五甲基槲皮素预处理对心肌细胞缺氧复氧损伤的保护机制研究

发布时间：2018-10-12 18:53

【摘要】：目的：槲皮素甲基化改造得到的五甲基槲皮素（PMQ），对心肌缺血再灌注损伤具有保护作用。本实验采用原代SD大鼠乳鼠心肌细胞，建立心肌缺氧/复氧（anoxia/re-oxygenation, A/R）损伤模型，探讨五甲基槲皮素预处理对大鼠心肌细胞缺氧/复氧损伤的保护作用及其对线粒体功能的影响。方法：培养原代SD大鼠乳鼠心肌细胞，随机分为8组，每组实验重复3次：（1）正常对照组（Cont）；（2）Cont+DMSO组；（3）缺氧/复氧组（A/R）；（4）A/R+DMSO组；（5）缺氧预适应的延迟相组（DPC）；（6）10μmol/L PMQ+A/R组；（7）30μmol/L PMQ+A/R组；（8）100μmol/L PMQ+A/R组。用MTT比色法检测细胞存活率,Beckman生化自动分析仪检测培养液中乳酸脱氢酶(LDH)活性，，酶联免疫检测仪测定各组脂质过氧化物丙二醛（MDA）含量、抗氧化物酶（SOD）活性、谷胱甘肽过氧化物酶（GSH-Px）的活性；流式细胞法检测线粒体膜电位（Δψm）以及细胞凋亡情况、线粒体肿胀法检测各组心肌细胞线粒体通透性转换孔(mPTP)开放情况。结果：与Cont组相比，A/R组的各项指标均显示细胞凋亡增加（P0.01）,说明A/R模型构建成功；与A/R组相比，DPC组的各项指标均显示细胞凋亡减少，说明DPC模型构建成功（P0.01）；与A/R组相比，不同剂量PMQ (10,30,100μmol/L)预处理24h组，其呈现剂量依赖性的使LDH活性降低、细胞存活率增加、MDA含量显著减少、SOD酶活性显著上升、GSH-Px酶活性显著上升、细胞凋亡减少（P0.05或P0.01）；与A/R组相比，30,100μmol/L PMQ预处理24h组，其线粒体膜电位更为稳定、mPTP开放减少（P0.05或P0.01）。结论：PMQ预处理24h后，可产生药理性延迟保护作用，其作用机制与抑制氧化应激、稳定线粒体膜电位、抑制mPTP开放，进而减少细胞凋亡有关。
[Abstract]:Aim: to study the protective effect of pentamethylquercetin (PMQ), on myocardial ischemia reperfusion injury. In this study, we established myocardial hypoxia / reoxygenation (anoxia/re-oxygenation, A / R) injury model by primary SD rat neonatal cardiomyocytes, and investigated the protective effect of pentamethylquercetin preconditioning on hypoxia / reoxygenation injury of rat cardiomyocytes and its effect on mitochondrial function. Methods: primary SD rat cardiomyocytes were cultured and randomly divided into 8 groups: (1) normal control group (Cont); (2) Cont DMSO group, (3) anoxic / reoxygenated group (A / R); (4) A / R DMSO group, (5) anoxic preconditioning delayed phase group (DPC); (6) 10 渭 mol/L PMQ A / R group, and (3) anoxia / reoxygenation group (A / R); (4) A / R DMSO group. (7) 30 渭 mol/L PMQ A / R group and (8) 100 渭 mol/L PMQ A / R group. The cell survival rate was detected by MTT colorimetry, the activity of lactate dehydrogenase (LDH) was detected by Beckman biochemical automatic analyzer, the content of lipid peroxide malondialdehyde (MDA) and the activity of antioxidant enzyme (SOD) (SOD) were measured by enzyme-linked immunosorbent assay (Elisa). The activity of glutathione peroxidase (GSH-Px), mitochondrial membrane potential (螖蠄 m) and apoptosis were detected by flow cytometry, and the opening of mitochondrial permeability transition pore (mPTP) was detected by mitochondrial swelling method. Results: compared with the Cont group, all the indexes in the A / R group showed an increase in apoptosis (P0.01), which indicated that the A / R model was successfully constructed, the DPC group showed a decrease in apoptosis, which indicated that the DPC model was successfully constructed (P0.01), and compared with the A / R group, all the indexes in the DPC group showed that the model was successfully constructed (P0.01). After pretreatment with different doses of PMQ (10 ~ 30100 渭 mol/L) for 24 h, the LDH activity decreased in a dose-dependent manner, the cell survival rate increased, the MDA content decreased, the activity of SOD increased significantly, the activity of GSH-Px increased significantly and the apoptosis of cells decreased in a dose-dependent manner (P0.05 or P0.01). Compared with the A / R group, the mitochondrial membrane potential of 30100 渭 mol/L PMQ pretreated for 24 hours was more stable and the opening of mPTP was decreased (P0.05 or P0.01). Conclusion: after pretreatment with PMQ for 24 h, the pharmacological delayed protective effect can be produced. The mechanism is related to the inhibition of oxidative stress, the stabilization of mitochondrial membrane potential, the inhibition of opening of mPTP, and the reduction of apoptosis.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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