姜春晖^,, 孙旭东^,, 唐燕, 罗胜缤, 徐闯^,, 陈媛媛^,黑龙江八一农垦大学动物科技学院,黑龙江大庆 163319

Curcumin Alleviates H₂O₂-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway

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

通讯作者: * 徐闯,E-mail： xuchuang7175@163.com; 陈媛媛,E-mail： 18249636785@163.com

姜春晖与孙旭东为同等贡献作者。
责任编辑: 林鉴非
收稿日期:2020-05-6接受日期:2021-01-25网络出版日期:2021-04-16

基金资助:

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

Received:2020-05-6Accepted:2021-01-25Online:2021-04-16
作者简介 About authors
姜春晖,E-mail： jiangchunh0606@163.com。

孙旭东,E-mail： sunxudong0323@163.com。







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H₂O₂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500 μmol·L^-1H₂O₂处理MAC-T细胞24 h后加入不同浓度的姜黄素（0、5、15或30 μmol·L^-1）处理3 h;MAC-T细胞转染Nrf2 siRNA 48 h后,用500 μmol·L ^-1 H₂O₂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30 μmol·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）H₂O₂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）。15 μmol·L^-1姜黄素+H₂O₂共同处理组和30 μmol·L^-1姜黄素+H₂O₂共同处理组MAC-T细胞中MDA的含量显著低于H₂O₂处理组（P<0.01）,而CAT、SOD和GSH-Px的活性显著高于H₂O₂处理组（P<0.05,P<0.01）。（2）H₂O₂处理组总Nrf2蛋白水平显著低于对照组（P<0.01）,H₂O₂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+H₂O₂处理组总Nrf2蛋白表达水平显著高于H₂O₂处理组（P<0.01）,姜黄素+H₂O₂处理组HO-1和NQO1的mRNA及蛋白表达量也显著高于H₂O₂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+H₂O₂+姜黄素组与si-Control+H₂O₂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+H₂O₂+姜黄素处理组与si-Control+H₂O₂+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H₂O₂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。
关键词： 姜黄素;奶牛;乳腺上皮细胞;氧化应激;Nrf2

Abstract
【Objective】The aim of this study was to investigate whether curcumin alleviated oxidative stress in bovine mammary epithelial cells induced by H₂O₂ via the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. 【Method】Bovine mammary epithelial cells MAC-T cells were treated with H₂O₂ (500 μmol·L^-1) for 24 h, followed by incubation of curcumin (0, 5, 15 or 30 μmol·L ^-1) for an additional 3 h; the MAC-T cells were transfected with Nrf2 siRNA for 48 h, followed by incubation of H₂O₂ (500 μmol·L^-1) for 24 h and then treated with curcumin (30 μmol·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, H₂O₂ 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 H₂O₂ group, the content of MDA in MAC-T cells in the 15 μmol·L^-1 or 30 μmol·L ^-1 curcumin with H₂O₂ 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, H₂O₂ 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 H₂O₂ 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+H₂O₂+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+H₂O₂+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 H₂O₂ 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.
Keywords：curcumin;bovine;mammary epithelial cells;oxidative stress;Nrf2


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本文引用格式
姜春晖, 孙旭东, 唐燕, 罗胜缤, 徐闯, 陈媛媛. 姜黄素通过Nrf2信号通路对H₂O₂诱导奶牛乳腺上皮细胞氧化应激的缓解[J]. 中国农业科学, 2021, 54(8): 1787-1794 doi:10.3864/j.issn.0578-1752.2021.08.017
JIANG ChunHui, SUN XuDong, TANG Yan, LUO ShengBin, XU Chuang, CHEN YuanYuan. Curcumin Alleviates H₂O₂-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway[J]. Scientia Acricultura Sinica, 2021, 54(8): 1787-1794 doi:10.3864/j.issn.0578-1752.2021.08.017


