壳寡糖对脂多糖诱导猪空肠上皮细胞氧化损伤的作用

摘要

本试验旨在通过脂多糖（ LPS）诱导猪空肠上皮细胞（ IPEC⁃J2）来建立氧化应激模型，探讨壳寡糖（ COS）的抗氧化作用效果。采用噻唑蓝（ MTT）法检测LPS和COS对IPEC⁃J2作用12、24、48 h后细胞增殖活性的变化；选择适宜浓度LPS（1．0μg／mL）和COS（200μg／mL）作用于IPEC⁃J2，分为对照组、LPS组、COS组、LPS＋COS组。采用Western Blot法检测核因子E2相关因子2（Nrf2）、血红素氧合酶－1（HO⁃1）蛋白的表达；试剂盒检测超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性及丙二醛（MDA）的含量。结果表明：1．0μg／mL的LPS对IPEC⁃J2作用24 h，细胞增殖的抑制率为41．6％，为造模的最适浓度和作用时间；COS在一定浓度范围内对IPEC⁃J2有增殖作用，其浓度为200μg／mL 时效果最佳，作用时间为24 h时，细胞增殖率达到126．3％。 LPS＋COS组较对照组的SOD、CAT活性和MDA含量差异不显著（P＞0．05），Nrf2和HO⁃1蛋白相对表达量显著升高（ P＜0．05）；LPS＋COS组较LPS组的Nrf2蛋白相对表达量显著升高（P＜0．05），HO⁃1有升高趋势，但差异不显著（P＞0．05），SOD、CAT 活性显著升高（P＜0．05），MDA含量显著降低（P＜0．05）。由此可见，LPS诱导IPEC⁃J2氧化应激，而COS可进一步促进Nrf2和HO⁃1蛋白的高表达，并提高抗氧化酶的活性，降低氧化产物含量，从而增强细胞对氧化应激的抵抗力，起到保护作用。%This study was designed to investigate the effect of chitooligosaccharide ( COS) on oxidative stress and used lipopolysaccharide ( LPS) induced epithelial cells of pig jejunum ( IPEC⁃J2) as an in vitro model of oxidative stress. The IPEC⁃J2 were incubated with LPS and COS for 12, 24 and 48 h,and then used methylthi⁃azolyldiphenyl⁃tetrazolium bromide ( MTT) method to detect the activity of cell proliferation; the appropriate concentrations of LPS (1.0 μg/mL) and COS (200 μg/mL) were chosen in the IPEC⁃J2 cells, divided into control group, LPS group, COS group and LPS+COS group, and then detected the expression of NF⁃E2⁃relat⁃ed factor 2 ( Nrf2 ) and heme oxygenase 1 ( HO⁃1 ) by Western Blot; and detected superoxide dismutase ( SOD) , catalase ( CAT) activities and malonaldehyde ( MDA) content by the kit. The results showed that the optimum concentration of LPS and incubated time for model was 1.0 μg/mL and 24 h, the inhibition of cell proliferation was 41.6%; COS promoted the proliferation of IPEC⁃J2, worked the best when its concentration was 200 μg/mL and incubated 24 h, the cell proliferation rate was 126.3%. Compared with the control group, LPS+COS group was not significantly different in SOD, CAT activities and MDA content (P>0.05), but Nrf2 and HO⁃1 protein expression was significantly increased (P<0.05); compared with LPS group, Nrf2 protein expression of LPS+COS group was significantly higher (P<0.05), and there was an increasing trend of HO⁃1 protein expression, but the difference was not significant ( P>0.05) , moreover, SOD, CAT activities were significantly increased and MDA content was significantly reduced (P<0.05). It is concluded that LPS induce oxidative stress of IPEC⁃J2, but COS can induce high expression of Nrf2 and HO⁃1 protein, and im⁃prove the activity of antioxidant enzymes, reduce the content of oxidative products, so increase cellular resist⁃ance to oxidative stress, and play a protective role.