醋炙降低芫花二氯甲烷部位对大鼠小肠隐窝上皮细胞IEC-6毒性的研究

Vinegar processing attenuates toxicity on IEC-6 cells caused by chloroform extraction of Daphne genkwa

该实验以大鼠小肠隐窝上皮细胞IEC-6为研究对象,比较醋炙前后芫花二氯甲烷部位对IEC-6细胞的毒性差异,初步探讨芫花醋炙减毒的机制。通过MTT法筛选芫花各极性部位对于IEC-6细胞毒性,比较醋炙前后芫花毒性部位对IEC-6细胞活性的影响;通过测定IEC-6细胞中的乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、Na~＋-K~＋-ATP酶,Ca^（2＋）-Mg^（2＋）-ATP酶等酶的活力和谷胱甘肽（GSH）、丙二醛（MDA）的含量评价IEC-6细胞氧化损伤程度;使用流式细胞术检测IEC-6细胞的细胞周期和细胞凋亡情况。结果表明芫花二氯甲烷部位为致IEC-6细胞的毒性部位,醋炙后二氯甲烷部位毒性显著降低（P〈0.01）。与空白组相比,芫花二氯甲烷部位可显著降低IEC-6细胞中Na~＋-K~＋-ATP酶、Ca^（2＋）-Mg^（2＋）-ATP酶、SOD活力（P〈0.01）,显著提高细胞上清液LDH,MDA含量,显著降低细胞中GSH的含量（P〈0.01）。同时,芫花二氯甲烷部位能显著提高IEC-6细胞的早期与晚期凋亡率,显著降低G1期细胞百分比,增加S与M期细胞百分比（P〈0.01）。醋炙后,与相应芫花生品二氯甲烷组相比,醋芫花二氯甲烷组可显著提高IEC-6细胞内Na~＋-K~＋-ATP酶、Ca^（2＋）-Mg^（2＋）-ATP酶、SOD活力（P〈0.01）,显著降低细胞中LDH,MDA含量（P〈0.01）,显著提高细胞中GSH含量（P〈0.01）。显著降低IEC-6细胞的早期凋亡率、晚期凋亡率（P〈0.01）,显著增加G1期细胞百分比,减少S与M期细胞百分比（P〈0.01）。提示醋炙可降低芫花二氯甲烷部位致IEC-6细胞的毒性,其可能机制为提高细胞中抗氧化酶的活性与细胞膜的通透性,降低氧化损伤而实现。

To screen the toxic polar fractions of Daphne genkwa,compare the toxicity of D. genkwa on crypts epithelial cells IEC-6 before and after vinegar processing,and preliminarily investigate the mechanism of D. genkwa vinegar processing on toxicity reducing. The proliferation of IEC-6 cells was observed by MTT. The levels of superoxide dismutase（ SOD）,malondialdehyde（ MDA）,glutathione（ GSH）,lactate dehydrogenase（ LDH）,as well as the enzyme activity of Na~＋-K~＋-ATPase and Ca^（2＋）-Mg^（2＋）-ATPase were determined in IEC-6 cells to evaluate the oxidative damages degree of IEC-6 cells. The apoptosis and cell cycle were analyzed by Flow Cytometry. The results showed that the dichloromethane extraction was the toxic polar fraction of D. genkwa,and after vinegar processing,the toxicity of dichloromethane fraction was significantly reduced（ P 0. 01）. As compared with the blank control group,the dichloromethane fraction of D. genkwa can obviously decrease the levels of SOD,Na~＋-K~＋-ATPase,Ca^（2＋）-Mg^（2＋）-ATPase（ P 0. 01）and content of GSH,but increase the level of LDH and MDA in cell supernatant（ P 0. 01）. Besides,it obviously increased the early and late apoptotic rate of IEC-6 cells,obviously decreased the proportion of G1 stage cells,increased the ratio of S stage cells and M stage cells（ P 0. 01）. After vinegar processing,as compared with D. genkwa groups of various doses,it can significantly increase the levels of SOD,Na~＋-K~＋-ATPase,Ca^（2＋）-Mg^（2＋）-ATPase（ P 0. 01） and content of GSH,decrease the level of LDH,MDA（ P 0. 01）,significantly decrease the early and late apoptosis rate of IEC-6 cells（ P 0. 01）,increase the proportion of G1 stage cells,and decrease the ratio of S stage cells and M stage cells（ P 0. 01）. Vinegar processing can reduce the toxicity of dichloromethane fraction of D. genkwa,and its mechanism may be associated with improving the activity of antioxidant enzymes and permeability in IEC-6 cells,and decreasing the oxidative damage.