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0 引言

【研究意义】近些年来,随着我国奶牛养殖业不断向规模化、集约化发展,围产期奶牛代谢性疾病的发病率也随之增加。从妊娠后期到泌乳前期,随着胎儿的生长发育及泌乳启动,奶牛机体对能量和营养的需求增加,而采食量却减少,导致能量负平衡^[1]。此时奶牛启动脂肪动员,诱发高非酯化脂肪酸（nonesterified fatty acid,NEFA）血症和高酮血症,致使脂肪肝、酮病等代谢性疾病的发生^[2]。在负能量平衡期间,大量的NEFA和酮体随血液进入乳腺以满足产奶的能量需求^[3]。研究发现高NEFA血症导致奶牛乳腺组织产生大量的活性氧（reactive oxygen species,ROS）,过量的ROS会导致氧化应激,致使乳腺上皮细胞氧化损伤^[4]。作为维持奶牛泌乳的基本功能单位,乳腺上皮细胞氧化损伤会造成乳腺功能受损,产奶量显著降低,严重影响奶业健康发展。因此,如何缓解围产期奶牛代谢应激导致的乳腺上皮细胞氧化应激,提高其产奶量和生产性能,已成为当前我国奶牛生产中的一大焦点问题。【前人研究进展】转录因子E2相关因子2（nuclear factor E2-related factor 2,Nrf 2）是机体重要的内源性保护分子。该分子通过调节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）等氧化分子的表达从而发挥抗氧化应激作用^[5]。张倩研究发现,激活Nrf 2信号通路可以增加HO-1、超氧化物歧化酶（Superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（Glutathione peroxidase,GSH-Px）等抗氧化分子的表达水平从而缓解H₂O₂诱导的人肝细胞的氧化应激^[6]。此外,研究发现激活Nrf 2信号通路可以缓解H₂O₂诱导的奶牛乳腺上皮细胞的氧化应激^[7],表明Nrf 2信号通路可能是保护奶牛乳腺上皮细胞免受氧化损伤的潜在治疗靶标。因此,筛选Nrf 2信号通路的调节药物是防治围产期奶牛乳腺上皮细胞氧化损伤研究的必要环节。姜黄素[Curcumin,1,7-二-（4-羟基-5-甲氧基苯基）-1,6-庚二烯-3,5-二酮]是姜黄根茎的提取物,是姜黄的主要活性成分之一^[8,9]。因为姜黄素具有抗炎、抗氧化和抗肿瘤等生物活性,广泛应用于医药行业和兽医领域。研究发现姜黄素可抑制脂质过氧化并有效清除超氧阴离子和羟基自由基^[10,11,12]。除了其清除氧自由基的能力外,姜黄素还具有增强谷胱甘肽-S-转移酶活性的作用^[13]。这些研究表明姜黄素具有较强的抗氧化能力。此外,体内研究发现姜黄素可以通过激活Nrf2信号通路缓解肾功能不全大鼠肾脏的氧化应激^[14]。体外研究发现,姜黄素通过激活Nrf2信号通路增加SOD活性并降低丙二醛（malondialdehyde,MDA）的含量从而缓解H₂O₂诱导的软骨细胞氧化应激^[15]。【本研究切入点】这些研究说明姜黄素可以通过激活Nrf2信号通路缓解氧化应激,但姜黄素是否可以通过激活Nrf2信号通路缓解奶牛乳腺上皮细胞的氧化应激尚不清楚。【拟解决的关键问题】通过培养奶牛乳腺上皮细胞添加H₂O₂,建立氧化应激模型,转染Nrf2 siRNA并添加不同浓度的姜黄素,检测Nrf2及其下游基因：NQO1、HO-1的表达量,并检测SOD、GSH-Px、CAT和MDA等氧化应激相关指标的活性及含量,旨在探讨姜黄素对H₂O₂诱导奶牛乳腺上皮细胞氧化应激的影响及其分子机制,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。

1 材料与方法

1.1 试验时间及地点

试验于2019年8–12月在黑龙江八一农垦大学动物群发性普通病监控实验室完成。

1.2 主要试剂及仪器

Eagle培养基（HG-DMEM,Pierce Hyclone,Fremont,CA,美国）;胎牛血清（美国纽约州格兰德岛）;H₂O₂（Sigma）;SOD、GSH-Px、CAT和MDA检测试剂盒（均购自南京建成生物工程研究所）;Trizol（Takara Biotechnology Co.,Ltd,大连,中国）;K5500显微分光光度计（北京凯奥科技发展有限公司,北京,中国）;反转录试剂盒（TaKaRa生物技术有限公司,东京,日本）;SYBR green plus试剂盒（Roche,Norwalk,CT,美国）;BCA试剂盒（北京普利莱基因技术有限公司,北京,中国）;荧光定量PCR仪（7500 Real-Time PCR系统Applied Biosystems）;RIPA裂解液（Beyotime,江苏,中国）;ECL化学发光溶液（Pierce Biotechnology Inc.,美国芝加哥）;一抗Nrf2（Abcam;1﹕1 000）;HO-1（Abcam;1﹕1 000）;NQO1（Abcam;1﹕1 000）;β-actin（Abcam;1﹕1 000）;二抗（博士德生物,武汉,中国）。

