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及蛋白表达量也显著高于H_(2)O_(2)处理组(P<0.01)。(3)si-Nrf2组与对照组相比,Nrf2的mRNA表达水平显著降低(P<0.01)。si-Control+H_(2)O_(2)+姜黄素组与si-Control+H_(2)O_(2)组相比,MDA的含量显著降低(P<0.01),而CAT、SOD和GSH-Px的活性显著升高(P<0.01)。si-Nrf2+H_(2)O_(2)+姜黄素处理组与si-Control+H_(2)O_(2)+姜黄素组相比,MDA的含量显著升高(P<0.01),而CAT、SOD和GSH-Px活性显著降低(P<0.01)。【结论】姜黄素可以通过增加Nrf2的表达,诱导下游抗氧化分子的转录从而缓解H_(2)O_(2)诱导奶牛乳腺上皮细胞的氧化应激。本研究为防治围产期奶牛代谢紊乱导致的奶牛乳腺上皮细胞氧化损伤提供理论依据。
【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 content of MDA was significantly decreased in si-Control+H_(2)O_(2)+curcumin treatment group(P<0.01),while the activity of SOD,GSH-Px,and CAT were significantly increased(P<0.01).However,compared with si-Control+H2O2+curcum group,the content of MDA was significantly increased in si-Nrf2+H_(2)O_(2)+curcumin treatment group(P<0.01),while the activity of SOD,GSH-Px,and CAT were significantly decreased(P<0.01).【Conclusion】These results suggested that curcumin could alleviate the oxidative stress of bovine mammary epithelial cells induced by H_(2)O_(2)through increasing the expression of Nrf2 and inducing the transcription of downstream antioxidant molecules.This study provided a theoretical basis for the prevention and treatment of oxidative damage of mammary epithelial cells caused by metabolic disorderd in perinatal dairy cows.
作者
姜春晖
孙旭东
唐燕
罗胜缤
徐闯
陈媛媛
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)
出处
《中国农业科学》
CAS
CSCD
北大核心
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)。
作者简介
同等贡献作者/联系方式:姜春晖,E-mail:jiangchunh0606@163.com;同等贡献作者/联系方式:孙旭东,E-mail:sunxudong0323@163.com;通信作者:徐闯,E-mail:xuchuang7175@163.com;通信作者:陈媛媛,E-mail:18249636785@163.com。
