竹叶黄酮对过氧化氢诱导的奶牛乳腺上皮细胞氧化损伤的保护作用

Protective Effect of Bamboo Leaf Flavonoids on Oxidative Damage of Bovine Mammary Epithelial Cells Induced by Hydrogen Peroxide

本试验旨在筛选建立过氧化氢(H_(2)O_(2))诱导的奶牛乳腺上皮细胞氧化应激模型的最佳条件,并探究竹叶黄酮(BLF)对奶牛乳腺上皮细胞氧化损伤的保护作用。以奶牛乳腺上皮细胞MAC-T细胞系为研究对象,将维持培养基中处理的MAC-T设为对照组,在维持培养基中添加不同浓度(200~1000μmol/L)H_(2)O_(2)处理的MAC-T设为氧化损伤组,在维持培养基中添加80μg/mL BLF和800μmol/L H_(2)O_(2)处理的MAC-T设为BLF预处理组。采用细胞增殖试剂盒(CCK-8)法检测细胞活力,采用乳酸脱氢酶(LDH)法检测细胞凋亡率,每次试验平行孔6个,重复试验3次。利用倒置荧光显微镜观察细胞内活性氧(ROS)含量的变化,利用试剂盒法检测细胞内抗氧化指标的变化,利用实时荧光定量PCR法检测细胞线粒体损伤相关基因表达的变化,利用流式细胞仪检测细胞线粒体膜电位(MMP)的变化。结果显示:1)600和800μmol/L H_(2)O_(2)刺激细胞6和8 h符合建模条件,细胞活力为80.77%~72.81%,细胞凋亡率为20.79%~43.52%;2)与对照组相比,800μmol/L H_(2)O_(2)刺激细胞8 h可显著提升ROS含量(P<0.05),显著抑制过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和总超氧化物歧化酶(T-SOD)活性(P<0.05);3)与对照组相比,800μmol/L H_(2)O_(2)刺激细胞8 h对B淋巴细胞瘤/白血病-2(Bcl-2)的mRNA相对表达量无显著影响(P>0.05),但可显著增加B淋巴细胞瘤/白血病-2相关X蛋白(Bax)的mRNA相对表达量(P<0.05),并使Bax/Bcl-2比值显著增加(P<0.05),同时显著增加MMP去极化细胞比例(P<0.05);4)与氧化损伤组相比,80μg/mL BLF预处理24 h能显著提高细胞活力(P<0.05),显著降低ROS含量(P<0.05),显著提高CAT和GSH-Px活性(P<0.05),显著降低Bax的mRNA相对表达量(P<0.05),显著提高Bcl-2的mRNA相对表达量(P<0.05),使Bax/Bcl-2比值显著降低(P<0.05)。综上所述,本试验成功建立了H_(2)O_(2)诱导的MAC-T氧化应激模型,最佳条件为800μmol/L H_(2)O_(2)处理8 h。BLF可以抑制H_(2)O_(2)诱导的MAC-T凋亡和ROS生成,缓解线粒体损伤并恢复抗氧化能力,保护MAC-T抵御H_(2)O_(2)诱导的氧化损伤。

The purpose of this study was to screen the best conditions for the establishment of oxidative stress model of bovine mammary epithelial cells induced by hydrogen peroxide(H_(2)O_(2)),and to explore the protective effect of bamboo leaf flavonoids(BLF)on oxidative damage of bovine mammary epithelial cells.Experiments used bovine mammary epithelial cells MAC-T cell line as research object.MAC-T treated with the maintenance medium as control group,different concentrations(200 to 1000μmol/L)of H_(2)O_(2) were added into the maintenance medium as oxidative damage group,80μg/mL BLF and 800μmol/L H_(2)O_(2) were added into the maintenance medium as BLF pretreatment group.The cell viability of MAC-T was detected by CCK-8 assay and the cell apoptosis rate was detected by lactate dehydrogenase(LDH)method.There were 6 parallel multiple holes in each test and repeated three times.The changes of intracellular ROS content were observed by inverted fluorescence microscope,antioxidant indexes were detected by kit method.Flow cytometry were used to analyze the mitochondrial membrane potential(MMP) changes,and the changes of the expression of mitochondria damage related genes were detected by real-time fluorescence quantitative PCR.The results showed as follows:1)the cells stimulated by 600 and 800μmol/L H_(2)O_(2) for 6 and 8 h were in accordance with the modeling conditions,the cell viability was 80.77%to 72.81%,and the cell apoptosis rate was 20.79%to 43.52%,respectively.2)Compared with the control group,800μmol/L H_(2)O_(2) significantly increased the content of intracellular ROS and significantly inhibited the activities of catalase(CAT),glutathione peroxidase(GSH-Px)and total superoxide dismutase(T-SOD)after 8 h treatment(P<0.05).3)Compared with the control group,800μmol/L H_(2)O_(2) had no significant effect on the mRNA relative expression level of B-cell lymphoma/leukemia-2(Bcl-2)(P>0.05),but significantly increased the mRNA relative expression level of Bcl-2 associated X protein(Bax)and the ratio of Bax/Bcl-2(P<0.05),and significantly increased the proportion of MMP depolarized cells after 8 h treatment(P<0.05).4)Compared with oxidative damage group,treatment with 80μg/mL BLF for 24 h could significantly increase cell viability(P<0.05),significantly decreased the content of intracellular ROS(P<0.05),significantly increased the activities of CAT and GSH-Px(P<0.05),significantly decreased the mRNA relative expression level of Bax and the ratio of Bax/Bcl-2(P<0.05),while significantly increased the mRNA relative expression level of Bcl-2(P<0.05).In conclusion,the oxidative stress model of MAC-T induced by H_(2)O_(2) is successfully established in this experiment,and the best treatment conditions are 800μmol/L H_(2)O_(2) treat for 8 h.BLF pretreatment can inhibit apoptosis and ROS production of MAC-T induced by H_(2)O_(2),alleviate mitochondrial damage and restore antioxidant capacity,and protect MAC-T from oxidative damage induced by H_(2)O_(2).