1.3 细胞培养及处理

奶牛乳腺上皮细胞系（MAC-T）培养在含100 U·mL^-1青霉素,100 g·mL^-1链霉素和10％（v/v）胎牛血清（美国纽约州格兰德岛）的DMEM培养基中,在37 ℃,5% CO₂的条件下培养。每隔24 h换液一次。根据先前的研究选择了H₂O₂的浓度和处理时间^[4]。用500 μmol·L^-1H₂O₂处理MAC-T细胞24 h后加入不同浓度的姜黄素（0、5、15或30 μmol·L^-1）处理3 h。姜黄素的浓度和处理时间参照文献[16]处理。

1.4 Nrf2 siRNA转染及细胞处理

根据文献[7]方法设计靶向Nrf 2编码区和非靶向阴性对照的siRNA,并由上海基因化学有限公司（中国上海）合成。Nrf 2 siRNA引物序列为：CTGGAGCA AGATTTAGATCAT,ATGATCTAAATCTTGCTCCAG。胰蛋白酶消化细胞,将2×10⁶细胞/mL在不含抗生素的培养基中培养24 h。在37 ℃下孵育5 min后,将siRNA和lipofectamine混合,在室温下再孵育20 min,然后添加到每个孔中。转染12 h后更换培养基。继续培养36 h后,将细胞用于随后的分析或处理。

1.5 检测指标

1.5.1 氧化应激指标 弃去培养液,每孔加入1 mL磷酸盐缓冲液（phosphate buffered saline,PBS）清洗后,收集细胞。根据制造商的说明书测定细胞中SOD、GSH-Px和CAT的活性以及MDA的含量,在560 nm（SOD）、420 nm（GSH-Px）、405 nm（CAT）和532 nm（MDA）读取吸光值。

1.5.2 RNA分离和实时定量PCR（qPCR） 培养结束后收集细胞,采用RNAiso Plus（TaKaRa,D9108A）总RNA提取试剂盒提取RNA,将1 μmol·L^-1 RNA样品反转录为cDNA。使用SYBR green plus试剂盒在7500 Real-Time PCR系统检查靶基因的表达量;用2^-ΔΔCT方法计算各基因的相对表达量。本研究中所用引物见表1。

Table 1
表1
表1Real-time PCR分析所用引物
Table 1Gene-specific primers of Real-time PCR

基因 Gene	引物序列 Primer sequences (5′-3′)	产物大小 Length (bp)
Nrf2	F GCCCTCACTGGATAAAGAA	202
	R CATGCCGTTGCTGGTAC
NQO1	F AACCAACAGACCAGCCAATC	154
	R CACAGTGACCTCCCATCCTT
HO-1	F CAAGGAGAACCCCGTCTACA	225
	R CCAGACAGGTCTCCCAGGTA
β-Actin	F GCCCTGAGGCTCTCTTCCA	101
	R GCGGATGTCGACGTCACA
GAPDH	F TCTTCACTACCATGGAGAAGG	197
	R TCATGGATGACCTTGGCCAG

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1.5.3 Western blot 使用RIPA裂解液（Beyotime,江苏,中国）提取MAC-T细胞总蛋白,并用BCA试剂盒（北京普利莱基因技术有限公司,中国,北京）测定蛋白含量。将30 μg的蛋白经过12% 的SDS-PAGE电泳分离后,将目的蛋白转至PVDF膜上。转膜结束后,将膜放入配置好的3%BSA溶液中室温下封闭4 h。4 ℃条件下摇床孵育一抗（Nrf2、HO-1、NQO1和β-actin;抗体均按照1﹕1 000稀释）过夜,然后室温条件下孵育二抗（辣根过氧化物酶标记的羊抗兔IgG和辣根过氧化物酶标记的羊抗鼠IgG抗体;均按照1﹕5 000稀释）40 min。使用ECL发光液在凝胶成像系统中曝光。

