摘要
目的:探讨灯盏乙素苄酯衍生物对乳鼠心肌细胞缺血损伤的保护作用及其抗凋亡机制。方法:体外原代培养SD乳鼠的心肌细胞,将搏动状态良好且生长密度无明显差异的心肌细胞随机分为5组:正常对照组、缺血模型组、灯盏乙素苄酯衍生物组(高、中、低剂量分别为100、50、25μmol/L,缺血造模前加入干预药物)。通过氧糖剥夺模型体外模拟心肌缺血损伤,缺血6h后,通过MTT法测定各组细胞代谢活力,采用生化手段检测各实验组培养基中乳酸脱氢酶活性,采用Hoechst-碘化丙啶双染色法观察各组心肌细胞核的形态改变,采用流式细胞术测定各组心肌细胞凋亡率,通过逆转录聚合酶链反应技术检测各组细胞的细胞色素c和caspase-3在mRNA水平的表达,通过蛋白质印迹技术检测各组细胞的细胞色素c和caspase-3在蛋白水平的表达。结果:与正常对照组相比,缺血后模型组心肌细胞的代谢活力显著下降(P<0.01)。与模型组相比,灯盏乙素苄酯衍生物可明显减轻缺血损伤心肌细胞代谢活力的丧失(高剂量P<0.01,中剂量P<0.05);各剂量灯盏乙素苄酯衍生物可显著减少缺血损伤心肌细胞乳酸脱氢酶的外漏量(P<0.01);形态学方面,灯盏乙素苄酯衍生物可有效减轻缺血损伤心肌细胞的染色质凝集、细胞核固缩等凋亡形态学改变;灯盏乙素苄酯衍生物能明显降低缺血损伤心肌细胞的凋亡百分率(P<0.01);灯盏乙素苄酯衍生物可显著抑制缺血损伤心肌细胞的细胞色素c和caspase-3在mRNA及蛋白水平的表达(P<0.01)。结论:灯盏乙素苄酯衍生物对体外培养心肌细胞的缺血损伤具有显著的保护作用。这种保护作用的机制可能是通过抑制细胞色素c释放和caspase-3活化从而阻断细胞凋亡的途径而实现的。
Objective： To investigate the protective effects of scutellarin benzyl ester on neonatal rats＇ cardiomyocytes injured by ischemia and its anti-apoptosis mechanism.Methods： The cardiomyocytes in primary culture were prepared from ventricular tissue of 1-to 3-day-old Sprague-Dawley rats and the cells in good condition were assigned to five groups： control group, ischemic model group and three scutellarin benzyl ester groups （doses of 100, 50 and 25 μmol/L, respectively）. The model of ischemic injury was established in the primary culture of cardiomyocytes under glucose-free anoxic condition. After ischemia for 6 h, the metabolic ability of the cells was detected by MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide） assay and the activity of lactate dehydrogenase （LDH） in the media was measured by biochemistry approaches. The nuclear damage was revealed by Hoechst-propidium iodide staining. The percentage of apoptotic cells was monitored by flow cytometry. The expression levels of cytochrome c and caspase-3 mRNAs and proteins were determined by reverse transcription-polymerase chain reaction and Western blotting, respectively.Results： After exposure to ischemic condition, the cell viability of the model group was degraded compared with that of the control group （P0.01） and scutellarin benzyl ester （high and medium doses） could attenuate the loss of cell viability induced by ischemia （P0.01 and P0.05）. In addition, each dose of scutellarin benzyl ester could significantly reduce the release of LDH from cardiomyocytes injured by ischemia （P0.01）. In morphology, the injured nuclei presented significant changes such as condensation of chromatin, and shrinkage and fragmentation of nuclei, which could be attenuated remarkably by pretreatment with scutellarin benzyl ester. Furthermore, scutellarin benzyl ester could significantly decrease the percentage of apoptosis induced by ischemia （P0.01） and inhibit the increased expression levels of cytochrome c and caspase-3 mRNAs and proteins （P0.01）.Conclusion： Scutellarin benzyl ester has a remarkable protective effect against myocardial ischemic injury and the protective mechanism may associate with its anti-apoptosis effect by inhibiting cytochrome c release and caspase-3 activation.
出处
《中西医结合学报》
CAS
2011年第9期1014-1021,共8页
Journal of Chinese Integrative Medicine
基金
国家自然科学基金资助项目(No.20902110)
天津市应用基础及前沿技术研究计划资助项目(No.10JCYBJC14700)
武警医学院重点资助项目(No.WYZ201101)
武警医学院面上资助项目(No.WY200908)
武警医学院重点实验室开放基金资助项目(No.WZK200901)~~
