| Part1Effect of lobelia extract LOB on ischemia-reperfusioninjury in isolated rat heartOBJECTIVE:To observe the influence of LOB on rat heart with ischemia/reperfusion injury in vitro using Langendorff-perfused heart model.METHODS:Sixty SD rats were equally randomized into control group, model group, positive drug (verapamil20μg/L) treatment group and three concentration LOB (100,300and1000μg/L) treatment group. Under the Langendorff-perfused heart models, the left ventricular systolic pressure (LVSP), the maximal rate of the change of intraventricular pressure (±dp/dtmax), heart rate (HR) and coronary flow of the isolated rat hearts of each group were measured. In addition, the creatine kinase (CK) and lactic dehydrogenase (LDH) levels in the coronary effluent volume were determined.RESULTS:Compared with model group, in the three concentrations of LOB groups, the LVSP and±dp/dtmax of the isolated hearts were increased significantly, and meanwhile the HR was reduced and CF was elevated significantly, and all above effects presented a concentration-dependent manner. Compared with control group, the CK and LDH levels in model group were significantly increased, while the three concentrations of LOB significantly inhibited the both increases in a concentration-dependent manner. CONCLUSION:LOB has protective effect against cardiac ischemia-reperfusion injury in vitro, and the mechanism may be probably associated with its improving haemodynamics, reducing oxygen consumption of heart and stabilizing cardiomyocyte membrane.Part2Protective effect of lobelia extract LOB on myocardialischemia-reperfusion injury in vivoOBJECTIVE:To establish rat myocardial ischemia-reperfusion injury animal model using coronary artery ligation. The protection and its mechanism of lobelia extract LOB on myocardial ischemia-reperfusion injury in rats were studied.METHODS:The rat myocardial ischemia-reperfusion injury animal model were established, dynamic ECG situation were observed.10min before the sublingual artery ligation,5mg/kg,10mg/kg,20mg/kg LOB were intravenously injected, respectively, the positive and sham controls were set. At the end of the experiment, the levels of LDH, IL-6in the serum were detected, myocardial infarct sizes were measured and pathological morphological changes were observed, and the MDA content of myocardial tissue was examined.RESULTS:In normal control group, there was no infarction myocardial tissue; in the sham group, there was no significant effect on the myocardial tissue. In the myocardial ischemia-reperfusion group, visible part of the infarct area was observed in myocardial tissue sections, accouts for32.1±3.5%of the total area; however,10min before ischemia, different doses of LOB injected by sublingual vein can significantly lower ischemia-reperfusion injury induced infarct size, compared with the model group, the infarct sizes in the middle、 high-dose groups were smaller, which were of significantly differences. HE staining showed that in the normal control group, structure of myocardial tissue was integrated, clear, the cell morphology and size was regular, no apoptosis and necrosis; in the ischemia-reperfusion injury group, myocardial tissue structure disordered, myocardial cell necrosed.10min before ischemia, different doses of LOB injected by sublingual vein can significantly lower ischemia-reperfusion injury induced pathological morphological changes. After ischemia-reperfusion injury, the levels of LDH, IL-6increased significantly, which had significant differences. Different doses of LOB injected by sublingual vein before ischemia-reperfusion injury could lower ischemia-reperfusion injury induced the increase of LDH activities. Besides, the levels of MDA in the myocardial tissues increased after ischemia-reperfusion injury, compared with normal group, the differences were significant, different doses of LOB could lower ischemia-reperfusion injury induced the increase of MDA levels.CONCLUSION:The rat ischemia-reperfusion model was successfully established, and the protective effects of lobelia extract LOB on myocardial injury were initially confirmed, which may be associated with decreased LDH, IL-6levels and MDA release.Part3Mechanisms of the protective effect of LOB myocardialischemia-reperfusion injuryOBJECTIVE:Myocardial cell hypoxia/reoxygenation injury model was established, and the protective effect of LOB on myocardial injury was observed, and its possible mechanism was preliminary studied.METHODS:H9C2(2-1) myocardial cells were normally cultured, and the myocardial hypoxia/reoxygenation model was established.1h after different doses of LOB were added, the myocardial hypoxia/ reoxygenation injury was carried out, which was the experimental group, the normal controls and positive controls were set. MTT assay was used to detect the cell viability; the LDH, IL-6, MDA levels were detected by LDH, IL-6, MDA kits, respectively; Cell apoptosis of each group was detected by TUNEL assay; the activity of Caspase-3were detected using Caspase-3detection kit; while, the expression of IL-6mRNA were detected by RT-PCR method.RESULTS:After hypoxia/reoxygenation injury, compared with the normal control group, myocardial cell viability decreased significantly, three different concentrations of LOB can minimize the decrease of hypoxia/reoxygenation-induced myocardial cell viability; After hypoxia/reoxygenation injury, the levels of LDH, MDA increased significantly, compared with normal controls, the difference was significant;1h before the hypoxia/reoxygenation injury, three concentrations of LOB (10-6,3×10-6and10-5M) can lower the increase of hypoxia/reoxygenation-induced LDH activity, MDA content. We detected the expression of IL-6mRNA in the myocardial cells and IL-6levels in the culture medium, we found that compared with the normal control group, hypoxia reoxygenation injury could induce the elevation of IL-6mRNA and IL-6levels significantly.1h before the hypoxia/reoxygenation injury, three concentrations of LOB (10-6,3×10-6and10-5M) can lower the increase of hypoxia/reoxygenation-induced IL-6levels. The cell apoptosis was detected by TUNEL assay, and the activity of caspase-3was also detected, we found that compared with the normal control group, hypoxia reoxygenation injury could induce the elevation of cell apoptosis rate and the activities of caspase-3significantly.1h before the hypoxia/reoxygenation injury, three concentrations of LOB (10-6,3×10-6and10-5M) can lower the increase of hypoxia/reoxygenation-induced cell apoptosis and activities of Caspase-3. CONCLUSION:LOB can protect myocardial cells, which may be associated with decrease of LDH activity and MDA content; reduction of the expression and secretion of IL-6; reduction of myocardial cell apoptosis. |
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