摘要
本试验旨在探究二烯丙基二硫醚(DADS)对过氧化氢(H_(2)O_(2))诱导的湖羊瘤胃上皮细胞(RECs)自由基的清除能力及其对瘤胃上皮细胞氧化应激的保护作用。分别使用不同浓度(0、50、100、150、200、250μmol/L)的H_(2)O_(2)和不同浓度(0、25、50、75、100、150、200μmol/L)的DADS处理湖羊瘤胃上皮细胞24 h,使用CCK-8法检测细胞活力,荧光探针法检测细胞内活性氧(ROS)含量,筛选构建湖羊瘤胃上皮细胞氧化应激模型的适宜H_(2)O_(2)浓度和DADS安全浓度范围。随后先用不同浓度(0、25、75、150μmol/L)的DADS预处理湖羊瘤胃上皮细胞2 h,对其进行保护,再使用200μmol/L H_(2)O_(2)处理湖羊瘤胃上皮细胞24 h,检测细胞活力以及细胞内ROS含量,采用试剂盒法检测细胞内总抗氧化能力(T-AOC)、丙二醛(MDA)含量及过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPX)活性,并采用实时荧光定量PCR(qRT-PCR)检测SOD、CAT和GPX的mRNA相对表达量。结果显示:与空白对照组(H_(2)O_(2)浓度为0μmol/L)相比,200μmol/L的H_(2)O_(2)可显著降低湖羊瘤胃上皮细胞活力(P<0.05),显著增加细胞内ROS含量(P<0.05),成功诱导湖羊瘤胃上皮细胞氧化应激。与空白对照组(DADS浓度为0μmol/L)相比,25~150μmol/L的DADS不会对湖羊瘤胃上皮细胞活力产生显著影响(P>0.05)。与空白对照组(H_(2)O_(2)和DADS浓度均为0μmol/L)相比,200μmol/L的H_(2)O_(2)显著降低细胞活力(P<0.05),显著增加细胞内ROS和MDA含量(P<0.05),显著降低T-AOC和SOD活性与mRNA相对表达量(P<0.05);与氧化应激模型组(经200μmol/L H_(2)O_(2)诱导)相比,使用75、150μmol/L DADS预处理湖羊瘤胃上皮细胞可显著降低细胞内ROS和MDA含量(P<0.05),显著增加T-AOC和SOD活性与mRNA相对表达量(P<0.05)。综上所述,适量的DADS可有效缓解H_(2)O_(2)诱导的湖羊瘤胃上皮细胞氧化应激。
This study aimed to investigate the free radical scavenging ability of diallyl disulfide(DADS)and its protective effect against hydrogen peroxide(H_(2)O_(2))-induced oxidative stress in Hu sheep rumen epithelial cells(RECs).Hu sheep RECs were treated with different concentrations(0,50,100,150,200 and 250μmol/L)of H_(2)O_(2)and different concentrations(0,25,50,75,100,150 and 200μmol/L)of DADS for 24 hours.Cell viability was assessed using the CCK-8 assay,and intracellular reactive oxygen species(ROS)content was measured using fluorescent probes.Appropriate concentrations of H_(2)O_(2)for establishing an oxidative stress model in Hu sheep RECs and safe concentration range of DADS were determined.Subsequently,the RECs were pretreated with different concentrations(0,25,75 and 150μmol/L)of DADS for 2 hours,followed by treatment with 200μmol/L H_(2)O_(2)for 24 hours.Cell viability and intracellular ROS content were assessed.The total antioxidant capacity(T-AOC),the content of malondialdehyde(MDA),and the activities of catalase(CAT),superoxide dismutase(SOD)and glutathione peroxidase(GPX)in Hu sheep RECs were measured using assay kits.Real-time fluorescence quantitative PCR(qRT-PCR)was performed to evaluate the mRNA relative expression levels of SOD,CAT and GPX.The results showed as follows:compared with the blank control group(0μmol/L H_(2)O_(2)),200μmol/L H_(2)O_(2)significantly decreased cell viability and significantly increased intracellular ROS content(P<0.05),inducing oxidative stress in Hu sheep RECs.Compared with the blank control group(0μmol/L DADS),DADS at concentrations ranging from 25 to 150μmol/L did not significantly affect the cell viability(P>0.05).Compared to the blank control group(0μmol/L H_(2)O_(2)and 0μmol/L DADS),200μmol/L H_(2)O_(2)significantly decreased cell viability(P<0.05),significantly increased intracellular ROS and MDA contents(P<0.05),and significantly reduced intracellular T-AOC,the mRNA relative expression level and the activity of SOD(P<0.05);compared with the oxidative stress model group(induced by 200μmol/L H_(2)O_(2)),pretreatment of Hu sheep RECs with 75 and 150μmol/L DADS significantly reduced intracellular ROS and MDA contents(P<0.05),and significantly increased intracellular T-AOC,the mRNA relative expression level and the activity of SOD(P<0.05).In conclusion,appropriate concentrations of DADS can effectively alleviate H_(2)O_(2)-induced oxidative stress in Hu sheep RECs.
作者
张庆月
黄李
王玉梅
汤莹莹
杨春蕾
张子军
朱雯
ZHANG Qingyue;HUANG Li;WANG Yumei;TANG Yingying;YANG Chunlei;ZHANG Zijun;ZHU Wen(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;College of Biotechnology and Bioengineering,Zhejiang University of Technology,Hangzhou 310014,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2023年第12期8036-8045,共10页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金项目(21239013)
安徽省大学生创新创业训练计划项目(X202210364111)
安徽省牛羊产业技术体系(160607)。