二烯丙基二硫醚对过氧化氢诱导的湖羊瘤胃上皮细胞氧化应激的缓解作用

Alleviation Effect of Diallyl Disulfide on Oxidative Stress Damage in Hu Sheep Rumen Epithelial Cells Induced by Hydrogen Peroxide

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.