维生素A对一氧化氮诱导损伤的奶牛乳腺上皮细胞内乳脂合成下降的缓解作用

Alleviative Effect of Vitamin A on Milk Fat Synthesis Reduction in Bovine Mammary Epithelial Cells Injured by Nitric Oxide

本试验旨在研究维生素A对一氧化氮(NO)诱导损伤的奶牛乳腺上皮细胞(BMECs)内乳脂合成相关酶活性和基因表达下降的缓解作用,并从中筛选出适宜的维生素A添加量。本试验采用单因子完全随机试验设计,以二乙烯三胺/一氧化氮聚合物(DETA/NO)作为氧化应激源,对照组不添加DETA/NO和维生素A培养30 h;试验1组(记为NO组)不添加维生素A培养24 h后添加DETA/NO继续培养6 h;试验2~8组(记为NOA0.05、NOA0.1、NOA0.2、NOA0.5、NOA1、NOA2和NOA4组)分别添加0.05、0.10、0.20、0.50、1.00、2.00和4.00μg/mL维生素A培养24 h后添加DETA/NO继续培养6 h。每组6个重复。结果表明:与对照组相比,NO组细胞活力,谷胱甘肽过氧化物酶(GPx)、过氧化氢酶(CAT)、硫氧还蛋白还原酶(TrxR)、脂肪酸合成酶(FASN)、脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶(ACC)和硬脂酰辅酶A去饱和酶(SCD)活性,甘油三酯(TG)含量,总抗氧化能力(T-AOC)及GPx、TrxR、过氧化酶体增殖物激活受体γ(PPARγ)、固醇调节元件结合蛋白1(SREBP1)、ACC和FASN mRNA相对表达显著下降(P<0.05),活性氧(ROS)、诱导型一氧化氮合酶(iNOS)活性和一氧化氮(NO)含量显著增加(P<0.05)。与NO组相比,NOA0.1~NOA4组细胞活力、T-AOC与ACC、FASN、LPL活性以及GPx1、TrxR、FASN、LPL、PPARγ和SREBP1 mRNA相对表达量显著增加(P<0.05);NOA0.2~NOA2组TG含量,TrxR、CAT、GPx和SCD活性及SCD mRNA相对表达量显著增加(P<0.05),而iNOS活性、NO含量显著降低(P<0.05)。综合上述结果分析得出,NO诱导BMECs损伤导致抗氧化功能及乳脂合成相关酶活性及基因表达、TG合成下降;维生素A缓解了NO诱导损伤的BMECs抗氧化功能和乳脂合成的下降,以0.20~2.00μg/mL的效果较好,尤以1.00μg/mL效果最好。

This study was conducted to investigate the alleviative effect of vitamin A(VA)on the enzyme activities and gene expression relate to milk fat synthesis in bovine mammary epithelial cells(BMECs)injured by nitric oxide(NO),and to screen the optimum supplemental level of VA.The single-factor completely randomized trial design was used in this study.The diethylenetriamine/nitric oxide adduct(DETA/NO)was used to injure cells.The control group cells were cultured without VA and DETA/NO for 30 h.The test group 1(named as NO group)was treated without VA for 24 h and added DETA/NO for another 6 h.The test groups2 to 8 were treated with 0.05,0.10,0.20,0.50,1.00,2.00 and 4.00μg/mL VA for 24 h and added DETA/NO for another 6 h,they were named NOA0.05,NOA0.1,NOA0.2,NOA0.5,NOA1,NOA2 and NOA4groups.Each group had 6 replicates.The results showed that,compared with the control group,the cell viability,the activities of glutathione peroxidase(GPx),catalase(CAT),thioredoxin(TrxR),fatty acid synthetase(FASN),lipoprotein lipase(LPL),acetyl-CoA carboxylase(ACC)and stearyl CoA desaturase(SCD),the content of triglyceride(TG),total antioxidant capacity(T-AOC)and the mRNA relative expression levels of GPx,TrxR,peroxidase proliferators activate the receptorγ(PPARγ),sterol regulatory element binding protein 1(SREBP1),ACC and FASN of the NO group were significantly decreased(P<0.05),but the ROS and iNOS activities and NO content were significantly increased(P<0.05).Compared with the NO group,the cell viability,T-AOC,the activities of ACC,FASN,LPL,and the mRNA relative expression levels of GPx1,TrxR,FASN,LPL,PPARγand SREBP1 of the NOA0.1 to NOA4 groups were significantly increased(P<0.05);the TG content,the activities of TrxR,CAT,GPx and SCD,and the SCD mRNA relative expression level of the NOA0.2 to NOA2 groups were significantly increased(P<0.05),while the iNOS activity and NO content were significantly decreased(P<0.05).These results indicate that BMECs injury induced by NO result the reduction of antioxidant function,milk fat synthesis related enzyme activities and gene expression and TG synthesis.VA can alleviate the reduction of antioxidant function and milk fat synthesis,and0.20 to 2.00μg/mL VA have better effect,especially 1.00μg/mL VA has the best effect.