Jiang Zhiguo, Li Li, Hou Lianjie, Zhou Zhuoqiang, Wang Chong
National Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University Guangzhou 510642, China.
Guangzhou Technician College No. 68, Huangshi East Road, Baiyun District, Guangzhou 510642, China.
Int J Clin Exp Pathol. 2020 Mar 1;13(3):447-455. eCollection 2020.
This study aims to study the protective effect and mechanism of carnosol on intestinal oxidative stress. Porcine intestinal epithelial cells (ZYM-SIEC02) were pretreated with carnosol. Tert-butyl hydroperoxide (t-BHP) was added to stimulate the cells. The cell colonization and viability were detected by Edu staining, MTT, and cell counting kit-8 (CCK8) assays. The expressions of reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD), and malondialdehyde (MDA) in intracellular and oxidative stress were detected. The expression of related genes and proteins in cells was detected by real-time PCR and western blot. The regulatory mechanisms were identified by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (CHIP) assays. The results showed that t-BHP reduced cell proliferation and viability, while cells pretreated with carnosol had resistance to t-BHP, decreased intracellular ROS, MDA and NO levels, and increased SOD content. The mRNA and protein levels of heme oxygenase 1/Nuclear respiratory factor 2 (HO-1/Nrf-2) in ZYM-SIEC02 cells treated with carnosol were significantly increased. Nrf2 was able to bind to cell cycle negative regulatory protein p21 Nrf2 could bind to the promoter regions of () and genes. In conclusion, carnosol has a protective effect on intestinal epithelial cells by up-regulating the expression of Nrf2 and inhibiting p21 protein to promote the expression of CCND1 and SOD.
本研究旨在探讨鼠尾草酸对肠道氧化应激的保护作用及机制。用鼠尾草酸预处理猪小肠上皮细胞（ZYM-SIEC02）。加入叔丁基过氧化氢（t-BHP）刺激细胞。通过Edu染色、MTT和细胞计数试剂盒-8（CCK8）检测细胞增殖和活力。检测细胞内活性氧（ROS）、一氧化氮（NO）、超氧化物歧化酶（SOD）和丙二醛（MDA）的表达及氧化应激情况。通过实时PCR和蛋白质印迹法检测细胞中相关基因和蛋白质的表达。通过免疫共沉淀（Co-IP）和染色质免疫沉淀（CHIP）实验确定调控机制。结果表明，t-BHP降低细胞增殖和活力，而经鼠尾草酸预处理的细胞对t-BHP具有抗性，细胞内ROS、MDA和NO水平降低，SOD含量增加。用鼠尾草酸处理的ZYM-SIEC02细胞中血红素加氧酶1/核呼吸因子2（HO-1/Nrf-2）的mRNA和蛋白质水平显著升高。Nrf2能够结合细胞周期负调控蛋白p21，Nrf2可与（）和基因的启动子区域结合。总之，鼠尾草酸通过上调Nrf2表达并抑制p21蛋白以促进CCND1和SOD的表达，对肠上皮细胞具有保护作用。