1.6 统计学分析

结果表示为均值±标准差（Mean±SD）。采用SPSS 22.0软件进行单因素方差分析（One-way ANOVA）。

*表示差异显著（P<0.05）,**表示差异极显著（P<0.01）。

2 结果

2.1 姜黄素缓解H₂O₂引起的氧化应激

用不同浓度姜黄素作用于经H₂O₂处理过的MAC-T细胞,继续培养3 h,使用试剂盒检测MDA含量及CAT,SOD和GSH-Px的活性。与对照组相比,H₂O₂处理组MAC-T细胞中MDA含量显著升高（图1-A;P<0.01）,而CAT、SOD和GSH-Px的活性显著降低（图1-B—D;P<0.01）。与H₂O₂处理组相比,15 μmol·L^-1或30 μmol·L^-1姜黄素和H₂O₂共同处理组MAC-T细胞中MDA（图1-A;P<0.01）的含量显著降低,而CAT,SOD和GSH-Px的活性显著升高（图1-B—D;P<0.05,P<0.01）。

图1

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图1姜黄素激活抗氧化酶系统和减弱毒性标志物

A：MDA含量;B：CAT活性;C：SOD活性;D：GSH-P_X活性。*表示差异显著（P<0.05）,**表示差异极显著（P<0.01）。下同
Fig. 1Curcumin activates the antioxidant enzyme system and reduces toxicity markers

A: MDA content; B: CAT activity; C: SOD activity; D: GSH-P_X activity. * Means differ significantly (P<0.05), ** Means differ highly significantly (P<0.01). The same as below

2.2 姜黄素激活了Nrf2信号通路

将经H₂O₂处理好的细胞加入经上一试验所示的最佳浓度姜黄素（30 μmol·L^-1）,继续培养3 h后,检测总Nrf2蛋白和下游基因HO-1和NQO1的mRNA和蛋白表达水平。结果如图2所示,与对照组相比,H₂O₂处理组总Nrf2的蛋白水平显著降低（图2-A和B;P<0.01）。然而,与对照组相比,姜黄素处理组总Nrf2蛋白表达水平显著升高（图2-A和B;P<0.01）。与H₂O₂处理组相比,姜黄素+H₂O₂处理组总Nrf2蛋白表达水平显著升高（图2-A和B;P<0.01）。与对照组相比,H₂O₂处理组Nrf2的下游基因HO-1和NQO1的mRNA和蛋白表达量显著降低（图3-A—E;P< 0.01）。与对照组相比,姜黄素处理组的HO-1和NQO1的mRNA和蛋白的表达量显著升高（图3-A—E;P<0.01）;与H₂O₂处理组相比,姜黄素+H₂O₂处理组的HO-1和NQO1的mRNA和蛋白表达量显著升高（图3-A—E;P<0.01）。

图2

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图2姜黄素增加Nrf2蛋白水平表达

A：Nrf2蛋白条带;B：Nrf2蛋白表达水平
Fig. 2Curcumin induced Nrf2 expression

A: Western blot analysis of Nrf2; B: Relative protein level of Nrf2

图3

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图3姜黄素增加Nrf2下游基因HO-1和NQO1 mRNA及蛋白表达

A：HO-1 mRNA表达水平;B：NQO1 mRNA表达水平;C：HO-1和NQO1蛋白条带;D：HO-1蛋白表达水平;E：NQO1蛋白表达水平
Fig. 3Curcumin induced Nrf2 target genes HO-1 and NQO1 mRNA and protein expression

A: HO-1 mRNA level; B: NQO1 mRNA level; C: Western blot analysis of HO-1 and NQO1; D: Relative protein level of HO-1; E: Relative protein level of NQO1

2.3 姜黄素通过Nrf2缓解H₂O₂诱导的氧化应激

将细胞转染 si-Nrf2处理 48 h 后,用500 μmol·L^-1H₂O₂处理MAC-T细胞24 h后加入姜黄素（30 μmol·L^-1）处理3 h,采用qPCR方法检测Nrf2 mRNA表达水平;使用试剂盒检测MDA含量及CAT,SOD和GSH-PX的活性。结果如图4所示,与对照组相比,si-Nrf2组Nrf2的mRNA表达水平显著降低（图4-A;P<0.01）。与si-Control+H₂O₂组相比si-Control+H₂O₂+姜黄素组中MDA的含量显著降低（图4-B;P<0.01）,而CAT、SOD和GSH-Px的活性显著升高（图4-C—E;P<0.01）。然而,与si-Control+H₂O₂+姜黄素组相比si-Nrf2+H₂O₂+ 姜黄素处理组MDA的含量显著升高（图4-B;P<0.01）,而CAT、SOD和GSH-Px活性显著降低（图4-C—E;P<0.01）。

