| Background:Growth hormone deficiency(GHD)is due to partial or complete absence of synthetic and/or secreting growth hormone(GH)in the anterior pituitary gland.GHD not only affects the lifetime of children due to GH deficiency,but also affects a series of heart diseases,such as heart morphological changes,ventricular structural changes,vascular endothelial cells and arteriosclerotic cardiovascular diseases.There are various studies show that untreated GHD associated with varying degrees of cardiovascular diseases.As is known to all,Ghrelin is a peptide hormone which is an endogenous ligand for the growth hormone secretin receptor(GHSR).Ghrelin is not only with the biological functions of promoting GH release,promoting gastrointestinal motility,increasing appetite,decreasing blood pressure,inhibiting inflammatory factors release,but also with the function of protecting cardiovascular system.There are two forms of Ghrelin in human body:the one is acyl-ghrelin which catalyzed by ghrelin-acyltransferase(GOAT),while the other is desacyl-ghrelin which not catalyzed by GOAT.Myocardial cells have poor tolerance to hypoxic injury.Hypoxia/ischemia can lead to abnormal cardiac electrical activity,myocardial cell apoptosis,and induce various cardiovascular diseases.IGF-1 is an active protein polypeptide substances,so the expression of IGF-1 is an important index to measure the normal function of cardiac myocytes and tissues.In addition,AKT is considered as a Ser/Thr protein kinase and an important part of PI3K/AKT signaling pathway.As a classical signal pathway,PI3K/AKT signaling pathway plays an important role in many physiological and pathological processes by regulating gene expression,such as cell survival,differentiation,growth and apoptosis.In this investigation,conventional ultrasound and 3D-STI were used to detect the myocardial strain ability and structural changes in children with GHD,to study the degree of cardiac damage in children with GHD,and analyze the correlation between the levels of acyl-ghrelin,desacyl-ghrelin and total ghrelin and the changes of cardiac structure and function,and to explore the regulation of GH/IGF-1 axis on plasma ghrelin level.At the same time,the expression vector of ghrelin lentivirus was constructed and transfected into neonatal rat cardiomyocytes and myocardial tissues.The effects of exogenous ghrelin on the expression of GH,GHSR,IGF-1 and p-AKT proteins in cardiomyocytes and their mechanisms were verified by immunofluorescence,fluorescence quantitative PCR,Western Blot and immunohistochemistry.Part 1 Correlation between the Level of Surem Ghrelin and Change of Structure and Function of Cardiovascular in children with Growth Hormone DeficiencyObjective:The aim of our paper was to investigate changes of acyl-ghrelin and desacyl-ghrelin levels in GHD children with cardiac abnormalities,further more we analyzed and explored the possible mechanisms of protecting heart of these two types of ghrelin in GHD children.Methods:(1)We collected 35 cases of GHD children as GHD group and 30 healthy children as control group.Cardiac structure and function indexes were decected by conventional two-dimensional echocardiography in GHD group and control group,such as left ventricular end diastolic ventricular septal thickness(IVSd),left ventricular end diastolic posterior wall thickness(LVPWd),left ventricular end diastolic volume(LVPWTd),left ventricular end systolic volume(LVESV),left ventricular ejection dimension two fraction(2D-EF).(2)These cardiac indexes were decected by three-dimensional cardiac ultrasound speckle tracking imaging(3D-STI)in GHD group and control group,such as global longitudinal strain(GLS),global radial strain(GRS),circumferential strain(GCS),torsion angle of the overall(GTA)and left ventricular mass(LVM),left ventricular mass index(LVMI).(3)The levels of acyl-ghrelin and desacyl-ghrelin in GHD group and control group were tested through ELISA,then calculated total-ghrelin and ratio of A/D,tested IGF-1 level in GHD group as well.(4)Statistical software was used to analyze the total-ghrelin,acyl-ghrelin,desacyl-ghrelin and A/D ratio between GHD group and control group.(5)The correlation between the total-ghrelin,acyl-ghrelin,desacyl-ghrelin,A/D ratio and the cardiac indexes of echocardiographic and 3D-STI in GHD group was analyzed.