Ghrelin suppresses inflammation in HUVECs by inhibiting ubiquitin-mediated uncoupling protein 2 degradation

Abstract

Atherosclerosis is considered the major cause of heart attack, stroke and gangrene of the extremities, which is responsible for 50% of all mortality in Western countries. The pathogenesis and causes of atherosclerosis remain elusive. Recent studies highlight inflammation as a contributing factor for atherosclerosis in all stages of the disease process. In this study, we demonstrate that the treatment of human umbilical vein endothelial cells (HUVECs) with ghrelin inhibits the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response, In addition, treatment with ghrelin led to the accumulation of uncoupling protein 2 (UCP2) in the cells, thus decreasing reactive oxygen species (ROS) generation. Moreover, the siRNA-mediated knockdown of UCP2 expression suggested that the inhibitory effects of ghrelin on the inflammatory response relied on its ability to induce the accumulation of cellular UCP2 levels. Further analysis indicated that the accumulation of UCP2 in the ghrelin-treated cells was due to the ability of ghrelin to inhibit the ubiquitination of UCP2 and prevent UCP2 degradation, resulting in the extended protein half-life of UCP2. On the whole, our data indicate that ghrelin inhibits the oxLDL-induced inflammatory response in HUVECs, and may thus have potential for use as an anti-atherosclerotic agent. Our data may also provide valuable insight into the pathogenesis of atherosclerosis.

Figure 1

Ghrelin inhibits the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were either pre-treated with ghrelin or left untreated, followed by oxLDL stimulation. RT-qPCR was conducted to determine the mRNA levels of IL-6, CCL-2, ICAM-1 and VCAM-1. (B) HUVECs were pre-treated with gradiatint concentrations of ghrelin followed by oxLDL stimulation. RT-qPCR was conducted to monitor the mRNA levels of IL-6, CCL-2, ICAM-1 and VCAM-1. Quantitative experiments were repeated at least 3 times. ^*P<0.05, significant differences between different groups.

Figure 2

Ghrelin upregulates uncoupling protein 2 (UCP2) to decrease reactive oxygen species (ROS) generation. (A) Human umbilical vein endothelial cells (HUVECs) were either pre-treated with ghrelin or left untreated, followed by oxidized low-density lipoprotein (oxLDL) stimulation. The cellular ROS level was measured in indicated groups. (B) Western blot analysis for the UCP2 protein level in HUVECs treated with ghrelin. Untreated cells was included as a control. (C) Gradient treatment of HUVECs with ghrelin for the observed the dose-depended upregulation of UCP2 at the protein level. Quantitative experiments were repeated at least 3 times. ^*P<0.05, significant differences between different groups.

Figure 3

siRNA-mediated knockdown of uncoupling protein 2 (UCP2) expression antagonizing ghrelin caused reactive oxygen species (ROS) reduction. (A) Western blot analysis for UCP2 protein level in human umbilical vein endothelial cells (HUVECs) subjected to the indicated treatments. Cells were transfected with siRNA first, and then ghrelin was added to the siRNA-transfected cells. Cells from indicated groups were harvested 24 h later and examined to determine the UCP2 protein level. (B) Quantitative analysis of the ROS level in the indicated groups. (C) The ICAM-1 mRNA level in cells from indicated groups. Experiments were repeated 3 times. ^*P<0.05, significant differences between different groups.

Figure 4

Ghrelin extends the protein half-life of uncoupling protein 2 (UCP2) without affecting the UCP2 mRNA level. (A) Treatment with ghrelin did not alter the mRNA level in human umbilical vein endothelial cells (HUVECs). (B) UCP2 half-life assay. Cells were treated with ghrelin for 24 h first, then cycloheximide was added to the cells to block mRNA translation. Both the ghrelin-treated or untreated cells were harvested at different time points to monitor the changes in the UCP2 level for the determination of the UCP2 half-life. Quantitative experiments were repeated 3 times. ^*P<0.05, significant differences between different groups.

Figure 5

Ghrelin prevents uncoupling protein 2 (UCP2) degradation by inhibiting UCP2 ubiquitination. (A) Ubiquitination assay of UCP2: human umbilical vein endothelial cells (HUVECs) treated with ghrelin for 24 h, and cells were then lysed for immunoprecipitation (IP) by anti-UCP2 antibody. Anti-ubiquitin antibody was used for probing the ubiquitination of UCP2. (B) Western blot analysis of the total ubiqutination level in HUVECs; ghrelin untreated cells were used as controls.