| Part I The Preventive Effects of Orientin on Isolated and Ischemia-reperfusion Rat HeartsObjective:The objective of this experiment was to investigate the preventive effects of orientin on isolated and ischemia-reperfusion rat hearts.Methods:70mature male wistar rat were randomLy divided into7groups(n=10):normal control group (Con), ischemia-reperfusion group (Model group), resveratrol group (1.5mg/kg, Res), orientin low dosage group(1mg/kg Ori), orientin middle dosage group (2mg/kg Ori), orientin high dosage group (4mg/kg Ori) and2mg/kg Ori+wortmannin group (2mg/kg Ori+wortmannin15μg/kg, OW).Each group was Intraperitoneal injected of corresponding medicine for7days, Wortmannin was given30minutes before the experiment. Hemodynamic index was recorded by PowerLab during the experiment. Activities of cardiac enzyme creatine kinase-MB (CK-MB) and lactic dehydrogenase (LDH) were measured by automatic biochemical analyzer. Pathological changes were observed by a light microscopy and the ultrastruetural changes were examined with an electron microscopyobserve.Results:1. After ischemia for30min and reperfusion for120min, the CF, LVDP and+dp/dtmax were decreased compared with the control group (P<0.01); leakage rates of LDH and CK-MB were significantly higher than the control group (P<0.01). Observed under common microscope, the HE staining sections showed that the cardiac myocyte was hypertrophy, and cellinterstitial was severe edema in MERI rat hearts compared with those in control group; muscle fiber derangement and dissolve were also observed. Electron microscopy showed that muscle wires were disorganized and myofibrils were dissolved and fractured. Swelling of mitochondria and disarranged cristae could be observed as well.2. Intraperitoneal injection of Ori for7days significantly ameliorated hemodynamic indexes compared with the model group. Injection of4mg/kg Ori induced CF increased from (6.15±1.27) mL/min to (8.20±1.72) mL/min (P <0.05vs. Model). Injection of2mg/kg Ori and4mg/kg Ori induced LVDP increased from (32.73±.71) mmHg to (47.32±8.77) mmHg and (46.52±9.50) mmHg (P<0.01&P<0.01vs. Model). Injection of2mg/kg Ori and4mg/kg Ori induced+dp/dtmax increased from (1373.58±371.31) mmHg/s to (2127.41±314.25) mmHg/s and (1986.76±348.08) mmHg/s (P<0.001&P <0.01vs. Model). Injection of2mg/kg Ori and4mg/kg Ori induced-dp/dtmax increased from (-677.76±107.42) mmHg/s to (-956.30±186.71) mmHg/s and (-951.26±192.17) mmHg/s (P<0.01&P<0.01vs. Model)3. Orientin groups could reduce the enzyme leakage of LDH and CK-MB caused by the ischemia-reperfusion injury compared with the modle group during reperfusion. For reperfusion30min, injection of Ori(1mg/kg,2mg/kg and4mg/kg) induced enzyme leakage of LDH reduced from (53.40±7.54) U/L to (18.17±2.56,14.72±1.37&14.22±1.49) U/L (P<0.001, P<0.001&P<0.001vs. Model),and that of CK-MB reduced from (24.58±4.55) U/L to (7.33±0.84,7.87±1.50&7.40±1.74)U/L(P<0.001, P<0.001&P<0.001vs. Model). For reperfusion60min, injection of Ori(1mg/kg,2mg/kg and4mg/kg) induced enzyme leakage of LDH reduced from (42.50±6.53) U/L to (10.33±1.41,9.50±0.71&10.72±0.80) U/L (P<0.01, P<0.05&P<0.01vs. Model),and that of CK-MB reduced from (14.28±2.22) U/L to (3.97±1.27,4.08±0.96&6.08±1.47) U/L (P<0.01, P<0.01&P<0.01vs. Model). For reperfusion120min, injection of Ori(1mg/kg,2mg/kg and4mg/kg) induced enzyme leakage of LDH reduced from (83.75±10.12) U/L to (13.83±0.83,14.56±0.51&15.06±1.83) U/L(P<0.01, P<0.01&P<0.01vs. Model),and