(6)Correlation between the levels of acyl-ghrelin,desacyl-ghrelin,total-ghrelin,A/D ratio and the level of IGF-1 in GHD group was analyzed.(7)Finally,multiple linear regression analysis was used to identify as independent risk factors that resulted in changes of acyl-ghrelin and desacyl-ghrelin levels.Results:(1)The detected values of IVSd,LVPWd,LVPEDV,LVESV and 2D-EF in GHD group were statistically significant compared with control group(P<0.05).(2)The detected values of GLS,GRS,GCS and LVM in GHD group were significantly decreased compared with the control group(P<0.05),but GTA showed no significant difference between these two groups(P>0.05).(3)The levers of acyl-ghrelin,desacyl-ghrelin,total-ghrelin and A/D ratio in GHD group were higher than those in control group(P<0.05).(4)In GHD group,the levels of acyl-ghrelin,desacyl-ghrelin and total-ghrelin were negatively related to the values of GLS,GRS and GCS r=-0.548,P=0.001;r=-0.481,P=0.003;r=-0.411,P=0.014;r=-0.520,P=0.001;r=-0.444,P=0.007;r=-0.381,P=0.024;r=-0.536,P=0.001;r=-0.464,P=0.005;r=-0.397,P=0.018),while there was no correlation between the level of acyl-ghrelin,desacyl-ghrelin,total-ghrelin and GTA,LVM,IVSd,LVPWd,LVEDV,LVESV and 2D-EF(P>0.05);As well as,there was no correlation between the ratio of A/D and detected values through two-dimensional ultrasound and 3D-STI(P>0.05).(5)In GHD group,the levels of acyl-ghrelin,desacyl-ghrelin and total-ghrelin were negatively related to the level of IGF-1(r=-0.498,P=0.002;r=-0.498,P=0.002;r=-0.501,P=0.002),while there was no correlation between the ratio of A/D and the level of IGF-1(r=-0.070,P=0.703).(6)Multiple linear regression analysis showed that IGF-1 was the independent risk factor for acyl-ghrelin and desacyl-ghrelin(B=-4.680,95%CI-8.909 to-0.652,P=0.025)and(B=-5.873,95%CI-10.767 to-0.979,P=0.021).Conclusions:(1)Our results demonstrated that there were cardiac structural and functional changes in children with GHD,the levels of acyl-ghrelin,desacyl-ghrelin and total-ghrelin cwere orrelated with the change degree of cardiac function,but which were not related to cardiac structural change.(2)Human's body would increase the plasma levels of acyl-ghrelin and desacyl-ghrelin through secreting to inhibit cardiac changes in GHD children,dysfunction of GH/IGF-1 axis would increase the plasma levels of acyl-ghrelin and desacyl-ghrelin through negative feedback adjusting which play the role of protecting heart in GHD children.Part 2 The Protective Effect of Ghrelin on Myocardial Anoxia/Reoxygenation InjuryObjective:Ghrelin is an endogenous ligand of growth hormone secreting hormone receptor(GHSR).It can be widely found in various tissues and organs of human body and has various biological effects,which can promote accumulating fat and protect heart.In our paper,we investigated the expression of ghrelin in anoxia/reoxygenation cardiomyocytes,and explored its protective effects of hypoxia/reoxygenation cardiac myocytes,which would provide a theoretical basis for treatment of myocardial injury.Methods:(1)Ghrelin lentiviral expression vector was constructed,and plvx-puro vector was used to connect Ghrelin gene.Enzyme digestion and colony PCR were used to comfirm whether expression vector was successfully constructed or not.(2)Cardiomyocytes from neonatal SD rats were isolated successfully.Immunofluorescence was used to identify whether the cardiomyocytes were correctly separated.Then the ghrelin lentiviral expression vector was transfected into cardiomyocytes,and myocardial cell hypoxia/reoxygenation model was established.(3)The transfected cardiomyocytes were divided into four groups:(A)control group;(B)blank group:hypoxia/reoxygenation;(C)transfection group-1;expression no load+hypoxia/reoxygenation;(D)transfection group-2:Ghrelin expression vector+hypoxia/reoxygenation.CCK-8 cell toxicity test was carried out in each group,and the protective effect of ghrelin on myocardial cell was verified.(4)The isolated rat heart perfusion model was constructed,then divided into four groups:(A)normal group;(B)sham operation group(Group sham);(C)hypoxia reoxygenation model group;(D)drug treatment+hypoxia reoxygenation group.(5)Real time PCR was used to detect the mRNA expression of GH,GHSR,IGF-1 and AKT in neonatal rat cardiomyocytes and rat myocardium.The expression level of mRNA in hypoxia/reoxygenation myocardium was detected after ghrelin transfection,and the protective effect of ghrelin on myocardial cell was determined.