图4

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图4沉默Nrf2减弱了姜黄素的抗氧化应激作用

A：Nrf2 mRNA表达水平;B：MDA含量;C：CAT活性;D：SOD活性;E：GSH-P_X活性
Fig. 4Silence of Nrf2 attenuated the beneficial effects of curcumin on H₂O₂-induced oxidative stress

A: Nrf2 mRNA level; B: MDA content; C: CAT activity; D: SOD activity; E: GSH-P_X activity

3 讨论

围产期对于奶牛的健康、生产和盈利能力至关重要^[17]。在此期间,由于奶牛存在严重的代谢应激导致脂肪肝、酮病、乳腺炎和子宫内膜炎等生产性疾病的发病率升高。在产后,由于奶牛开始泌乳,大量的NEFA随血液进入使乳腺组织难以满足能量需求。而过量的NEFA可以增加活性氧代谢产物ROS的产生^[18]。当ROS的生成量超过ROS的清除能力时,就会发生氧化应激,导致细胞脂质、蛋白质和DNA的过氧化诱发乳腺组织损伤,致使奶牛泌乳能力降低。因此,提高奶牛乳腺上皮细胞的抗氧化能力对防治围产期奶牛乳腺组织氧化应激,维持奶牛的健康和产奶性能有着至关重要的意义^[19]。

近年来,研究发现中草药对氧化应激具有明显的治疗效果,而且能避免药物残留等问题,因而应用中草药治疗奶牛乳腺组织氧化损伤一直被寄予厚望。姜黄素是姜黄的主要活性成分,作为香料和着色剂广泛应用于食品中^[20]。体内和体外研究发现,姜黄素具有抗氧化、抗炎和抗肿瘤等多种药理作用和生物学活性^[21]。由于姜黄素具有类黄酮化学结构,姜黄素可以通过抑制脂质过氧化并中和超氧化物、羟基自由基和一氧化氮等ROS而发挥抗氧化作用。据报道,姜黄素可抑制脂质过氧化,从而缓解H₂O₂诱导的肾上皮细胞的氧化应激^[22]。此外,郑媛媛等研究发现,姜黄素通过降低MDA含量并增加GSH从而缓解大鼠肝脏星状细胞的氧化应激^[16]。与郑媛媛等报道一致,本试验结果显示,与H₂O₂处理组相比,姜黄素和H₂O₂共同处理组MAC-T细胞中MDA的含量显著降低,而CAT,SOD和GSH-Px的活性显著升高,说明姜黄素是通过增强细胞抗氧化的能力来缓解奶牛乳腺上皮细胞的氧化应激。但本研究采用的H₂O₂处理诱导的细胞氧化应激模型是否可以模拟围产期奶牛体内条件尚不清楚,因此需进一步研究姜黄素对围产期奶牛乳腺组织氧化应激的作用。

Nrf 2是抗氧化应激最重要转录因子之一,通过调节抗氧化分子的表达而发挥抗氧化作用^[23]。正常生理条件下,Nrf 2通过与细胞质中Kelch样ECH相关蛋白1（Keap1）形成复合物而呈失活状态^[24]。当细胞处于氧化应激状态下,细胞质中的Nrf2与Keap1解离,随后Nrf2转移至细胞核中与ARE结合,从而调节NQO1和HO-1等抗氧化剂基因转录^[25,26]。BALOGUN等报道,姜黄素导致Nrf2与Keap1解离,促进猪肾上皮近端小管细胞中HO-1的表达^[27]。此外,饲料中添加亚麻粉可以增加奶牛的乳腺组织中Nrf2 mRNA表达量,说明亚麻粉通过增加Nrf2表达而增强奶牛乳腺上皮细胞的抗氧化能力^[28]。MA等研究发现,过表达Nrf2通过增加SOD和GSH-Px活性并降低ROS含量缓解H₂O₂诱导的奶牛乳腺上皮细胞氧化应激^[7],表明Nrf2信号通路在调节奶牛乳腺上皮细胞氧化应激中发挥重要作用。本研究发现,姜黄素处理可以增加MAC-T细胞中Nrf2蛋白表达量,并增加及其下游基因HO-1和NQO1的mRNA和蛋白表达量,说明姜黄素可以激活Nrf 2信号通路,试验结果与其他报道一致^[29,30]。此外,本研究发现通过降低Nrf 2,增加MAC-T细胞中MDA的含量,降低CAT,SOD和GSH-Px的活性,从而抑制姜黄素的抗氧化功能,表明姜黄素通过Nrf 2信号通路缓解H₂O₂诱导的奶牛乳腺上皮细胞氧化应激。

