姜黄素通过Nrf2信号通路对H_(2)O_(2)诱导奶牛乳腺上皮细胞氧化应激的缓解  

Curcumin Alleviates H_(2)O_(2)-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway

作　　者：姜春晖 孙旭东[1] 唐燕 罗胜缤 徐闯[1] 陈媛媛[1] JIANG ChunHui;SUN XuDong;TANG Yan;LUO ShengBin;XU Chuang;CHEN YuanYuan(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国农业科学》2021年第8期1787-1794,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31672622);中国博士后科学基金(2019M661316);黑龙江博士后科学基金(LBH-Z19090);黑龙江八一农业大学学成、引进人才科研启动计划项目(XYB201909)。

摘　　要：【目的】验证姜黄素是否能够通过转录因子E2相关因子2(Nuclear factor E2-related factor 2,Nrf2)信号通路减轻H_(2)O_(2)诱导的奶牛乳腺上皮细胞的氧化应激,为防治围产期奶牛代谢紊乱导致的氧化损伤提供理论依据。【方法】培养奶牛乳腺上皮细胞(MAC-T),用500μmol·L^(-1)H_(2)O_(2)处理MAC-T细胞24 h后加入不同浓度的姜黄素(0、5、15或30μmol·L^(-1))处理3 h;MAC-T细胞转染Nrf2 siRNA 48 h后,用500μmol·L^(-1)H_(2)O_(2)处理奶牛乳腺上皮细胞系MAC-T细胞24 h后加入不同浓度的姜黄素(0、5、15或30μmol·L^(-1))处理3 h。采用实时定量PCR(Quantitative real-time PCR,qPCR)、蛋白免疫印迹(Western blot,WB)和试剂盒等方法,检测Nrf2的蛋白表达量及下游靶基因NAD(P)H醌氧化还原酶1(NAD(P)H quinone oxidoreductase 1,NQO1)和血红素加氧酶1(Heme oxygenase 1,HO-1)的mRNA及蛋白表达量和超氧化物歧化酶(Superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)、过氧化氢酶(Catalase,CAT)和丙二醛(Malondialdehyde,MDA)等氧化应激相关指标的活性及含量。【结果】(1)H_(2)O_(2)处理组MAC-T细胞中MDA含量显著高于对照组(P<0.01),而CAT、SOD和GSH-Px的活性显著低于对照组(P<0.01)。15μmol·L^(-1)姜黄素+H_(2)O_(2)共同处理组和30μmol·L^(-1)姜黄素+H_(2)O_(2)共同处理组MAC-T细胞中MDA的含量显著低于H_(2)O_(2)处理组(P<0.01),而CAT、SOD和GSH-Px的活性显著高于H_(2)O_(2)处理组(P<0.05,P<0.01)。(2)H_(2)O_(2)处理组总Nrf2蛋白水平显著低于对照组(P<0.01),H_(2)O_(2)处理组Nrf2下游基因HO-1和NQO1的mRNA及蛋白表达量也显著低于对照组(P<0.01)。姜黄素处理组总Nrf2蛋白表达水平显著高于对照组(P<0.01),姜黄素处理组HO-1和NQO1的mRNA及蛋白表达量也显著高于对照组(P<0.01)。姜黄素+H_(2)O_(2)处理组总Nrf2蛋白表达水平显著高于H_(2)O_(2)处理组(P<0.01),姜黄素+H_(2)O_(2)处理组HO-1和NQO1的mRNA及蛋白表达量也�【Objective】The aim of this study was to investigate whether curcumin alleviated oxidative stress in bovine mammary epithelial cells induced by H_(2)O_(2)via the nuclear factor E2-related factor 2(Nrf2)signaling pathway.【Method】Bovine mammary epithelial cells MAC-T cells were treated with H2O2(500μmol·L^(-1))for 24 h,followed by incubation of curcumin(0,5,15 or 30μmol·L^(-1))for an additional 3 h;the MAC-T cells were transfected with Nrf2 siRNA for 48 h,followed by incubation of H2O2(500μmol·L^(-1))for 24 h and then treated with curcumin(30μmol·L^(-1))for an additional 3 h.Real-time quantitative PCR(qPCR),Western blot(WB)were used to detect the protein abundance of Nrf2 and the mRNA and protein abundance of NAD(P)H quinone oxidoreductase 1(NQO1)and Heme oxygenase 1(HO-1),the activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)and the content of malondialdehyde(MDA).【Result】(1)Compared with the control group,H_(2)O_(2)treatment significantly increased MDA content(P<0.01),while it decreased the activity of SOD,GSH-Px,and CAT(P<0.01).Compared with the H_(2)O_(2)group,the content of MDA in MAC-T cells in the 15μmol·L^(-1)or 30μmol·L^(-1)curcumin with H_(2)O_(2)treatment groups was significantly decreased(P<0.01),while the activity of SOD,GSH-Px,and CAT were significantly increased(P<0.05,P<0.01).(2)Compared with the control group,H_(2)O_(2)treatment significantly decreased Nrf2 protein abundance(P<0.01)and decreased HO-1 and NQO1 their mRNA and protein abundance(P<0.01).However,compared with the control group,curcumin treatment significantly increased Nrf2 protein abundance(P<0.01)and increased HO-1 and NQO1 mRNA and protein abundance(P<0.01).Compared with the H_(2)O_(2)group,H2O2+curcumin treatment significantly increased Nrf2 protein abundance(P<0.01)and increased HO-1 and NQO1 mRNA and protein abundance(P<0.01).(3)Compared with the control group,si-Nrf2 treatment group significantly decreased Nrf2 mRNA abundance(P<0.01).Compared with si-Control+H2O group,the con

关 键 词：姜黄素 奶牛 乳腺上皮细胞 氧化应激 NRF2 

分 类 号：S823[农业科学—畜牧学]