| Chapter I:The regulation of sphingolipids signaling in patients with chronic heart failureObjective:The aim of this study was to observe the relationship between the changes of sphingomyelin (SPM), Ceramide and secretory acid sphingomyelinase(S-ASMae) activity in plasma of patients with chronic heart failure and to study the relationship between ceramide and symptom severity and prognosis.Methods:This study enrolled423patients with chronic heart failure and104healthy people, collecting clinical data and resluts of echocardiography and other detection, then finished4years of follow-up. The level of SPM was detected by chemiluminescence, the level of ceramide by liquid chromatography-mass spectrometry (LS-MS), plasma S-ASMase activity was detected by high performance liquid chromatography detection;], plasma tumor necrosis factor alpha (TNF-α), soluble FAS (sFas), soluble Fas ligand (sFasL) and amino-terminal pro brain natriuretic peptide (NT-proBNP) level were detected by enzyme linked immunosorbent assay (ELISA method); high sensitive C reactive protein (hsCRP) level was detected by turbidimetry; left ventricular ejection fraction (LVEF) and left ventricular end diastolic diameter (LVED) were detected by echocardiogram. The plasma levels of SPM, Ceramide, TNF-α, hsCRP, NT-proBNP, ASMase activity, combined with the general results were analyzed. Patients were divided into three tertiles on the basis of ceramide level. Kaplan-Meier analysis was performed to compare the survival rates of the three groups. A multivariate Cox proportional hazard model was performed to identify the prognostic value of plasma ceramide level.Result:(1) In patients with chronic heart failure, SPM, Ceramide, TNF-α, hsCRP, sFas, NT-proBNP, LVED and ASMase activity was higher than those of control group (P<0.05), SBP, DBP, LVEF lower than that of control group (P<0.05)(2) The levels of plasma ceramide with NYHA cardiac functional classes and gradually increased, the differences were statistically significant (P<0.05);(3) Plasma level of ceramide were positively correlated with LVED, NT-proBNP, TNF-α, sFas, SMase (P<0.05), and negatively correlated with LVEF (P＜0.05)(4) After median follow-up of4years,200patients died in423patients, the cumulative mortality rate47.2%, the risk of death in patients with the highest levels of ceramide group was about2.5times the minimum group (HR:2.48,95%CI:1.74-3.55, P＜0.001);(5) Cox regression analysis showed that, BMI, LVEF, Log (NT-proBNP), atrial fibrillation, and Ceramide levels are independent predictors of death in patients with chronic heart failure.Conclusion:Plasma ceramide increased with chronic heart failure disease severity increased, and is an independent risk factor for predicting mortality in patients with chronic cardiac insufficiency, suggesting that the sphingomyelin signaling pathway plays an important role in development of chronic heart failure. Chapter Ⅱ:The role of sphingolipids signaling in ischemia/reperfusion injury in ratsObjective:To observe the changes of sphingomyelinase (SMase) activity, ceramidase (CDase) activity, ceramide level and sphingosine level in myocardial ischemia/reperfusion injury in rats, and to test whether neutral ceramidase (NCDase) might mediate preconditioning/postconditioning protection.Methods:32Sprague Dawley (SD) rats were randomly divided into4groups:control group (Control), ischemia/reperfusion group (IR), NCDase preconditioning group (IPC), NCDase postconditioning group (IPost),8rats in each group. Connect isolated heart with a Langendorff device fulled with K-H buffer. After30min’s continuous perfusion by95%O2and5%CO2mixed gas saturation (Krebs-Henseleit, K-H) constant temperature (37℃), isolated hearts were treated according to groups. Control group:perfusion with150min; IR group:stop filling30min,120min K-H liquid reperfusion; IPC group:K-H CDase solution containing10min perfusion, stop filling30min, reperfusion120min; IPost group:to stop filling30min, K-H solution with CDase perfusion10min, then routine K-H perfusion110min. Heart rate (HR), left ventricular pressure (LVP), left ventricular pressure increase/decrease rate (±dP/dtmax) and left ventricular end diastolic pressure (LVEDP) and coronary flow (CF), were detected in the baseline and during reperfsion. 5min after reperfusion for IR group and IPC group, or15min for IPost group,5ml coronary flow was collected to detect creatine kinase (CK) and lactate dehydrogenase (LDH) level. Areas of myocardial infarction were measured, myocardial tissue observed under a microscope by HE staining, tissue homogenate for detecting SMae activity, CDase activity, ceramide level and sphingosine level.Result:(1) Compared with the control group,+dP/dtmax and-dP/dtmax, LVP in IPC group and IPost group significantly decreased, LVEDP significantly increased (P<0.05); HR, CF no significant difference;(2) Compared with the control group, myocardio infarction area, perfusion fluid CK and LDH levels were significantly increased in IR gourp, IPC group and IPost group(P<0.05), and compared with the IR group, myocardio infarction area, CK perfusion and LDH significantly decreased in IPC group and IPost group (P<0.05);(3) Compared with Control group, ASMase activity, NSMase activity and ceramide levels in tissue homogenate significantly increased in IR group (P<0.05), the activity of NCDase in tissue homogenate was significantly decreased in IR group(P<0.05), ASMase activity, NSMase activity, NCDase activity and sphingosine levels in tissue homogenate were significantly increased IPC group and IPost group (P<0.05); compared with IR group, ASMase activity, NSMase activity and ceramide levels in tissue homogenate significantly decreased in IPC group and IPost group (P<0.05), the activity of NCDase and sphingosine levels in tissue homogenate significantly increased in IPC group and IPost group (P＜0.05). Conclusion:Ischemia reperfusion injury through upregulation of ASMase activity and NSMase activity, while the down-regulation of NCDase activity, resulting in increased ceramide levels in myocardium, ultimately causing a heart cell damage, increase infarct size, cardiac diastolic function, and give the NCDase preconditioning and postconditioning can significantly reverse the ischemia reperfusion injury, suggesting that the pathophysiological mechanism of sphingomyelin pathway participate in ischemia reperfusion injury, which may become a new therapeutic target heart failure after myocardial infarction. |
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