4 结论

本研究发现姜黄素可以缓解H₂O₂诱导的奶牛乳腺上皮细胞的氧化应激。姜黄素通过增加抗氧化酶的活性,降低细胞毒性产物丙二醛的含量,增加总Nrf2蛋白的表达量及其下游基因HO-1和NQO1的mRNA和蛋白表达量,发挥抗氧化作用。沉默Nrf 2后降低姜黄素对奶牛乳腺上皮细胞的氧化应激保护作用。说明姜黄素可通过激活Nrf 2信号通路,增加抗氧化分子的表达,缓解H₂O₂诱导的奶牛乳腺上皮细胞氧化应激。

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SCOPE: We hypothesized that curcumin, by increasing the expression of nuclear factor-erythroid-2-related factor 2 (Nrf2), could reduce oxidative stress, inflammation, and renal fibrosis in remnant kidney. METHODS AND RESULTS: Sprague-Dawley rats were subjected to 5/6 nephrectomy and randomly assigned to untreated (Nx), curcumin-treated (75 mg/kg/day, orally), and telmisartan-treated groups (10 mg/kg/day, orally; as positive control). Sham-operated rats also served as controls. Five/sixth nephrectomy caused renal dysfunction, as evidenced by elevated proteinuria, blood urea nitrogen, and plasma creatinine, and decreased creatinine clearance that were ameliorated by curcumin or telmisartan treatment. The Nx rats demonstrated reduced Nrf2 protein expression, whereas the Kelch-like ECH-associated protein 1 was upregulated and heme oxygenase-1 level was significantly diminished. Consequently, Nx animals had significantly higher kidney malondialdehyde concentration and lower glutathione peroxidase activity, which was associated with the upregulation of nicotinamide adenine dinucleotide phosphatase oxidase subunit (p67(phox) and p22(phox) ), NF-kappaB p65, TNF-alpha, TGF-beta1, cyclooxygenase-2, and fibronectin accumulation in remnant kidney. Interestingly, all of these changes were ameliorated by curcumin or telmisartan. CONCLUSION: These findings demonstrate that, by modulating Nrf2-Keap1 pathway, the curcumin effectively attenuates oxidative stress, inflammation, and renal fibrosis, which suggest that curcumin hold promising potential for safe treatment of chronic kidney disease.

[15] 宋永周, 关健, 李明, 马维, 袁鹤, 王斌, 童九辉. Nrf2介导姜黄素对软骨细胞氧化应激损伤的保护作用
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SONG Y Z, GUAN J, LI M, MA W, YUAN H, WANG B, TONG J H. Nrf2 mediates the protective effect of curcumin on oxidative stress injury of chondrocytes
Chinese Journal of Tissue Engineering Research, 2015,19(51):8218-8222. (in Chinese)
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[16] 郑媛媛, 刘震雄, 赵曙光, 王旭霞, 崔曼丽, 闻勤生. 姜黄素对氧化应激中肝星状细胞活化及细胞外基质分泌的影响
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[19] HOLBEN D H, SMITH A M. The diverse role of selenium within selenoproteins: a review
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Selenium functions within mammalian systems primarily in the form of selenoproteins. Selenoproteins contain selenium as selenocysteine and perform a variety of physiological roles. Eleven selenoproteins have been identified: cellular or classical glutathione peroxidase; plasma (or extracellular) glutathione peroxidase; phospholipid hydroperoxide glutathione peroxidase; gastrointestinal glutathione peroxidase; selenoprotein P; types 1, 2, and 3 iodothyronine deiodinase; selenoprotein W; thioredoxin reductase; and selenophosphate synthetase. Of these, cellular and plasma glutathione peroxidase are the functional parameters used for the assessment of selenium status. Glutathione peroxidases catalyze the reduction of peroxides that can cause cellular damage. Thioredoxin reductase provides reducing power for several biochemical processes and defends against oxidative stress. Selenoprotein P appears to play a role in oxidant defense. Selenoprotein W may play a role in oxidant defense and be involved with muscle metabolism. Thyroid deiodinases function in the formation and regulation of active thyroid hormone. Selenophosphate synthetase is an enzyme required for the incorporation of selenocysteine into selenoproteins. In addition, a protein in the sperm mitochondrial capsule, which is vital to the integrity of sperm flagella, may be a unique selenoprotein. Recommended intakes, food sources, and status assessment of selenium, as well as selenium's role in health and disease processes, are reviewed.

