| Objective: We researched the protective effects and its Effects on Alteration of Protein Expression Pattern of n-butanol fraction of Potentilla anserine L following acute myocardial ischemia and reperfusion in Rats,futher more we study its mechanisms of n-butanol fraction of Potentilla anserine L on myocardial cells.Methods: Male Sprague-Dawley rats were randomly divided into control group and injury groups which were subjected to coronary occlusion lasting for 30min and reperfusion for 120min and then subdivided into model group and intervention group of n-butanol fraction of Potentilla anserine L (49.3mg?kg-1, 98.6mg?kg-1, 197.2mg?kg-1). Retrograde inserting into the left ventrical through a cannula and fixed in the right common carotid arter, recording the indexs of haemonamics. Activities of LDH, CK,SOD and the content of MDA in serum were measured.Observe the change of mitochondrion and glycogen in myocardial cell by electron microscope.The effect of different doses of Potentilla anserine L on acute myocardial ischemia and reperfusion injury of myocardial cell were observed by H.E staining;The protein samples, extracted from rat myocardium of different groups , were applied to 2D electrophoresis. The 2D images were analyzed by PDQuest7.0 analysis software. Selected the differentially expressed protein spots were digested by trypsin, analyzed by mass spectrometry, and then searched against the NCB Inr database using Mascot software. The types and roles of the different expressed proteins which if that could anti ischemia-reperfusion could be ascertained preliminarily. As for these proteins whether or not expressed in rat myocardium, which would be checked via the method of Western-blot in the end.Results: 1 The protective effects of n-butanol fraction of Potentilla anserine L on acute myocardial ischemia and reperfusion injury in rats: (1)The change of ST-segment of ECG on groups: the ST-segment of model group changed obviously. But the change of ST-segment decrease on the high dose group, the middle dose group and the fufangdansen group .(2)HE staining: Model group, which histopathological changes significantly, inflammatory cells infilter in myocardial interstitial, acidophilia myocardial fibers change, sarcomere fault, disappeared necrosis obviously. Effect of Potentilla anserine L high and middle dose group significantly reduced the injury compared with model group. Effect of low-dose group have not any change of the model group. (3)Myocardial ultra-structure changes: electron microscopy showed that the treatment group concentration Effect of Potentilla anserine L to reduce myocardial ischemia-reperfusion injury caused by swelling of mitochondria, vacuoles, such as changes in the ultra-structure of injury. (4) Serum Biochemical Indicators: Compared with model group, The intermediate and low doses of Potentilla anserine L could decreased the activities of LDH(P<0.01)and CK(P<0.01). (5) Serum MDA, SOD detection: Compared with the model group,each dose of Potentilla anserine L could significantly enhance SOD activity in rats serum (P<0.01), reduce the production of MDA (P<0.01).2 The results of 2-D following ischemia-reperfusion injury group and other groups.1003±23,918±65,877±72,1046±43,891±23,738±69 protein spots were detected respectively in control,danshen,low dose,middle dose,high dose and model groups via PDquest software. Furthermore, most of these spots were dispersed into the scopes of PI 4.8-9.0 and MW 30kDa-70kDa.Compared with the control group(P<0.05), the model group included 5 up-regulated spots,39 down-regulated spots and 45 protein-spots couldn't be detected after analysis.In the model group , there is 1protein-spot can be down-regulated in the 5 protein-spots compared with the middle, high and the fufang danshen group. In the n-butanol fraction of Potentilla anserine L groups 6 protein-spots were up-regulated in the 45 protein-spots, and have 3 protein-spots which same with the fufang danshen group.At the same time,4 protein-spots of the medicine intervention groups were up-regulated in the 39 spots. 3 Identification of the different displayed protein spots via MALDI-TOF-MS. Seven protein spots were analyzed via MALDI-TOF-MS, which scores were more than 61 (p<0.05).They are alpha B-crystallin;phosphatidylethanolamine binding protein ; NADH dehydrogenase 1 alpha 10 ;apolipoprotein E;myosin, light chain 3;Myosin regulatory light chain 2;crystallin, alpha B, isoform CRA_b.4 The result of Western-blot NADH dehydrogenase 1 alpha 10 were authenticated via western blot. The result demonstrated that NADH dehydrogenase 1 alpha 10 has expressed in rat myocardium,and have the same tendency of protein change with the 2D images which were analyzed by PDQuest7.0 software.Conclusion: 1 n-butanol fraction of Potentilla anserine L could significantly protect acute myocardial ischemia- reperfusion induced myocardial injury, the myocardial tissue ultra-structure.Elevate the cleaning ability to oxygen free radical and improve cardiac function. 2 Myocardial injury can cause protein expression profile changes. Protein expression profiles are different in different treatment groups, suggesting that the protein may be some differences in myocardial ischemic and reperfusion injury-induced gene expression products and drug targets.3 After verification, n-butanol fraction of Potentilla anserine L could up-regulate NADH dehydrogenase 1 alpha 10, reduce the production of oxygen free radical, protect the cell membrane to against ischemia-reperfusion injury. 4 The distinct protein detected at different group may be the specific expression biomarkers of effect of protection for ischemia reperfusion injury. The further study could be useful for molecular mechanism, diagnosis or therapy of ischemia reperfusion injury.
|
PDF Full Text Request