酸枣仁总皂苷对过氧化氢损伤心肌细胞的保护作用及机制研究

Protective Effect and Mechanism of Total Saponin from Semen Ziziphi Spiosi in Damaged Myocardial Cells Induced by Hydrogen Peroxide

目的观察酸枣仁总皂苷对过氧化氢损伤心肌细胞的保护作用及其机制。方法采用差速贴壁法分离培养1～3 d Wistar新生乳鼠心肌细胞,以过氧化氢作用心肌细胞建立氧化损伤模型,将培养72 h的原代心肌细胞,选择搏动良好、生长密度无明显差异的细胞按孔随机分为以下4组:①空白对照组:细胞培养过程中不给予任何处理;②模型组:心肌细胞培养基中加终浓度为400μmol.L-1过氧化氢作用4 h;③酸枣仁总皂苷治疗组:操作同模型组,造模前予酸枣仁总皂苷(20 mg.L-1)干预24 h;④蛋白激酶C抑制剂组:操作同治疗组,但在造模前5 min予白屈菜红碱(5μmol.L-)1。倒置显微镜观察心肌细胞形态学变化;四甲基偶氮唑盐(MTT)比色法检测心肌细胞活力;流式细胞仪检测细胞凋亡率;免疫印迹技术检测心肌细胞膜蛋白中蛋白激酶C Eplison亚型(PKCε)的表达。结果与空白对照组比较,模型组经400μmol.L-1过氧化氢作用4 h后,细胞核暗淡,伪足显著变细,细胞间连接明显减少,搏动节律减慢,细胞活力显著下降(P<0.001),细胞凋亡率显著增高(P<0.001),PKCε的表达差异无统计学意义(P>0.05)。与模型组比较,酸枣仁总皂苷能使心肌细胞形态明显改善,显著提高细胞活力、降低细胞凋亡率及升高PKCε的表达,差异有统计学意义(P<0.001)。PKC抑制剂白屈菜红碱可显著减弱酸枣仁总皂苷的保护作用,差异有统计学意义(P<0.01)。结论酸枣仁总皂苷对过氧化氢损伤大鼠心肌细胞具有保护作用,其机制可能与激活PKCε有关。

Objective To study the protective effect and mechanism of total saponin from Semen Ziziphi Spiosi （SZS-TS） on the damage of myocardial cells induced by hydrogen peroxide. Methods Primary cultures of cardiac my- ocytes were prepared from the ventricles of neonate Wistar rats aged 1 - 3 days, and then cultured for 72 hours. Hy- drogen peroxide was used for the induction of oxidative injury myocardial cell models. The well-grown neonate rat my- ocardial cells were divided into four groups. The blank control group had no medication during the culture, the model group was treated with hydrogen peroxide at the final concentration of 400 txmol＇L-1 in the cell culture fluid for 4 hours. SZS-TS group was pretreated with 20 mg＇L-~ of SZS-TS for 24 h, and then was given the same treatment as the model group. Protein kinase C （PKC） inhibitor group was given 5 ~mol＂ L-~ of chelirubine 5 rain before the modeling. Mor- phological changes of primary cultured neonatal rat myocardial cells were observed under the inverted microscope. The viability of myocardial cell was assayed by methyl thiazoly｜ tetrazolium（MTT） method. Apoptotic rate was determined by flow cytometric analysis. Western blotting was used to analyze the expression of PKC Eplison subtype in the cytomem- brane of each group. Results In model group, most cells were crenulated, the nucleus became dim, the pseudopo- dia became significantly thin, cell-cell junction was less, the rhythm of cell beating was decreased significantly, cell viability decreased significantly （P 〈 0.001 ） and apoptotic rate increased significantly （P 〈 0.001 ）, but the expression level of PKC Eplison subtype in the cytomembrane remained unchanged as compared with the blank control group. Comparing with the model group, SZS-TS improved the morphological changes, increased cell viability, decreased apoptotic rate and increased the expression level of PKC Eplison subtype in the cytomembrane significantly（P 〈 0.001 ）. The PKC inhibitor significantly attenuated the protective effects of SZS-TS（P 〈 0.01 ）. Conclusion SZS-TS can reduce the damage of myocardial cells induced by hydrogen peroxide, and the mechanism may he related with the activation of protein kinase C Epsilon subtype.