| Cyclovirobuxine D (CVB-D) is effective monomer of Buxus alkaloids, the main composition of Huangyangning tablets.It’s mainly treatment for the clinical is myocardial ischemia, coronary heart disease and other cardiovascular diseases. In recent years, the research of Cyclovirobuxine D is mainly in treating cardiac and cerebral vascular diseases. Previous studies have shown that:CVB-D has a protective effect on heart failure, and this effect is related to the inhibition of cardiac Na+-K+-ATPase.This study explores whether the positive inotropic effect of CVB-D on heart failure is associated with cardiac Na+-K+-ATPase and the effect of Na+-K+-ATPase subunit al,a2,a3,β1.The experimental modeling methods:ligating the left anterior descending coronary artery, copy the rat model of heart failure.Detecting echocardiographic, viewing parameters, determines whether the model is successful.The standard of a successful model is EF<50%.After model established, grouping and administrating,60days later, general condition of the rats was observed (survival rate, diet, movement, etc.). Detecting echocardiographic once again, observes the improvemen of heart function on rats with heart failure.Hemodynamics:systolic pressure, diastolic pressure, mean pressure, changes in pulse pressure; left ventricular systolic pressure, the left ventricular end diastolic pressure, left ventricular pressure and the maximum change rate, left ventricular systolic beginning to change to the largest indoor pressure time, judging the role of CVB-D to cardiac contraction,heart function, blood pressure on rats with heart failure.Morphology:weighing animal ventricular mass, calculation of correlation index, making myocardial tissue sections, HE staining, observing lesions, severity of heart, and observing the ultrastructure changes through transmission electron microscope.The content change of heart failure myocardial tissue is detected by LDH, MDA, SOD.Cell experiment:tissue adherent culturing primary myocardial cells, identifying the purity of primary myocardial cell. Copying the model of hypoxia/reoxygenation injury in vivo, imitating the model of heart failure. Detecting cell survival rate with MTT method, detecting the content change of LDH, MDA, SOD of myocardial cells and the change of Na+-K+-ATPase activity is to observe the effect of CVB-D to myocardial cell of hypoxia/reoxygenation injury.Copying the model of heart failure, detecting Na+-K+-ATPase activity in myocardial tissue of rats with heart failure by Elisa, detecting the changes of protein expression of Na+-K+-ATPase α1, α2, α3, β1by Western Blot, detecting the changes of mRNA expression of Na+-K+-ATPase α1, α2, α3, β1subunit.The experimental results show: Echocardiography:compared with the sham group, the model group EF,LVFS,SV,CO, LVPWs decreased significantly; LVIDd, LVIDs increased significantly (P<0.05,P<0.001). Compared with the model group, high dose group of CVB-D of EF, LVFS increased significantly (P<0.05, P<0.01); low, middle dose group of CVB-D of SV increased (P<0.05); low, middle and high dose group of CVB-D, showed increasingly on CO (P<0.05). At the same time, compared with the model group, high dose group of CVB-D showed that LVPWs increased, but LVPWs decreased (P<0.05).Hemodynamics:compared with the sham group, the model group hemodynamic indexes decreased, including±dp/dtmax, LVSP, systolic pressure, diastolic pressure, diastolic pressure and mean pressure; at the same time, LVEDP (P<0.05,P<0.01) increased. Compared with model group,(0.5,1,2mg/kg) three dose groups of CVB-D increased±dp/dtmax, LVSP, systolic pressure, diastolic pressure, diastolic blood pressure, and LVEDP decreased significantly (P<0.05,P<0.01).Morphology:compared with the sham group, the model group, HMI and LVMI significantly increased, suggesting the heart of rats of the model group have a certain degree of hypertrophy. Each group of CVB-D decreased HMI and LVMI (P<0.05,P<0.01). HE staining showed:low, middle, high dose group of CVB-D myocardial fiber tapers, muscle fiber gradually tidy,"branches" and "vortex" shape gradually disappeared, nuclear staining shallowed relatively uniform and regular in shape. Transmission electron microscopy showed that each dose group of CVB-D muscle fibers were improved, muscle fiber fracture were reduced, the number of mitochondria were increased, reducing swelling and degeneration were reduced. Ridge increased, became clarity, and the density is improved.Assay:compared with the sham group, the model group reduced the content of SOD, LDH, and increased the content of MDA significantly (P<0.05,P<0.01). Three dose groups of CVB-D increased the content of SOD, decreased the content of MDA significantly, compared with the model group (P<0.05,P<0.01). Delivery only the high dose group of CVB-D decreased LDH (P<0.05).Hypoxia and reoxygenation injury:MTT results showed that CVB-D10-4,10-5,10-6mol/L had promoted the proliferation of primary myocardial cells. The effect of CVB-D on hypoxia/reoxygenation myocardial cell after12hours, was that increased the cell myocardial cell vitality and adherence. The content of SOD, MDA of CVB-D group, compared to the model group were significantly increased (P<0.05, P<0.01). The releasing of LDH of high dose CVB-D group, compared with the model group (P<0.05) was different.Compared with the model group, low dose CVB-D group increased Na+-K+-ATPase activity (P<0.05), and the middle, high dose CVB-D group increased Na+-K+-ATPase activity significantly (P<0.01). Experiment of animal heart failure showed:compared with the sham group, model group, the Na+-K+-ATPase activity was significantly decreased (P<0.01); compared with the model group, CVB-D on each group significantly increased the activity of Na+-K+-ATPase. Compared with the sham group, the protein expression of Na+-K+-ATPasea1, a2, a3, β1of the model group decreased significantly (P<0.01); compared with the model group, CVB-D on each group of Na+-K+-ATPase α1,α2,α3,β1protein expression was significantly increased (P <0.01).Compared with the sham group, the genes expression of model group, Na+-K+-ATPase a1α2, α3,β decreased significantly (P<0.01);Compared with model group, CVB-D on each group of Na+-K+-ATPase α1, α2, α3, β1gene expression was significantly increased (P<0.01)... |
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