| Objective:To investigate the effect of Ghrelin on function and morphology of ox-LDL-stimulated human umbilical vein endothelial cells (HUVECs), as well as the possible mechanism,which may have important implications concerning Ghrelin as a potential therapy for atherosclerosis.Methods:1. Endothelial cells were isolated from human umbilical cord veins and cultured in Medium 199 with 20% fetal bovine serum (FBS). After four to five serial passages, the HUVECs were analyzed by Wright's-Giemsa staining and immunohistochemical staining using an antibody to human anti-factor VIII-related antigen2. To investigate the influence of dose and time, HUVECs were separately divided into four groups. HUVECs were incubated for 24 hours, and then both endothelial cells and culture supernatants were collected to determine cell apoptosis, levels of caspase-3 , NO, IL-6, IL-8 and TNF-αby Hoechest 33258 staining, Western-blot , nitrate reductase assay ,ELISA assay respectively. The total RNA were extracted from the cells and detected by semi-quantitative RT-PCR method using the specific primer for intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1).Results:1. The antagonist effect of Ghrelin on ox-LDL-induced HUVECs apoptosis ①As shown in Hoechst 33258 staining assay, compared with the control group, the number of cells with nuclear condensation induced by ox-LDL (50mg/l) increased significantly. Pre-treatment with different concentrations (1, 10 or 100 ng/mL) of Ghrelin caused a decreased apoptotic proportion by 30%, 44%, 56%, respectively, compared with ox-LDL group (all p<0.05). The apoptotics proportion of the HUVECs pretreated with 10ng/mL Ghrelin for different times (1, 6, 12 hours) was lowered by 40%, 60%, 72%, respectively, in comparison with ox-LDL-treated group (all p<0.05).②Compared with the control group, expression of caspase-3 induced by ox-LDL (50mg/l) increased significantly. The expression levels with different concentrations (1, 10 or 100 ng/mL) of Ghrelin was decreased by 17%, 23%, 45%, respectively, and with 10ng/mL Ghrelin for different times (1, 6, 12 hours) was lowered by 32%, 47%, 52%, respectively, between each group the differences were significant (p<0.01 or 0.05).③As shown in nitrate reductase assay, compared with the control group, the NO production induced by ox-LDL (50mg/l) increased significantly (p<0.01). Pre-treatment with different concentrations (1, 10 or 100 ng/mL) of Ghrelin caused an increased NO production by 25%, 54%, 85%, respectively, and 30%, 46%, 68% respectively for different times (1, 6, 12 hours), compared with ox-LDL group (p<0.05).2. The inhibitory effect of Ghrelin on ox-LDL-mediated inflammatory cytokines expression in HUVECs①Compared with the control group, the expression levels of IL-6 in HUVECs treated with ox-LDL were significantly increased (p<0.01), which was notably inhibited by 19%, 35% and 60% decrease with different concentrations (1, 10 or 100 ng/mL) of Ghrelin, and by 36%, 45% and 56% decrease with different times (1, 6, 12 hours), between each group the differences were significant (p<0.01 or 0.05).②Compared with the control group, the expression levels of IL-8 in ox-LDL group were significantly increased (p<0.01). Pre-treament of Ghrelin significantly decreased the ox-LDL induced IL-8 expression by 31%, 52% and 84% with different concentrations (1, 10 or 100 ng/mL) of Ghrelin, and by 56%, 68% and 79% with different times (1, 6, 12 hours), between each group the differences were significant (p<0.01 or 0.05).③The treatment with ox-LDL induced the expression of TNF-αin control HUVEC cells (p<0.01). Pre-treament of Ghrelin significantly inhibit the TNF-αexpression by 7%, 19% and 30% decrease with different concentrations (1, 10 or 100 ng/mL) of Ghrelin (p<0.05), and by 18%, 28% and 33% with different times (1, 6, 12 hours), and there was no difference between 6 and 12 hours (P>0.05).3. The inhibitory effect of Ghrelin on ox-LDL-mediated adhesion molecule expression in HUVECs①The results of semi-quantitative RT-PCR showed that ox-LDL could increase the expression of VCAM-1 in control HUVEC (p<0.01), and the decrease in Ghrelin-pretreated HUVECs were 33%, 52% and 63% respectively with different concentrations (1, 10 or 100 ng/mL) of Ghrelin, compared with ox-LDL group, and 48%, 65% and 72% with different times (1, 6, 12 hours), but there was no difference between 10 and 100 ng/mL, 6 and 12 hours (P>0.05).②ox-LDL significantly increased ICAM-1 expression in HUVECs, compared with control group (p<0.01), which could be inhibited by pre-treatment of Ghrelin (p<0.05). The decrease in Ghrelin-pretreated group were 18%, 46% and 57% respectively with different concentrations (1, 10 or 100 ng/mL) of Ghrelin, and 48%, 63% and 69% with different times (1, 6, 12 hours), and no difference was observed between 10 and 100 ng/mL, 6 and 12 hours (P>0.05).Conclusion Ghrelin can inhibit apoptosis and dysfunction of HUVECs by ox-LDL inducted. May be relevant with up-regulated expression of caspase-3 and down-regulated NO production。Ghrelin can inhibit IL-6,IL-8,TNF-α,VCAM-1and ICAM-1 expression of HUVECs by ox-LDL inducted. |
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