[20] 乔青青, 任顺成, 吕真真. 姜黄色素稳定性研究
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[21] MENON V P, SUDHEER A R. Antioxidant and anti-inflammatory properties of curcumin
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[22] NITURE S K, KASPAR J W, SHEN J, JAISWAL A K. Nrf2 signaling and cell survival
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DOI:10.1016/j.taap.2009.06.009URLPMID:19538984 [本文引用: 1]
Nrf2:INrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2. Under basal conditions, cytosolic INrf2/Cul3-Rbx1 is constantly degrading Nrf2. When a cell encounters stress Nrf2 dissociates from the INrf2 and translocates into the nucleus. Oxidative/electrophilic stress induced modification of INrf2Cysteine151 and/or protein kinase C (PKC)-mediated phosphorylation of Nrf2Serine40 controls Nrf2 release from INrf2 followed by stabilization and nuclear translocation of Nrf2. Nrf2 binds to the antioxidant response element (ARE) and activates a myriad of genes that protect cells against oxidative/electrophilic stress and neoplasia. A delayed response of oxidative/electrophilic stress activates GSK-3beta that phosphorylates Fyn at unknown threonine residue(s). Phosphorylated Fyn translocates to the nucleus and phosphorylates Nrf2Tyrosine568 that leads to nuclear export and degradation of Nrf2. Prothymosin-alpha mediated nuclear translocation of INrf2 also degrades nuclear Nrf2. The degradation of Nrf2 both in cytosol and nuclear compartments rapidly brings down its levels to normal resulting in suppression of Nrf2 downstream gene expression. An auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it. In conclusion, switching on and off of Nrf2 combined with promoting an auto-regulatory loop between them regulates activation/deactivation of defensive genes leading to protection of cells against adverse effects of oxidative and electrophilic stress and promote cell survival.

[23] COHLY H H P, TAYLOR A, ANGEL M F, SALAHUDEEN A K. Effect of turmeric, turmerin and curcumin on H₂O₂-induced renal epithelial (LLC-PK1) cell injury
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[24] 林健, 文富强. Nrf2-Keap1抗氧化系统与慢性阻塞性肺疾病
西部医学, 2009,21(9):1590-1592.
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LIN J, WEN F Q. Nrf2-keap1 antioxidant system and chronic obstructive pulmonary disease
Western Medicine, 2009,21(9):1590-1592. (in Chinese)
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[25] HAYES J D, MCMAHON M. Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention
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This article provides an overview of the mechanisms by which cancer chemopreventive blocking agents increase the expression of detoxication and antioxidant genes. These agents all appear capable of transcriptionally activating a gene battery that includes NAD(P)H:quinone oxidoreductase, aldo-keto reductases, glutathione S-transferases, gamma-glutamylcysteine synthetase, glutathione synthetase and heme oxygenase. Gene induction occurs through the antioxidant responsive element (ARE), a process that is dependent on the Nuclear Factor-Erythroid 2p45-related factors, Nrf1 and Nrf2. Under basal conditions, these basic region leucine zipper (bZIP) transcription factors are located in the cytoplasm of the cell bound to Keap1, and upon challenge with inducing agents, they are released from Keap1 and translocate to the nucleus. Within the nucleus, Nrf1 and Nrf2 are recruited to the ARE as heterodimers with either small Maf proteins, FosB, c-Jun, JunD, activating transcription factor 2 (ATF2) or ATF4. The role of protein kinases in transducing chemical stress signals to the bZIP factors that affect gene induction through the ARE is discussed.

