Effects And Mechanisms Of Ghrelin On Cardiac Microvascular Endothelial Cells In Rats

Background and aimsGhrelin, a 28 amino acid acylated peptide released from the stomach, is discovered by a Japanese scientist. It is an endogenous ligand for growth hormone secretagogue receptor (GHSR), which includes two subtypes: GHSR-1a and GHSR-1b. Recent researches mainly focus on GHSR-1a, and there is few researches about GHSR-1b. GHS-R are expressed in the hypothalamus, pituitary gland, adrenal gland, stomach and intestinal tract and exert various physiological functions including regulating growth hormone (GH) secretion, stimulating prolactin, ACTH and cortisol secretion, enhancing food intake, promoting sleep and immunity, etc. Recently, GHS-R has been discovered to be highly expressed in cardiovascular system. Moreover, ghrelin was found to have direct effects on protecting cardiovascular system, including inhibiting apoptosis of vascular endothelial cell, promoting angiogenesis, and suppressing vascular inflammation reaction.Cardiac microvascular endothelial cells are one of the most important cell types with the secretion function, which are able to produce physiological functions such as regulating vascular metabolism and pharmaceutical response, as well as involving in angiogenesis. Moreover, cardiac microvascular endothelial cells injuries are the essential joint of atherosclerosis and vascular lesion in diabetes mellitus. Therefore medications that protect the morphology and function of cardiac microvascular endothelial cell may decrease atherosclerosis and vascular lesion of diabetes mellitus.our study intends to explore the effects of ghrelin on proliferation, migration and NO secretion of rat cardiac microvascular endothelial cells and its possible mechanisms.MethodsPartⅠ: Primary culture and identification of cardiac microvascular endothelial cell. 1. Cardiac microvascular endothelial cells (CMEC) were isolated by enzyme dissociation method as described from adult Sprague–Dawley rat hearts. Primary cells were seeded on tissue culture plates coated with fibronectin, maintained in DMEM supplemented with 15% fetal bovine serum at 37°C and 5% CO2. Cell medium were changed every 3 days. CMECs were applied to the treatment until 80% growth confluence (typically need 5–7 days culturing). 2. Rat cardiac microvascular endothelial cells were identified by morphological characteristics with microscopy and DiI-Ac-LDL intake assayPartⅡ: Effects of ghrelin with different doses on proliferation, migration and NO secretion of CMECs. CMECs were isolated from left ventricle of adult male Sprague–Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil-ac-LDL intake assay was applied to identify the CMECs. Cells were allocated into 4 groups: control group; different ghrelin dosage groups (1×10-9mol/L, 1×10-8mol/L, 1×10-7mol/L). After 24h treatment, cell proliferation capability was measured by MTT assay and immunofluorescent test for PCNA protein expression. Migration of CMECs was detected by transwell assay, NO (nitric oxide) secretion of CMECs was measured by nitrate reduction method. PartⅢ:mechanisms of ghrelin on cardiac microvascular endothelial cells. CMECs were allocated into 3 groups: A control group;B 1×10-7mol/L ghrelin group;C 1×10-7mol/L ghrelin+ 20 mol/L LY294002 group. After 24h treatment, cell proliferation capability was measured by MTT assay and immunofluorescent test for PCNA protein expression. Migration of CMECs was detected by transwell assay, NO (nitric oxide) secretion of CMECs was measured by nitrate reduction method. Protein expression of AKT and phosphorylated AKT of CMECs were measured by western blot after exposure to various concentrations of ghrelin and PI3K inhibitor LY294002.Data are presented as mean±S.D. Data were compared using standard or repeated measures ANOVA where appropriate. Pairwise comparisons were performed using a two-tailed Student's t–test. Differences were considered significant for P-values<0.05.Results:1. After cultured for 5 days, all cells assumed as typical confluent cobblestone appearance; more than 90% cells had showed positive reactions for the intake of Dil-ac-LDL.2. 10-8-10-7mol/L ghrelin promoted proliferation and migration of CMECs as well as increasing NO secretion of CMECs.3. 20 mol/L LY294002 inhibited ghrelin's effect on proliferation and migration of CMECs as well as increasing NO secretion of CMECs. Conclusion1.Ghrelin can promote proliferation and migration of CMECs as well as increasing NO secretion of CMECs at some dosage.2.LY294002 could inhibit ghrelin's effect on proliferation and migration of CMECs as well as increasing NO secretion of CMECs.