Effect Of Astragalus Membranaceus On Phospholamban And Sarcoplasmic Reticulum Ca～(2+)-ATPase Protein In The Myocardium In Spontaneously Hypertensive Rats

BackgroundAstragalus membranaceus(AM) is a traditional Chinese medicine as a kind drug of "Bu Qi".It has been treated various vascular diseases and has certainly curative effect .Acquired data show that AM can enhance the activity and the express of the gene in sarcoplasmic reticulum(SR) Ca²⁺-ATPase in the myocardium of the virus myocarditis; In hypertension sarcoplasmic reticulum Ca²⁺-ATPase and phosphlamban protein of the myocardium decrease remarkably. The change of intracellular calcium concentration play a central role in physiological function of cardiacmyocyte and cardiac dysfunction has been linked to Ca²⁺-cycling defect.It is noted calcium release activities are detemined by ryanodine receptor(RyR) and inositol triphosphate(IP3) receptor ,activity of the the cardiac sarcoplasmic reticulum Ca²⁺-ATPase(SERCA) is the rate-determining factor of Ca²⁺ reuptake into the SR, and SERCA activity in its regulated by phospholamban(PLB), PLB inhibits SERCA activity in its unphosphorylation form, whereas the phospholated form of PLB dissociates from SERCA .It has a great many researches that AM affect hypertension at present. There are a few reports that AM accurately improves the myocardiumfunction's mechanism and affects SERCA and PLB protein especially.Our experiment observe variation of quantity of SERCA and PLB of the myocardium SR in the early phase of the SHR's hypertension, also discuss that AM influence the myocardial PLB and SERCA protein in SHR, and progressively reveal that AM improve the possible mechanism of the heart function.Materials and MethodsEighteen Spontaneously Hypertensive Rats were randomly divided into control group(n=4) and AM groups.The AM groups were divided into four groups and intraperitoneal administrated AM parenteral solution 1.8g(n=5), 2.4g(n=4) and 3.6g(n=5) respectively once a day,the control group was given NS 0.9ml for eight weeks at twelfth week. Seventeen WKY(Wistar Kyoto,WKY) rats were divided into four groups and administrated as the same as SHR. At the end of eight weeks,animals were killed .Hearts were excised from SHR and WKY,trimmed free of atria , rinsed in cold 0.9% saline and freeze-clamped in liquid N₂, then powered in liquid N₂ cooled stainless steel mortar pestle . These ventricles were stored in -70℃ until needed for the experiment. On day of experiment, frozen ventricles were resuspended in buffer (100mg/1.0ml).The suspension was homogenized for 10-15 minutes, then centrifugated at 14000 ram at 4℃ for 15 minutes. Protein concentrations were determined by the method of Bradford , using BSA for the standard curve . 20ug(SERCA) and 2ug(PLB)cardiac homogenate proteins from rats were loaded on the same SDS-polyacrylamide gel and separated by electrophoresis (5% acrylamide stacking gel and 12% acylamide separating gel). Afterwards ,the proteins were electrophoretically transferred from the gel at 200mA at 4℃ for 90min onto nitrocellulose membranes with transfer buffer ,then the membranes were washed for one minutes in TBST and blocked with 5% defatted milk in TBST for one hour at room temperature .then the membranes were washed three times with TBST and incubated overnight with monoclonal antibody specific for PLB orSERCA at 1:1000 dilution in 5% defatted milk/TBST.The membranes were washed three times with TBST and incubated for one hour with a secondary anti-mouse antibody conjugated to horseradish peroxidase at 1:500 dilution in 5% defatted milk/TBST. Antigen-antibody complexes of all probed were detected by the enhanced chemiluminescence detection system.The intensity of each band was scanned by an imaging densitometer with the aid of Kodak image analysis system .Difference of "yes or no"is found in the expression of PLB, SERCA protein and ratio of PLB to SERCA between AM group and control group.Results1, Significant difference is found in the expression of PLB, SERCA of SHR andWKY that were given 0.9%-sodium chloride injection,but no significantdefference is found in the ratio of PLB to SERCA. 2, The p...