硒对脂多糖诱导的奶牛乳腺上皮细胞氧化损伤的保护作用

本试验旨在研究硒(Se)对脂多糖(LPS)诱导的奶牛乳腺上皮细胞(BMEC)氧化损伤的保护作用及其机制。将贴壁生长的第3代BMEC随机分为8组,每组6个重复,每个重复1个培养孔。对照(CON)组采用基础培养液,不添加Se和LPS,培养30h;LPS组和6个Se保护组在基础培养液中分别添加不同水平的Se(0、10、20、50、100、150和200nmol/L),培养24h后,加入1μg/mL LPS作为外源刺激作用6h。结果表明:1)与CON组相比,LPS组BMEC的相对增殖率显著下降(P0.05),谷胱甘肽过氧化物酶(GPx)、硫氧还蛋白还原酶(TrxR)、总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)活性和总抗氧化能力(T-AOC)均显著下降(P0.05),GPx1和TrxR1的基因和蛋白表达量、硒蛋白P(SelP)含量也显著下调(P0.05);而LPS组的一氧化氮(NO)含量,诱导型一氧化氮合酶(iNOS)活性及其基因和蛋白表达量,炎症因子肿瘤坏死因子-α(TNF-α)、白介素-1(IL-1)和白介素-6(IL-6)含量及其基因表达量,活性氧(ROS)活性,丙二醛(MDA)含量均显著升高(P0.05),丝裂原活化蛋白激酶(MAPK)信号通路相关因子p38丝裂原活化蛋白激酶(p38 MAPK)、c-Jun氨基端激酶(JNK)、细胞外信号调节激酶1/2(ERK1/2)的基因表达量呈相似变化。2)与LPS组相比,Se保护组随Se添加水平的增加,相对增殖率,T-SOD、CAT、GPx、TrxR活性,T-AOC,GPx1、TrxR1基因和蛋白表达量均呈先升高后下降趋势;而NO含量,iNOS活性及其基因和蛋白表达量,炎症因子TNF-α、IL-1、IL-6含量及其基因表达量,MAPK信号通路相关因子ERK1/2、JNK、p38 MAPK的基因表达量,ROS活性,MDA含量呈先降低后升高的趋势;以20~100nmol/L Se保护效果较好,综合来看50nmol/L Se保护效果最好。结果提示,Se可提高BMEC的抗氧化功能,对LPS引起的细胞氧化损伤具有保护作用,其机制是Se增强TrxR活性从而抑制MAPK信号通路的激活,最终减少NO的大量释放,但过高水平的Se会对细胞造成损伤。培养液中20~100nmol/L Se的保护作用较好,尤其以50nmol/L Se效果最好。

Protective Effects of Selenium on Bovine Mammary Epithelial Cells Oxidative Damaged by Lipopolysaccharide

This experiment was conducted to investigate the protective effects of selenium (Se) on bovine mammary epithelial cells (BMECs) oxidative damaged by lipopolysaccharide (LPS) and the mechanism.The third-generation attachment cultured BMECs were randomly divided into 8 groups with 6 replicates per group and 1 culture pore per replicate.BMECs in control (CON) group were cultured in a basal medium without Se and LPS for 30 h.BMECs in LPS group and Se protection groups were exposed in Se with different concentrations (0,10,20,50,100,150 and 200 nmol/L) for 24 h,and then treated with LPS (1 μg/mL) for 6 h.The results showed as follows: 1) compared with CON group,the relative proliferation rate in LPS group was significantly decreased (P<0.05),the activities of glutathione peroxidase (GPx),thioredoxin reductase (TrxR),superoxide dismutase (T-SOD) and catalase (CAT),and total antioxidant capacity (T-AOC) in LPS group were significantly decreased (P<0.05),and expressions of genes and proteins of GPx1 and TrxR1 and selenoprotein P (SelP) content in LPS group was significantly decreased (P<0.05);however,nitric oxide (NO) content,activity,gene and protein expressions of inducible nitric oxide synthase (iNOS),contents and gene expressions of inflammatory factors [tumor necrosis factor-alpha (TNF-α),interleukin-1 (IL-1) and interleukin-6 (IL-6)],active oxygen (ROS) activity,and malondialdehyde (MDA) content in LPS group were significantly increased (P<0.05),and gene expressions of factors related to the mitogen-activated protein kinase (MAPK) signaling pathway [p38 mitogen-activated protein kinase (p38MAPK),c-Jun N terminal kinase (JNK) and extracellular signal-regulated kinas 1/2 (ERK1/2)] in LPS group showed the same tendency.2) Compared with LPS group,with the increase of Se supplemental level in Se protection groups,relative proliferation rate,activities of T-SOD,CAT,GPx and TrxR,T-AOC,gene and protein expressions of GPx1 and TrxR1 were increased first and then decreased;while NO content,activity,gene and protein expressions of iNOS,contents and gene expressions of inflammatory factors (TNF-α,IL-1 and IL-6),gene expressions of factors related to MAPK signal pathway [ERK1/2,JNK and p38MAPK],ROS activity,and MDA content showed the opposite trend;Se at 20 to 100nmol/L showed better protection effects,and in general Se at 50nmol/L showed the best protection effects.The results indicate that Se improves the antioxidant function of BMECs,and protects cells from oxidative damage induced by LPS mainly by increasing TrxR activity,inhibiting the activation of MAPK signaling pathway,and then decreasing NO content,however,high level of Se can cause damage to cells.Se at 20 to 100 nmol/L in culture medium has a better protection effect,and Se at 50 nmol/L has the best effect.