that of CK-MB reduced from (29.30±4.01) U/L to (5.67±0.47,7.33±1.54&6.87±1.86) U/L (P<0.01, P<0.01&P<0.05vs. Model)4. After reperfusion120min, group the LVDP and±dp/dtmax of2mg/kg Ori were higher than the orientin+Wortmannin group (P<0.01),and enzyme leakage of LDH and CK-MB were lower than Wortmannin group (P<0.001)5. Compared the HE stained sections with model group, orientin reduced MIRI rat myocardial fibrosis and necrosis, as well as the extent of cell edema. Orientin also improved myocardial structural damage and arrangement of muscle fibers. Mitochondrial swelling and cristae disorder were improved under the electron microscopy.Conclusion:Orientin can ameliorate hemodynamic indexs, enzyme leakage rates of CK-MB and LDH and myocardial pathological damage caused by the ischemia-reperfusion injury. The preventive effects of orientin on isolated and ischemia-reperfusion rat hearts is notable, and those effects are greatly weakened by autophagy inhibitor Wortmannin. Part Ⅱ Cardioprotection Mechanism of Orientin on Isolated and Ischemia-reperfusion Rat Hearts via Autophagy Involving the AMPK-mTOR PathwayObjective:The objective of this experiment was to investigate the effects of orientin on levels of autophagy and the AMPK-mTOR pathway in isolated and ischemia-reperfusion rat hearts.Methods:Establish the Langendorff model of rats hearts, levels of expression and phosphorylation of microtubule-associate protein1light chain3(LC3),mammalian target of rapamycin (mTOR) and AMP-activated kinase (AMPK) in tissue homogenates of left ventricular were detected by western blot. Photo grayscales were determined and analyzed with software Image Lab. The morphous and the number of the autophagsome in isolated and ischemia-reperfusion rat heartss were observed with an electron microscope as wellResults:1. Ischemia-reperfusion induced changes with LC3、mTOR、 AMPK protein expression and phosphorylation on isolated rat hearts. The ratio of LC3-Ⅱ/LC3-I of the model group rised compared with the model group (P<0.01). The ratio of p-mTOR Ser2448/mTOR of the model group reduced compared with the model group (P<0.01). The ratio of p-AMPK Thr172/AMPK of the model group rised compared with the model group(P<0.01)2. Intraperitoneal injection of Ori induced changes with LC3、mTOR、 AMPK protein expression and phosphorylation on isolated rat hearts. Injection of Ori(1mg/kg,2mg/kg and4mg/kg) induced LC3-Ⅱ/LC3-Ⅰ raised from (0.58±0.12) to (0.71±0.11,0.74±0.17&0.81±0.13)(P<0.05, P<0.05&P <0.01vs. Model). Injection of Ori(1mg/kg,2mg/kg and4mg/kg) induced p-mTOR Ser2448/mTOR reduced from (0.17±0.02) to (0.13±0.03,0.12±0.01&0.11±0.02)(P<0.01, P<0.01&P<0.01vs. Model). Injection of Ori(1mg/kg,2mg/kg and4mg/kg) induced p-AMPK Thr172/AMPK raised from (0.26±0.03) to (0.33±0.03,0.35±0.04&0.38±0.05)(P<0.05, P<0.01&P<0.01vs. Model)3. Compared with the2mg/kg Ori group, the ratio of LC3-Ⅱ/LC3-Ⅰ of the OW group was reduced (P<0.01). The ratio of p-mTOR Ser2448/mTOR of the model group was rised compared with the OW group (P<0.01). The ratio of p-AMPK Thr172/AMPK of the model group was reduced compared with the OW group (P<0.01)4. Bilayer-membrane autophagosome of different periods could be observed under electron microscopy in each group.Conclusion:Orientin can upregulate the level of autophagy. The mechanism may relate to the activation of AMPK and inhibition of mTOR. |
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