(6)Western Blot was used to detect the expression level of GH,GHSR,IGF-1,AKT and p-AKT in cardiac myocytes and rat myocardium,and further determine the cardioprotective function of ghrelin through detecting expression of these above proteins.(7)The expression levels of GH,GHSR,IGF-1 and AKT protein in cardiac myocytes were characterized through immunohistochemistry.The repair effect of ghrelin on myocardial cells was revealed by comparing the model group with ghrelin treatment group.Results:(1)The ghrelin expression vector was successfully constructed and transfected into the neonatal rat cardiac myocytes,and the myocardial cells were identified by immunofluorescence.(2)The survival rate of myocardial cells were detected by fluorescence identification,after transfecting by ghrelin expression vector at 24 h,48 h,72 h.Our results showed,compared with control group,the cell survival rates of transfection group-2,transfection group-1 and blank group were all decreased(P<0.05),but compared with transfection group-1 and blank group,transfection group-2 showed higer repairing function of myocardial cell and cell survival rate(P<0.05).(3)Hoechst staining was used to detect the cell apoptosis rate,the results showed that the apoptosis rate increased significantly in blank control group and transfection group-1,which was significantly higher than that in control group(P<0.01),but after intervented by ghrelin,transfection group-2 showed lower cell apoptosis rate than blank control group and transfection group(P<0.01).(4)Myocardial cell was detected by fluorescence quantitative PCR,our results showed that the mRNA expression levels of GH,GHSR,IGF-1 in blank control group and transfection group-1 were significantly lower than those in control group(P<0.01);After ghrelin intervention,the mRNA expression levels of GH,GHSR and IGF-1 in transfection group-2 were higher than those in blank group and transfection group-1(P<0.01).(5)Western blot was used to detect protein level of myocardial cell,the results showed that the protein level of GH,GHSR,IGF-1 and p-AKT/AKT ratio decreased significantly in blank control group and transfection group-1 compared with the control group(P<0.01),After intervention of ghrelin,protein expression of GH,GHSR,IGF-1 and p-AKT/AKT ratio all significantly increased in transfection group-2 compared with the blank control group and transfection group-1(P<0.01).(6)According to the fluorescent quantitative PCR experiment of rat model,the mRNA expression levels of GH,GHSR,IGF-1 were lower in hypoxia/reoxygenation group than those in control group and sham operation group(P<0.01);After intervened ghrelin,the mRNA levels of GH,GHSR and IGF-1 significantly increased again in Ghrelin+hypoxia/reoxygenation group compared with hypoxia/reoxygenation group(P<0.01).(7)According to the western blot experiment of rat model,the protein levels of GH,GHSR,IGF-1 and p-AKT/AKT ratio were significantly lower in hypoxia/reoxygenation group than those in control group(P<0.01);After intervened ghrelin,the protein levels of GH,GHSR,IGF-1 and p-AKT/AKT ratio were increased significantly again in Ghrelin+hypoxia/reoxygenation group compared with hypoxia/reoxygenation group(P<0.01).(8)Immunohistochemical analysis of rat cardiac slices showed that the protein expression levels of GH,GHSR,IGF-1 and AKT in Ghrelin+hypoxia/reoxygenation group were significantly higher than those in anoxia/reoxygenation group.Conclusions:(1)The expression levels of GH,GHSR and IGF-1 in anoxic cardiomyocytes were significantly increased after transfection of ghrelin expression vector.(2)Ghrelin would inhibit cardiomyocyte apoptosis and repair myocardial tissue after hypoxia injury.(3)Ghrelin would improve the expression levels of GH,GHSR and IGF-1 in myocardial cell and repair hypoxic damaged myocardium.The mechanism of protective effect of ghrelin on heart is related to activation of GH/IGF-1 axis.(4)Ghrelin would inhibit the activity of protein kinase by activating the AMPK pathway,thereby inhibit the apoptosis of cardiomyocytes,which provides a new idea for intervention and management of ghrelin on myocardial hypoxia injury. |
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