| Objective:It has become a cardiovascular hot issue to take effective measures to prevent myocardial ischemia-reperfusion injury or reduce the damage degree.With tree shrews as laboratory animals,using Langendorff heart model,applying molecular biology method,the research had analyzed the mechanism in Resina Draconis against myocardial ischemia-reperfusion injury,as to provide theoretical basis for its wide application.Methods:1.The study was established myocardial ischemia reperfusion model to utilize the Langendorff heart perfusion in vitro system with laboratory tree shrews.The experimental groups were:the continuous perfusion control group(groupl),stop infusion 15-min and start reperfusion 30-min group(group2),stop infusion 15-min and start reperfusion 60-min group(group 3),stop infusion 30-min and start reperfusion 30-min group(group 4),stop infusion 30-min and start reperfusion 60-min group(group 5).It could build the reliable laboratory animal models through the comprehensive evaluating the stability of perfusion,the analysis of myocardial enzymology in perfusate and organization,calculating myocardial infarction area in biopsy and so on.Its optimization model would be as a foundation for subsequent experimental drug intervention research.2.We had carried out the drug intervention study with Resina Draconis on the basis of the myocardial ischemia reperfusion Langendorff model in laboratory tree shrew.The experiment was divided into:continuous perfusion in the control group(group 1),ischemia reperfusion group(group 2),ischemia reperfusion group contrasted to solute(group 3),low-dose Resina Draconis intervention group(group 4),medium-dose Resina Draconis intervention group(group 5),high-dose Resina Draconis intervention group(group 6).By means of the laboratory tests,TTC staining observation,TUNEL method and Western blot,the discussion had been probed further into the mechanisms of Resina Draconis against myocardial ischemia-reperfusion injury in studying on oxygen free radical generation,apoptosis,MAPK pathway and endoplasmic reticulum stress respectively.It would provide necessary guarantee for normalized Chinese drug use in domestic.Results:1.In the experiment in building Langendorff model in vitro model,by comparing with the ischemia-reperfusion model groups,we found there had been the extremely significant difference between the five groups in myocardial enzymes of the perfusion fluid and the organization,myocardial infarction area in TTC staining and the ECG situation(p<0.01).There was significantly higher laboratory index and infarction area than the other three groups after ischemia reperfusion in the stop infusion 30-min then reperfusion 30-min group and stop infusion 30-min then reperfusion 60-min group,the myocardial ischemia reperfusion damage was the most serious,but there was no evident difference between the two groups(p>0.05);Contrast to the stability in ECG among groups,the average heart rate of vitro model in the stop infusion 30-min and reperfusion 30-min was close to the control group,whose values was remarkably increased than the stop infusion 30-min and reperfusion 60-min group's(p<0.01).In order to further explore the sources from the difference indexes,we proceeded with the double-factor analysis in each factor level and their interaction,then we found that both the ischemia time and reperfusion time had the outstanding influence in myocardial enzyme levels,although their interaction of the impact was not big,compared to ischemia time,the reperfusion time after ischemia appeared more important.Contrast to different phases of ischemia and reperfusion in these five groups,we found that there had been the extremely differences in myocardial enzymes of perfusion liquid and the tissue,including the infarction area(p<0.05).Both the stop infusion 30-min then reperfusion 30-min group and stop infusion 30-min then reperfusion 60-min group could fully expose the myocardial ischemia-reperfusion injury.2.In the drug intervention experiment with Resina Draconis,there were extremely differences on the levels of myocardial enzymes among these six groups,not only in the perfusion fluid,but also in myocardial tissue(p<0.01).The indexes were significantly higher than the control group's after drug intervention(p<0.05),the medication effect both in medium-dose Resina Draconis intervention group and high-dose Resina Draconis intervention group were better than that in low-dose group,however there was no clear difference between them(p>0.05).The MDA and SOD contents in myocardial tissue had the evident difference among these six groups(p<0.01).The high-dose Resina Draconis intervention group had obvious advantage(p<0.05).Through the TTC staining tissue slices,the infarction areas in ischemia reperfusion group were increased significantly(p<0.05),but that in Resina Draconis intervention groups were decreased distinctly(p<0.05).Comparison with TUNEL apoptosis rate,the high-dose Resina Draconis intervention group's had been declined clearly(p<0.05).Compared to myocardial tissue proteins,the relative quantity of BCL-2 showed a trend of increasing gradually(p>0.05);Bax protein expression and the level of phosphorylated P38 could reveal situation to increase after ischemia reperfusion then decline after drug intervention(p<0.05);the relative phosphorylation level of JNK were found that it would been dropped when finishing drug reperfusion;all the tendency of ERK phosphorylation level,GRP78 and CHOP-1 protein expression were the same as increasing dramatically after ischemia reperfusion and then declining inch by inch after drug intervention(p<0.05);however there was no remarkable difference in these protein contents among the low-dose,medium-dose and high-dose groups(p>0.05).Conclusion:1.It was successful to build myocardial ischemia reperfusion model in vitro with the laboratory tree shrews,which was advisable to stop pouring 30 minutes and start reperfusion 30 minutes.2.In the preparation of ischemia-reperfusion injury model,both ischemia and reperfusion time were the key which would influence the model whether to be successful,in specially the reperfusion time seemed to be more particularly important.3.Chinese medicine of Resina Draconis had the role of clearing oxygen free radicals,improving cell apoptosis and inhibiting endoplasmic reticulum stress,thus it could protect the damaged heart cells.4.The therapy of Resina Draconis existed dose relativity,this research recommended to treat with 0.5mg/ml using Resina Draconis,and there will be a great clinical significance to seek appropriate drug concentration. |
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