| Objectives: To evaluate the protective effect of Cuscuta chinensis Lam. Flavonoids(CCLF) on the ischemia-reperfusion injury in isolated working rat hearts, and the interactive functional relationship between left ventricle systolic function, necrosis,apoptosis, antioxidant capacity and energy metabolism.Methods: 30 Sprague-Dawley rats were randomly divided into three groups(n=10): model group(MOD), control group(CK) and CCLF group. The ischemia-reperfusion injury model that use the isolated working rat hearts were reperfused with Krebs-Henseleit buffer solution 40 min after global ischemia 30 min. For CK group, the hearts were perfused with Krebs-Henseleit solution last for about 90 min. For CCLF group, the CCLF was added to the Krebs-Henseleit solution(25mg/L) before global ischemia 10 min. The pressure sensing probe attached to the physiological recorder was inserted into the left ventricle though the aortic valve to monitor the left ventricle systolic function. The aortic blood flow and coronary blood flow were measured during perfusion. Before experiment end, the coronary effluent was saved for creatine kinase(CK) measurement. The left ventricle tissue that taken down after reperfusion weighed for myocardial index(HMI), fixed for histopathology HE coloration and TUNEL, cryopreserved for detection of biochemical indexes such as cytochrome C, caspase-9, ATPase, ATP, glycogen, SOD and MDA. In addition,Using different models explored the method of acute separation of rat cardiomyocytes.Results:(1) The improved Langendorff perfusion apparatus can make the hearts survived more than 90 min, which satisfied the requirement of the experiment.(2) HMI and myocardial enzyme:Compared to CK group hearts, both of HMI and creatine kinase of MOD group hearts have a significant rise(p<0.01).(3) Left ventricle systolic function: CCLF can increase the decline of The Leftventricle maximum systolic Pressure(Max pressure), Left ventricular pressure maximum increase/decrease rate(±dP/dt), Aortic blood Flow(AF), Coronary blood Flow(CF), Cardiac Out-put(CO) and Stroke Volume(SV) which caused by ischemia-reperfusion injury. Meanwhile, reduced the raise of Heart Rate(HR), The left ventricular end diastolic pressure(LVEDP), Tau, Squeezing Duration(SD), caused by damage, all had significant difference(p<0.05, p<0.01).(4) Antioxidant capacity: Compared to CK group hearts, the MDA in myocardial tissue of MOD group hearts have a significant rise which reduced by the administration of CCLF. The activity of superoxide dismutase(SOD) including T-SOD, Cu-Zn-SOD and Mn-SOD have a significant decline which recovered by CCLF(p<0.05, p<0.01).(5) Compared to CK group hearts, the activity of ATPase, the content of ATP and glycogen declined significantly, and CCLF group hearts have a recovery phenomena(p<0.05, p<0.01).(6) Apoptosis index: The result of TUNEL show that there was no appearance of apoptotic cells. Compared to CK group hearts, the release of cytochrome c and activation level of caspase-9 were significant raised, the CCLF group have a declining trend(p<0.05, p<0.01).(7) The cardiomyocytes isolated by enzymolysis of collagenase can be used for giga seal, satisfied the basic requirements of patch clamp experiment.Conclusion: Comprehensive analysis of the experimental results show that CCLF has certain protective effect on myocardial tissue cells after suffering ischemia-reperfusion injury. The effect performance in reducing the HMI and seepage of CK, enhancing the cardiac systolic function, improving the integrity of cardiac muscle fibers. The probable mechanism is that CCLF can raise the level of endogenous antioxidant, decrease the production of ROS, reduce calcium overload, inhibit the activation rate of caspase family which has a big damage on cell structure and function. |
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