[26] KANSANEN E, KUOSMANEN S M, LEINONEN H, LEVONEN A. The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer
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[27] SCHOGOR A L B, PALIN M F, DOS SANTOS G T, BENCHAAR C, LACASSE P, PETIT H V. Mammary gene expression and activity of antioxidant enzymes and oxidative indicators in the blood, milk, mammary tissue and ruminal fluid of dairy cows fed flax meal
British Journal of Nutrition, 2013,110(10):1743-1750.
DOI:10.1017/S0007114513001220URL [本文引用: 1]
The effects of flax meal (FM) on the activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)) in the blood, mammary tissue and ruminal fluid, and oxidative stress indicators (thiobarbituric acid-reactive substances (TBARS) and 1,1-diphenyl-2-picrylhydrazyl-scavenging activity) in the milk, plasma and ruminal fluid of dairy cows were determined. The mRNA abundance of the antioxidant enzymes and oxidative stress-related genes was assessed in mammary tissue. A total of eight Holstein cows were used in a double 4 x 4 Latin square design. There were four treatments in the diet: control with no FM (CON) or 5% FM (5FM), 10% FM (10FM) and 15% FM (15FM). There was an interaction between treatment and time for plasma GPx and CAT activities. Cows supplemented with FM had a linear reduction in TBARS at 2 h after feeding, and there was no treatment effect at 0, 4 and 6 h after feeding. TBARS production decreased in the milk of cows fed the 5FM and 10FM diets. There was a linear increase in nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) mRNA abundance in mammary tissue with FM supplementation. A linear trend for increased mRNA abundance of the CAT gene was observed with higher concentrations of FM. The mRNA abundance of CAT, GPx1, GPx3, SOD1, SOD2, SOD3 and nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFKB) genes was not affected by the treatment. These findings suggest that FM supplementation can improve the oxidative status of Holstein cows as suggested by decreased TBARS production in ruminal fluid 2 h post-feeding and increased NFE2L2/nuclear factor-E2-related factor 2 (Nrf2) mRNA abundance in mammary tissue.

[28] BALOGUN E, HOQUE M, GONG P, KILLEEN E, GREEN C, FORESTI R, ALAM J, MOTTERLINI R. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant- responsive element
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[29] 黎珊珊, 徐兆发. 姜黄素激活Nrf2-ARE通路抑制镉肾毒性的研究进展
环境与健康杂志, 2014 (1):94-97.
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LI S S, XU Z F. Research progress of curcumin activating Nrf2-ARE pathway to inhibit cadmium nephrotoxicity
Journal of Environment and Health, 2014(1):94-97. (in Chinese)
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[30] 展平. 姜黄素激活Nrf2系统缓解线粒体损伤和胰岛素抵抗作用机制研究
[D]. 福州: 福建中医药大学, 2017.
DOI:10.1186/1749-8546-6-27URLPMID:21777476 [本文引用: 1]
In recent years, a number of natural products isolated from Chinese herbs have been found to inhibit proliferation, induce apoptosis, suppress angiogenesis, retard metastasis and enhance chemotherapy, exhibiting anti-cancer potential both in vitro and in vivo. This article summarizes recent advances in in vitro and in vivo research on the anti-cancer effects and related mechanisms of some promising natural products. These natural products are also reviewed for their therapeutic potentials, including flavonoids (gambogic acid, curcumin, wogonin and silibinin), alkaloids (berberine), terpenes (artemisinin, beta-elemene, oridonin, triptolide, and ursolic acid), quinones (shikonin and emodin) and saponins (ginsenoside Rg3), which are isolated from Chinese medicinal herbs. In particular, the discovery of the new use of artemisinin derivatives as excellent anti-cancer drugs is also reviewed.
ZHAN P. Study on the mechanism of curcumin-activates Nrf2 System to Alleviate mitochondrial damage and Insulin Resistance
[D]. Fuzhou: Fujian University of Traditional Chinese Medicine, 2017. (in Chinese)
DOI:10.1186/1749-8546-6-27URLPMID:21777476 [本文引用: 1]
In recent years, a number of natural products isolated from Chinese herbs have been found to inhibit proliferation, induce apoptosis, suppress angiogenesis, retard metastasis and enhance chemotherapy, exhibiting anti-cancer potential both in vitro and in vivo. This article summarizes recent advances in in vitro and in vivo research on the anti-cancer effects and related mechanisms of some promising natural products. These natural products are also reviewed for their therapeutic potentials, including flavonoids (gambogic acid, curcumin, wogonin and silibinin), alkaloids (berberine), terpenes (artemisinin, beta-elemene, oridonin, triptolide, and ursolic acid), quinones (shikonin and emodin) and saponins (ginsenoside Rg3), which are isolated from Chinese medicinal herbs. In particular, the discovery of the new use of artemisinin derivatives as excellent anti-cancer drugs is also reviewed.