黑果枸杞花青素单体PrG对H_(2)O_(2)诱导的小鼠颗粒细胞氧化损伤的保护机制研究  

Study on the protective mechanism from Lycium ruthenium Murray anthocyanin monomer Pr G against H_2O_2induced oxidative damage in the granulosa cells of mice

作　　者：马荣花 张璐瑶[3] 潘波 赵婧 易毅 马海秀 孙静薇 齐佳瑞 曹成珠[6] 马雪曼 苏占海[6] MA Ronghua;ZHANG Luyao;PAN Bo;ZHAO Jing;YI Yi;MA Haixiu;SUN Jingwei;QI Jiarui;CAO Chengzhu;MA Xueman;SU Zhanhai(Research Center for High Altitude Medicine,Qinghai University,Xining 810001,China;Center of Reproduc-tive Medicine,Qinghai Provincial People′s Hospital,Xining 810007,China;Northwest Institute of Plateau Biology,Chinese Academy of Sciences,Xining 810001,China;College of Animal Science and Technology,Sichuan Agricul-tural University,Chengdu 611130,China;Xinjiang University of Science and Technology,Wulumuqi 830000,China;Medical College of Qinghai University,Xining 810001,China)

机构地区：[1]青海大学高原医学研究中心,西宁810001 [2]青海省人民医院生殖医学中心,西宁810007 [3]中国科学院西北高原生物研究所,西宁810001 [4]四川农业大学动物科技学院,成都611130 [5]新疆科技学院,乌鲁木齐830000 [6]青海大学医学部,西宁810001

出　　处：《中国高原医学与生物学杂志》2024年第2期98-106,共9页Journal of Chinese High Altitude Medicine & Biology

基　　金：国家自然科学基金项目(32360242);青海省自然科学基金-面上项目(2023-ZJ-932M)。

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H_(2)O_(2)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(H_(2)O_(2)组)、PrG处理组(H_(2)O_(2)+PrG组)。首先,构建了由不同浓度(0μmol/mL、25μmol/mL、50μmol/mL、100μmol/mL、200μmol/mL、400μmol/mL)、不同作用时间(30 min、60 min、120 min、180 min)H_(2)O_(2)诱导的GCs氧化应激损伤模型。随后应用PrG不同浓度(20μg/mL、40μg/mL、80μg/mL)、不同作用时间(12 h、24 h、48 h)处理氧化损伤后的GCs。采用CCK8法筛选H_(2)O_(2)与PrG的最佳作用浓度与作用时间,采用EdU法检测H_(2)O_(2)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H_(2)O_(2)在作用浓度为200μmol/mL、作用时间为2 h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80μg/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H_(2)O_(2)诱导的小鼠GCs氧化损伤发挥保护作用。Objdctive To investigate the protective mechanism of PrG,an anthocyanin monomer of Lycium ruthenium Murray,against H_2O_2-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(H_(2)O_(2)group)and PrG-treated group(H_2O_2+PrG group).Firstly,an oxidative 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 H_2O_2was 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 H_2O_2and PrG were selected by the CCK8 method.The proliferation of GCs induced by H_2O_2was detected by the EdU method.Reactive oxygen fluorescence probe was used to detect reactive oxygen species(ROS)levels in GCs.Mmitochondrial 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 200μM 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 distributed(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 H_2O_2-induced oxidative damage of mouse GCs by scavenging ROS free radicals and enhancing mitochondrial function.

关 键 词：枸杞 花青素 矮牵牛花色素苷 过氧化氢 颗粒细胞 氧化损伤 

分 类 号：Q95[生物学—动物学]