords: Ghrelin; NF-κB and MAPK pathways; atherosclerosis; inflammation; interleukin-8; monocyte chemoattractant protein-1; tumor necrosis factor-α.

Figure 1

The effect of ghrelin on AngII-induced mRNA expression of IL-8 (A), MCP-1 (B) and TNF-α(C). Difference between groups was analyzed by one-way ANOVA, *compared with control, P < 0.05; **compared with control, P < 0.01.

Figure 2

The effect of different inhibitors on the expression of IL-8 (A), MCP-1 (B) and TNF-α (C) mRNAs. AngII: 10^-6 mol/L; ghrelin: 10^-6 mol/L; [d-Lys³] GHRP: 625 μmol/L; PDTC: 1 μmol/L; PD98059: 25 μmol/L. *Compared with control, P < 0.05; #compared with AngII, P < 0.05; ◊compared with AngII + ghrelin or AngII + PD98059, P < 0.05.

Figure 3

The effect of ghrelin (A) and different inhibitors (B) on AngII-induced IL-8, MCP-1 and TNF-α protein expression in the culture medium. HUVECs were pretreated with 10, 100, 1000 nmol/L ghrelin, or 1000 nmol/L ghrelin + 25 μmol/L [Dys3] GHRP6 for 1 h, then treated with AngII (10^-6 mol/L) for 24 h. The concentration of TNF-α, IL-8 and MCP-1 was measured by ELISA. *Compared with control, P < 0.05; #compared with AngII, P < 0.05, ◊compared with AngII + G1000, P < 0.05. Data were presented as mean ± SD (n=4).

Figure 4

The effect of AngII and ghrelin on NF-κB and ERK1/2 activation. A: AngII activates NF-κB in a time-dependent manner. *Compared with 0 min group, P < 0.05. B: AngII activates ERK1/2 in a time-dependent manner. *compared with 0 min group, P < 0.05. C: Ghrelin inhibits ERK activation in a time-dependent manner. 1, AngII (10^-6 mol/L); 2-6, ghrelin (10^-6 mol/L) pretreatment for 5, 10, 20, 30 and 60 min, then treated with AngII. *compared with AngII group, P < 0.05. D: Ghrelin inhibits NF-κB activation in a time-dependent manner. 1, AngII (10^-6 mol/L); 2-6, ghrelin (10^-6 mol/L) pretreatment for 5, 10, 20, 30 and 60 min, then treated with AngII. *Compared with AngII group, P < 0.05.