摘要
目的 探讨黑果枸杞花青素单体矮牵牛花色素-3-O-(6-O-对香豆酰)芸香糖苷-5-O-葡萄糖苷(PrG)对H2O2诱导的小鼠颗粒细胞(GCs)氧化损伤的保护机制.方法 将20只ICR雌性小鼠的GCs进行原代培养.将GCs随机分为对照组(Control组)、过氧化氢处理组(H2O2组)、PrG处理组(H2O2+PrG组).首先,构建了由不同浓度(0 μmol/mL、25 μmol/mL、50 μmol/mL、100 μmol/mL、200 μmol/mL、400 μmol/mL)、不同作用时间(30 min、60 min、120 min、180 min)H2O2诱导的GCs氧化应激损伤模型.随后应用PrG不同浓度(20 μg/mL、40 μg/mL、80 μg/mL)、不同作用时间(12 h、24 h、48h)处理氧化损伤后的GCs.采用CCK8法筛选H2O2与PrG的最佳作用浓度与作用时间,采用EdU法检测H2O2诱导的GCs增殖情况,使用DCFH-DA活性氧荧光探针检测GCs内的ROS水平,使用JC-1探针和Mito-tracker Red CMXRos活细胞染料检测线粒体膜电位和线粒体分布.结果 H2O2在作用浓度为200 μmol/mL、作用时间为2h时小鼠GCs存活率下降约60%.GCs活力随着PrG浓度的升高而显著升高(P<0.01),且在浓度为80 μg/mL时对活性影响最佳.PrG能显著促进GCs增殖能力,降低活性氧水平(P<0.01),增加线粒体分布数量(P<0.01),恢复线粒体膜电位(P<0.01).结论 黑果枸杞花青素单体PrG通过清除ROS自由基、增强线粒体功能对H2O2诱导的小鼠GCs氧化损伤发挥保护作用.
Abstract
Objdctive To investigate the protective mechanism of PrG,an anthocyanin monomer of Lycium ru-thenium Murray,against H2O2-induced oxidative damage in the granulosa cells(GCs)of mice.Methods The GCs from 20 female ICR mice were subjected to primary culture.Then,these mice were randomly divided into control group,hydrogen peroxide-treated group(H2O2group)and PrG-treated group(H2O2+PrG group).Firstly,an oxida-tive stress model of GCs induced by different concentrations(0 μmol/mL,25 μmol/mL,50 μmol/mL,100 μmol/mL,200 μmol/mL,400 μmol/mL)and different action times(30 min,60 min,120 min,180 min)of H2O2 was constructed.Subsequently,different concentrations of PrG(20 μg/mL,40 μg/mL,80 μg/mL)and different action times(12 h,24 h,48 h)were applied to treat GCs after oxidative damage.The optimal concentration and action time of H2O2 and PrG were selected by the CCK8 method.The proliferation of GCs induced by H2O2 was detected by the EdU method.Reactive oxygen fluorescence probe was used to detect reactive oxygen species(ROS)levels in GCs.Mmitochondri-al membrane potential and mitochondrial distribution were detected by using the JC-1 probe and Mito-tracker Red CMXRos live cell dye.Results The survival rate of mice GCs was decreased by about 60%at a concentration of 200 μM and a time of 2 h.The viability of GCs was significantly increased with the rising of PrG concentrations(P<0.01),and the optimal effect on the viability was observed at a concentration of 80 μg/mL.PrG significantly promoted the proliferative capacity of GCs,decreased the level of ROS(P<0.01),increased the number of mitochondria distribut-ed(P<0.01)and restored mitochondrial membrane potential(P<0.01).Conclusion The anthocyanin monomer PrG of Lycium ruthenium Murray exerted a protective effect on H2O2-induced oxidative damage of mouse GCs by scav-enging ROS free radicals and enhancing mitochondrial function.
基金项目
国家自然科学基金(32360242)
青海省自然科学基金面上项目(2023-ZJ-932M)
NETL
NSTL
万方数据