| Part I The Therapeutic Effects of orientin on Post-myocardial infarction ratsObjectiveThe objective of this experiment was to investigate the therapeutic effects of orientin on post-myocardial infarction rats.MethodsThe rat MI models were established by ligating of the left anterior descending coronary artery. Twenty-four hours later after modeling, the survival model rats were randomly divided into MI group(Model group, n=12), resveratrol group (2.5mg/kg, Res, n=7), orientin low dosage group(1.0mg/kg Ori, n=8), orientin middle dosage group(2.0mg/kg Ori, n=10), and orientin high dosage group(4.0mg/kg Ori, n=7), with another sham-operated group(Sham group, n=11)as control. After3weeks, electrocardiogram (ECG) and hemodynamic index were recorded by PowerLab, activities of cardiac enzyme CK-MB, superoxide dismutase (SOD) and glutathione were measured by colorimetry, left ventricular weight(LVW), the left ventricular weight index(LVWI) and lung weight were calculated. The tissue sample were taken from the left ventricle wall and was stained with hematoxylin and eosin(HE) and examined with a light microscope. The ultrastructure of infarcted region were examined by electric scope.Results1. Compared with the sham-operated group, ligating of the left anterior descending coronary artery induced ST segment elevation(P<0.001vs. Sham); and caused the+dp/dt max and contraction index (CI) decreased, left ventricular end diastolic pressure (LVEDP),-dp/dt max and left ventricular ventricular relaxation time constant (ι) increased (P<0.01or P<0.001vs. Sham). Compared with the sham-operated group, heart weight, left ventricular weight/body weight ratio and lung/body weight ratio increased in MI group (P <0.05or P<0.01vs. Sham). Serum Creatine kinase-MB (CK-MB) activity was significantly increased (P<0.01vs. Sham); heart of rats in model group showed myocardial fiber degeneration and necrosis, telangiectasia, myocardial interstitial edema compared to the Sham-operated group; serum SOD and GSH activity decreased (P<0.05or P<0.01vs. Sham). Observed under common microscope, the HE staining showed that, in the MI rats compared with those in sham-operated group, ventricle wall of the infarct region was very thin and significantly reduced cardiocytes that were substituted by numerous strip-like fasciculation collagen fiber and structurally disordered and infiltrated with considerable inflammatory cells, and in the non-infarcted region of MI rats, compensatory hypertrophy of the survival cardiocytes were noted.2. Intraperitoneal injection of2.0mg/kg Ori for21days induced ST segment height from (0.164±0.022) mV reduced to (0.059±0.028)mV(P <0.05vs. Model),while4.0mg/kg Ori for21days induced ST segment height from (0.164±0.022) mV reduced to (0.046±0.024) mV (P<0.01vs. Model). In rats treated by Ori(1.0mg/kg,2.0mg/kg and4.0mg/kg) compared with those in MI group, hemodynamic indexes were significantly ameliorated (P<0.05vs. Model). Ori(1.0mg/kg,2.0mg/kg and4.0mg/kg) improve hemodynamic parameters in rats,2.0mg/kg and4.0mg/kg Ori increased+dp/dt max from (2185±494) mmHg/s to (5634±391) mmHg/s and (5824±1106) mmHg/s (P<0.01vs. Model), increased the CI from (65±15)s-1to(81±5)s-1and(93±20) s-1(P<0.05or P<0.01vs. Model), while decreased-dp/dt max from (-1782±127) mmHg/s to (-4957±917) mmHg/s,(-5553±550) mmHg/s and (-5783±797) mmHg/s (P<0.01vs. Model), decreased τ value from (0.037±0.013) s to (0.025±0.004) s,(0.024±0.007) sand (0.021±0.009) s (P<0.05or P<0.01vs. Model); therapic given4.0mg/kg Ori also decreased LVEDP from (20±3) mmHg to (11±2) mmHg (P<0.05vs. Model). LVW, LVWI and lung weight in the MI group were significantly elevated compared with the Sham-operated group(P<0.05), therapic given4.0mg/kg Ori also decreased heart weight from (1.20±0.09) g to (0.98±0.14)g (P<0.05vs. Model). therapic given2.0mg/kg and4.0mg/kg Ori decreased heart weight from (3.65±0.01) mg to (3.35±0.12) and (3.26±0.08) mg (P<0.05or P<0.01vs. Model).while ventricular weight index decreased from (0.36±0.02) to (0.31±0.02) and (0.29±0.02)(P<0.05or P<0.01vs. Model) and lung weight index decreased from (4.82±0.34) to (4.27±0.45) and (3.63±0.24)(P<0.05or P<0.01vs. Model). There are no difference in body weight and left ventricular weight between every group (P>0.05vs. Model).3. As compared with the model group, CK-MB decreased markedly in the Ori treated groups(P<0.05vs. Model),2.0mg/kg and4.0mg/kg Ori decreased CK-MB activity from (476±98) U/L to (359±51) U/L and (351±6) U/L (P<0.01vs. Model). Ori (1.0mg/kg,2.0mg/kg and4.0mg/kg) also promoted serum antioxidant enzymes activities,4.0mg/kg Ori increased SOD activity from (63±17) U/mg to(98±4) U/mg(P<0.01vs. Model), while4.0mg/kg Ori increased GSH activity from (12±1) μ mol/L to (17±2) μmol/L (P<0.001vs. Model) 4. As compared with the model group, Ori-treated groups could significantly attenuate myocardial structures, reduce myocardial fiber degeneration and necrosis of edema.ConclusionOri can ameliorate ECG changes, hemodynamics, serum antioxidant enzymes activities, cardiac injury enzymes and myocardial structures in MI rats. Within a concentration of4.0mg/kg, the curative efficacy of orientin may increase as the accumulative dose of the drug increased. Part Ⅱ Influence of orientin on Beclin1/Bcl-2involvement in myocardial infarctionObjectiveTo investigate the effects of orientin on levels of autophagosome and Beclinl/Bcl-2of left ventricle in myocardial infarction rats.MethodsMale Wistar rats were subjected to ligate the anterior descending branch of coronary artery and treated with orientin or physiological saline(intraperitoneal injection) for3weeks. To explore the expression and significance of autophagy-related gene Beclin-1and Microtubule-associated Protein1Light Chain3(LC3) in MI rats, we prove that autophagy take part in the development of ventricular remodeling after MI in rats, and explore the mechanism. So as to provide experimental and theoretical evidence for preventing and treating the development of ventricular remodeling after MI. All the data were analyzed using the software SPSS17.0. The protein expressions of LC3, Beclin1and Bcl-2were measured by western blot in left venticular. The expression of LC3, Beclin1and Bcl-2were assessed by immunohistochemical staining in the non-infarcted region. A part of sample were observed the morphous and the number of the autophagsome by electron microscope as well.Results1. As compared with the Sham-operated group, the expression of LC3, Beclin1in the MI group increased significantly, and the protein of Bcl-2decreased significantly, which demonstrate autophagy related gene Beclin1 increased and the anti-apoptosis gene Bcl-2decreased.2. As compared with the Sham-operated group, the ratio of LC3/β-actin, Beclin1/β-actin in the MI group increased significantly(P<0.001vs. Sham), and the ratio of Bcl-2/p-actin decreased significantly(P<0.001vs. Sham). Observed by electron microscope, the number of the autophagsome of myocardial group increased significantly compared with the Sham-operated group.3. Intraperitoneal injection of Ori for21days decreased the ratio of LC3/β-actin, Beclin1/β-actin and increased the ratio of Bcl-2/β-actin.2.0mg/kg and4.0mg/kg Ori induced LC3/p-actin decreased from (1.98±0.28) to (1.58±0.42)and(1.37±0.16)(P<0.05or P<0.01vs. Mod), while4.0mg/kg Ori induced Beclin1/β-actin decreased from (2.98±0.31) to (2.37±0.40)(P<0.01vs. Mod).2.0mg/kg and4.0mg/kg Ori induced Bc)-2/p-actin increased from (1.97±0.31) to (2.65±0.33) and (2.82±0.37)(P<0.01or P<0.001vs. Mod).4. The result of immunohistochemisty was similar with western blot, which has differences between model and Ori-treated group(P<0.05vs. Mod).Conclusion1. MI is capable of inducing a robust autophagic response in cardiac myocytes, and autophagy may be a pathological change in the process.2. Orientin may ameliorate myocardial infartion through inhibiting autophagic activity, which may act through inhibiting Beclin1and increasing Bcl-2expression. |
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