The Roles And Mechanisms Of Ghrelin In Coronary Artery Disease

Part One:Changes of circulating ghrelin levels in elderly patients with coronary artery disease and ischemic heart failure[Objective] To investigate changes of circulating ghrelin levels in elderly coronary artery disease (CAD) patients with or without ischemic heart failure (IHF).[Methods] Plasma total ghrelin levels were measured by enzyme linked immunosorbent assay (ELISA) in 56 CAD with IHF patients, 60 CAD without IHF patients and 100 healthy elderly subjects. Serum concentrations of lipids, creatinine, urea, fasting plasma glucose (FBS) and fasting insulin (FINS) were also detected. Height, weight, waist circumference and hip circumference were measured.[Results] There were no significant differences between the CAD group and control group regarding age, sex distribution, diastolic blood pressure, creatinine, cholesterol, triacylglycerol, HDL cholesterol (HDL-C), LDL cholesterol (LDL-C) and ghrelin levels. However, CAD patients had higher rate of smoking, systolic blood pressure, body mass index (BMI), waist circumference, waist to hip ratio (WHR), urea, FBS, FINS, homeostasis model assessment of insulin resistance (HOMA-IR) and lower left ventricular ejection fraction (LVEF) compared to control subjects. As expected, compared with the CAD without IHF group, the CAD with IHF group had lower LVEF (P<0.001) and ghrelin level (P=0.027). No other significant differences were observed between these two groups.[Conclusion] The plasma total ghrelin levels are increased in CAD with IHF patients. Ghrelin may have a protective effect on cardiovascular disease.Part Two:The association of ghrelin polymorphisms with coronary artery disease and ischemic chronic heart failure in an elderly Chinese population[Objective] To investigate the association of CAD and IHF with polymorphisms of the ghrelin gene in elderly Chinese patients.[Methods] Fifty-six patients of CAD with IHF, sixty patients of CAD without IHF, and one hundred healthy control subjects participated in the study. Three common single nucleotide polymorphisms (SNP) were selected in the coding region of the preproghrelin gene:the SNPs Arg51Gln (G346A, rs34911341) and Leu72Met (C408A, rs696217) in exon 2, and the SNP GIn90Leu (A3412T, rs4684677) in exon 3. The polymorphisms were evaluated by polymerase chain reaction (PCR), sequencing, and restriction fragment length polymorphism analysis (RFLP).[Results] Only one SNP Leu72Met (C408A, rs696217) was observed across all samples. Gene frequencies of CC and allele frequencies of C were significantly greater in the CAD with IHF group than those in the CAD without IHF group (P= 0.025, P= 0.011). There was no significant association between the Leu72Met SNP with coronary artery disease risk factors.[Conclusion] Our results suggest that CC gene and C allele at position 408 of the ghrelin gene is associated with genetic susceptibility to IHF in Chinese elders.Part Three:Effects and mechanisms of ghrelin on H9c2 cells apoptosis induced by hydrogen peroxide [Objective] To investigate the protective effects of ghrelin on H9c2 cells apoptosis induced by hydrogen peroxide (H2O2) and the possible molecular mechanisms.[Methods] To study apoptosis, the cells were assessed by morphologic examination, MTS assay, Annexin?-propidium iodide dual staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis. To investigate the underlying molecular mechanisms, the expression of Bcl-2, Bax, active cysteine aspartic acid specific protease-9 (caspase-9) and nuclear factor?B (NF-?B) were assessed by western blotting, and cysteine aspartic acid specific protease-3 (caspase-3) activity was determined by a colorimetric activity assay kit.[Results] After stimulation with H2O2 for 18 h, H9c2 cells viability decreased significantly; a large fraction of cells underwent apoptosis. We observed a dose-dependent rescue of H9c2 cells from H2O2-induced apoptosis in the presence of different ghrelin concentrations. Moreover, ghrelin decreased H2O2-induced Bax production and caspase-9 activation, and increased Bcl-2 levels. NF-?B phosphorylation was also significantly inhibited by ghrelin in H2O2-treated cells. Caspase-3 activation was suppressed by ghrelin in H2O2-treated H9c2 cells in a dose-dependent manner[Conclusion] Ghrelin protects H9c2 cells from oxidative stress induced apoptosis through downregulation of Bax expression, caspase-9 activation and NF-?B phosphorylation, and upregulation of Bcl-2 expression. Caspase-3 activation was also reduced in a dose-dependent manner. These data suggest that ghrelin might protect against cardiovascular disease through NF-?B and mitochondria-mediated signaling.