| Objective1. To investigate the difference of cardioprotective effect of ischemic preconditioning in adult and aged rat hearts, and prove that adiponectin mediates the effect of IPC through angiogenesis.2. To investigate the effect of adiponectin siRNA transfection on expression of adiponectin protein, angiogenesis in ischemia myocardium24h after IPC and the heart function after acute myocardial infarct, and to further verify the role of adiponectin in cardioprotection of IPC.MethodsIschemic preconditioning (IPC) and acute myocardial infarction model was set up.78adult and aged rats were randomly divided into5groups:IPC group (4cycles of ischemic for6min and reperfusion for6min), sham group (thracotomy was done without blood flow blocking), IPC+AMI group (coronary arteries in IPC group were ligated24h later), AMI group (coronary arteries in sham group were ligated24h later) and control group (nothing was done). The protective effect of IPC was studied by M-mode ultrasound and Masson's Tri chrome staining. The expression of protein and mRNA of adiponectin Oh,6h,12h and24h after IPC was examined by western blot, immunohistochemistry and quantitative real time RT-PCR. And the plasma levels of adiponectin at four time points after IPC was also detected by ELISA. The angiogenesis in ischemia myocardium24h after IPC was examined by immunohistochemistry.50adult rats were assigned randomly to5groups:adiponectin siRNA+IPC group, scramble siRNA+IPC group, saline+IPC group, adiponectin siRNA+IPC+AMI group and scramble siRNA+IPC+AMI group. After anesthesia and intercostal thoracotomy, prepared saline or complexes of siRNA and transfection reagent (jetPEI) were injected directly into myocardium on symmetric points along two sides of LAD. The scramble siRNA was used in control group. The delayed IPC procedure was performed24h after siRNA transfection or saline administration. In the first three groups,24h after IPC, myocardial samples were harvested from the ischemic-reperfused region for western blotting analysis (n=5in siRNA group and sham group respectively) to prove the successful transfection. And the other ischemia myocardium were buried by OCT and cut to thin sections, then examined by immunohistochemistry to detect angiogenesis. In the last two groups, myocardial infarction model was set up24h after IPC as described above. Fourteen days after ligation, the rats were anesthetized and before the hearts were harvested, cardiac function was also measured by M-mode echocardiograms. Myocardial infarction size measured at the level of midpapillary muscle was scored by Masson's trichrome staining and expressed as a percentage of the total left ventricular myocardial area.ResultsIPC in adult rats increased left ventricular fractional shortening (23.99%±1.14%vs.33.10%±1.16%, p<0.05) and reduced infarct size (31.73±3.98%LV area vs.22.45±2.67%LV area, p<0.05) compared with AMI group. In aged rats, however, the excessive mortality of myocardial infarct rats after IPC made it impossible to evaluate heart function and defect infart size. In adult rats, the numbers of capillary in ischemia myocardium24h after IPC was significantly more than which in sham group. And the expression of adiponectin mRNA6h and12h after the IPC was2.2and2.1times greater than the sham group (p<0.05) and the expression of protein also increased significantly (p<0.05). Immunohistochemistry shows that staining in the ischemia area was deeper and the expression of adiponectin was siginificantly higher than non-ischemic area (p<0.05). And, compared to the sham groups, the plasma level of adiponectin increased significantly Oh.6h and12h after IPC (0h:7.40±0.47vs.10.90±1.74;6h:8.18±1.41vs.10.98±1.74;12h:6.97±1.02vs.9.31±0.96, p<0.05). But in aged rats, the capillary numbers and expression of adiponectin mRNA and protein after IPC showed no difference with those in control group, and the plasma concentration increased until24h after IPC.Adiponectin siRNA transfection inhibited adiponectin protein expression after IPC in the adult rats as showed by Western blotting detection that24h after IPC, the expression of adiponectin protein in adiponectin siRNA+IPC group was significantly higher than scramble siRNA+IPC group and saline+IPC group. Specific knockdown of adiponectin by siRNA in vivo ameliorated the protective effect of IPC, as demonstrated by FS (scrambled siRNA vs. adiponectin siRNA:31.1±3.05%vs.22.42±1.28%, p<0.05) and infarct size (22.35±1.29%LVAvs.30.17±2.44%LVA, p<0.05). And24h after IPC, the numbers of capillaries in ischemic myocardium after adiponectin knockdown significantly decreased than which in scramble siRNA group.ConclusionsIPC in adult rats improved heart function, promoted angiogenesis and induced the expression of adiponectin mRNA and protein in myocardium, and also increased the adiponectin level in plasma, which shows that the adiponecitn may play a role in the protective effect of IPC. But in aged rats, the protection of IPC weakened, angiogenesis was not induced and the effect on expression of adiponectin also weakened, further confirmed that adiponectin played a role in the protective effect of IPC. Injection of adiponectin siRNA in local myocardium could successfully knockdown the expression of aiponectin protein. And the knockdown of adiponectin by siRNA induced attenuate of angiogenesis and cardioprotection of IPC, which strongly suggests a contribution of myocardium-derived adiponectin to the protective effect of IPC against myocardial injury through angiogenesis. Objective1. To investigate the relationship between serum adiponectin and high sensitivity C-reactive protein (hs CRP) as well as risk factors and severity in patients with coronary artery disease.2. To investigate the effect of prodrome angina48h before acute myocardial infarctioin on serum adiponectin levels.MethodsSerum adiponectin was measured by enzyme linked immunosorbent assay (ELISA) in patients with coronary heart disease and normal control group. Their serum hsCRP were measured by scatter velocity turbidimethy. All subjects were confirmed by coronary angiography, and a survey was done for cardiovascular risk factors.ResultsSerum adiponectin concentrations in ACS group and SAP group were significantly lower than those in NC group (p<0.05), accompanied with significantly higher hsCRP (p<0.05). And subjects in ACS group had lower serum adiponectin level and higher hsCRP than those in SAP group (p<0.05). Serum adiponectin concentrations were correlated negatively with hsCRP (r=-0.6, p<0.05) and TIMI scroe (r=-0.332, p<0.05) in ACS group. Moreover, the more risk factors, the lower serum adiponectin concentration in coronary artery disease. Subjects with prodrome angina48h before AMI in AMI group had lower adiponectin than those without prodrome angina.ConclusionsSerum adiponectin was associated with severity of coronary artery disease and cardiovascular risk factors. Adiponectin had anti-inflammatory and anti-athero genic properties. IPC in clinics may lower serum adiponectin levels. |
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