| Cardiovascular disease is one of the most dangerous threats to human health, which associated with many cardiovascular active substances. Salusins are newly found endogenous polypeptides, which consist of 28 or 20 amino acids designated Salusin and Salusinβ, respectively. Recent investigations have revealed that they increase intracellular calcium ([Ca2+]i), induce the expression of growth-associated genes such as c-myc and c-fos, and stimulate the proliferation of VSMCs and fibroblasts, cause rapid and profound decreases blood pressure and heart rate, and they may be regulatory factors for myocardial growth, hypertrophy and antiapoptosis, Salusinβcan alleviate ischemia-reperfusion injury and improve heart function, and they act as cardiac depressors with negative inotropic and chronotropic actions in rats. However, the cardiovascular effects of Salusins are not fully understood, the paper taking Salusinβas the research objects, discusses it from two parts, to deep our knowledge of the new cardiovascular regulatory polypeptides, to expect to find out a possible target of cardiovascular treatment, and to provide a new ideal for the cardiovascular treatment.Part One: Effects of Salusinβon vascular smooth muscle cells proliferation Aims:To observe the effects of Salusinβon c-Fos, c-Jun and PCNA in VSMCs in rats.Methods:Fifty Wistar rats were randomly distributed into Salusin-βgroup (n=25) and control group (n=25),there are five Wistar rats for 0.5h, 1h, 2h, 4h, 24h in two groups.The rats in Salusinβgroup were injected Salusinβ(5nmol/kg) via the femoral vein,while the rats in control group were injected aequales saline. The aorta pectoralis were removed after the rats had been perfused with paraform at 0.5, 1, 2, 4, 24h. The expression of c-fos, c-jun at 0.5h, 1h, 2h, 4h and PCNA at 24h in VSMCs of aorta pectoralis in rats were studied by immunocytochemistry and image analysis.Results:1. The expression of c-Fos: In Salusin-βgroup, the expression of c-Fos began to increase at 0.5h, and peaked at 2h and fell to control at 4h. The expression was not a statistically significant difference between 0.5h and 4h(P>0.05),but a significant difference among the 0.5h,4h and 1h,2h (P<0.01).In control group, there was no difference of the expression of c-Fos at different time(P>0.05). There was no difference of the expression of c-Fos between Salusinβgroup and control group at 0.5h and 4h(P>0.05),but was different significantly at 1h and2h (P<0.01). 2. The expression of c-Jun: In Salusinβgroup, the expression of c-Jun began to increase at 0.5h, and peaked at 2h and fell to approximately level of 1h at 4h. The expression was a statistically significant difference among the 0.5h,1h,2h and 4h (P<0.01). In control group, there was no difference of the expression of c-Jun at different time(P>0.05). There was no difference of the expression of c-Jun between Salusinβgroup and control group at 0.5h (P>0.05),but was different significantly at 1h ,2h and 4h (P<0.01). 3. The expression of PCNA: There was a statistically significant difference between Salusinβgroup and control group at 24h (P<0.01).Part Two: Proteomic profiling of heart treated with SalusinβAims:Proteomic analysis, including two-dimensional gel electrophoresis(2-DE) combined with mass spectrometry(MS), was used to compare the protein profile of heart samples from rats treated with Salusinβand saline as Control. The differential proteins identified in this study were classified by bioinformatics analysis to investigate the function of Salusinβin heart.Methods:Ten adult male Wistar rats were randomly divided into a control group(n=5) and an Salusinβgroup (n=5). Salusinβ(5nmol/kg) was injected into rat femoral veins. As controls, rats received injection of Saline before total protein preparation. After 24 hours, the rats in the control group and the Salusinβgroups were all sacrificed. For each rat, the heart sample was collected for subsequent analysis. Briefly, total protein were isolated by 2-DE and protein spots were stained with silver for clearer visualization. Then image analysis was performed using the ImageMaster 2D Platinum 5.0 software according to the protocols provided by the manufacturer. The differential protein spots were excised and digested by bovine trypsin dissolved in 50mM NH4HCO3 digestion buffer. Peptide mixtures were deposited on the stainless steel MALDI probe to dry slowly at ambient temperature. MS was performed using a Bruker Daltonics autoflex MALDI-TOF-MS. To enhance the results obtained by MALDI-TOF MS method, tandem mass spectrometry was performed on a Bruker ultraflex III TOF/TOF mass spectrometer for MS/MS analysis. Finally, Protein identification by MS data was accomplished using the Mascot search engine with the Rodentia category of the SwissProt database.Results:There were approximately 1828.33土29.14 protein spots in each 2-DE gel of the heart samples of control group visualized by silver staining, whereas 1851.33土23.01 protein spots in that of Salusinβ.Comparing the different groups, 12 protein spots analyzed by the ImageMaster software were significant difference. Among the 12 protein spots, 8 were down-regulated and 4 were up-regulated in heart after the treatment of Salusinβ.These proteins identified by MALDI-TOF-MS and MALDI-TOF/TOF,and 13 protein were identified,which are involved in many biological processes, including biological oxidation, energy metabolism, stress, development and binding.Conclusions:The results suggest that the effects of Salusinβon heart might be related to the identified 13 proteins which are involved in many biological processes,including biological oxidation, energy metabolism,stress,development and binding, especially biological oxidation. |
