| Part IEffect of ghrelin on biological activity of bone-marrow-derived endothelial progenitor cells in vitroObjective To investigate the effects of ghrelin on bone-marrow-derived endothelial progenitor cells (EPC) biological activityMethods EPC were isolated from bone marrow of Sprague Dawleyrats by using density gradient centrifugation method, and were identifiedby double positive of FITC-UEA-1binding and acLDL-Dil uptake，andimmunocytochemistry for CD34, CD133, vWF and Flk-1, EPC weretreated with various concentration of ghrelin (10-9～10-6M)， EPCsproliferation were assayed by WST-8assay, EPCs migration were assayedby Transwell chamber, tube formation were assayed on Matrigel， theconcentrations of the supernatant NO were assayed by Griess method.Results EPC were successfully obtained by density gradientcentrifugation method. Newly EPCs cultured for1days were nearlycircular, convert to Clostridium after4to7days, and cord-like arrangementfor10days of growth. FITC-UEA-1and Dil-acLDL were double positive stained, CD34, CD133, vWF and Flk-1immune staining were positive too.10-8M and10-7M ghrelin promoted EPCs proliferation,migration,tubeformation and NO secretion.Conclusions These results suggest that ghrelin can stimulate EPCsproliferation、migration、tube formation and NO secretion. It indicates thatghrelin may be used as a therapeutic strategy to ischemic diseases bypromoting EPC biological activity. Part IIThe molecular mechanisms of ghrelin promote biologicalactivity of endothelial progenitor cellsObjective To investigate the underlying molecular signal pathwaymechanisms of ghrelin on EPCs biological activityMethods EPC were treated with various concentrations(10-9～10-6M)of ghrelin with or without GHSR1a specific inhibitor [D-Lys3]-GHRP-6,PI3K specific inhibitor LY294002and endothelial nitric oxide synthase(eNOS) specific inhibitor L-NAME, phosphorylated and total Akt andeNOS were detected by Western-blot method. The EPCs proliferation，migration，tube formation and NO secretion were detected at the sametime.Results Ghrelin was time-dependent and dose-dependent manner to promote EPCs expression of Akt and eNOS phosphorylation; The levels ofphosphorylation Akt and eNOS induced by ghrelin were blocked byGHSR-1a specific blocker [D-Lys3]-GHRP-6and PI3K specific inhibitorLY294002. eNOS inhibitor L-NAME inhibited phosphorylation of eNOS,but had not any effect on Akt phosphorylation. At the same time,[D-Lys3]-GHRP-6, LY294002and L-NAME all could significantly inhibitghrelin-induced EPCs proliferation, migration, tube formation and NOsecretionConclusions These results suggest that ghrelin stimulates EPCbiological activity via GHSR1a/PI3K/Akt/eNOS signal pathwaymechanism. Objective：To investigate the changes of plasma growth hormonesecretions receptor endogenous ligand (ghrelin) in the critically ill patients,and its mechanism.Methods：plasma active ghrelin，TNF-α, IL-6levels of127cases ofcritically ill patients and30healthy were measured by enzyme-linkeimmunosorbent assay， and clinical data were recorded. Results：(1)Critically ill patients plasma active ghrelin levels weresignificantly higher than the healthy control group (P <0.01)(2)According to the APACHE lI score≥15and <15,patients were divided intohigh-risk group and low-risk groups; active ghrelin levels of the high-riskgroup were higher than the low-risk group (P <0.05);(3) In critically illpatients, plasma active ghrelin levels and body mass index (BMI) wasnegatively correlated (r=-0.536， P <0.01); with TNF-α, IL-6werepositively correlation (r=0.275, P <0.05； r=0.452, P <0.01). Multiplelinear regression analysis prompted IL-6levels were plasma active ghrelinindependently associated factor.Conclusion：Plasma active ghrelin is highly expressed in critically illpatients，may be related to malnutrition and inflammatory response incritical illness，Its determination helps to assess of the severity of theillness。... |
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