AbstractThe present study was conducted to observe the protective effects of c9, t11- conjugated linoleic acid (CLA) on oxidative stress and inflammation in bovine mammary epithelial cells (BMECs) exposed to H2O2. The BMECs were treated with different concentrations of H2O2 for 8 h, 600 µmol/L was determined to be the damage concentration.

摘要本研究旨在观察c9，t11-共轭亚油酸（CLA）对暴露于H2O2的牛乳腺上皮细胞（BMEC）氧化应激和炎症的保护作用。用不同浓度的H2O2处理BMEC 8小时，确定600μmol/L为损伤浓度。

Using different concentrations of c9, t11-CLA to process BMECs for 24 h, 50 and 100 µmol/L were determined to be the effective concentrations for subsequent analyses. Thus, four BMEC groups were established: Control group; H2O2 group; 50 µmol/L c9, t11-CLA + H2O2 group; 100 µmol/L c9, t11-CLA + H2O2 group.

使用不同浓度的c9，t11-CLA处理BMEC 24小时，确定50和100µmol/L是后续分析的有效浓度。因此，建立了四个BMEC组：对照组；H2O2组；50μmol/L c9，t11 CLA+ H2O2组；100µmol/L c9，t11 CLA+ H2O2组。

We observed that the H2O2 group exhibited significantly lower total antioxidant activity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and significantly higher secretions of malondialdehyde (MDA), interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α and expressions of IL-1β, IL-6, and IL-8 than the control group (p < 0.05).

我们观察到H2O2组的总抗氧化活性（T-AOC），超氧化物歧化酶（SOD），过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx）活性显着降低，丙二醛（MDA），白细胞介素（IL）-1β，IL-6，IL-8和肿瘤坏死因子（TNF）-α的分泌以及IL-1β，IL-6和IL-8的表达显着高于对照组（p<0.05）。

Pretreatment with c9, t11-CLA enhanced SOD, CAT, and GPx activities and SOD mRNA expression and repressed IL-6 and IL-8 secretion and expression in H2O2-treated BMECs (p < 0.05). In conclusion, c9, t11-CLA treatment efficiently enhanced antioxidant capacity and decreased inflammation induced by H2O2 in BMECs..

用c9，t11-CLA预处理增强了H2O2处理的BMEC中SOD，CAT和GPx活性以及SOD mRNA的表达，并抑制了IL-6和IL-8的分泌和表达（p<0.05）。总之，c9，t11-CLA处理有效增强了BMEC中H2O2诱导的抗氧化能力并减少了炎症。。

IntroductionIn recent years, the continuous advancement in modern feeding has significantly enhanced the production performance of dairy cows in China. However, during the perinatal period and the peak of lactation, the metabolic rate of mammary cells increases obviously, and the enhancement of metabolic activity leads to the accumulation of free radicals in the mammary tissues of dairy cows1,2.

引言近年来，现代饲养的不断进步显着提高了中国奶牛的生产性能。然而，在围产期和泌乳高峰期，乳腺细胞的代谢率明显增加，代谢活性的增强导致奶牛乳腺组织中自由基的积累1,2。

Free radical accumulation could reduce the feed utilization rate and milk yield in dairy cows3. The reduced antioxidant defense in cows makes them prone to oxidative stress, leading to the incidence of several diseases, such as bovine mastitis. Metabolic stress in dairy cows leads to lipid mobilization, inflammation, oxidative stress, and subsequent metabolic stress in the prenatal period, which affects milk production and health of dairy cows, resulting in enormous economic losses to the dairy industry4,5.

自由基积累会降低奶牛的饲料利用率和产奶量3。奶牛抗氧化防御能力的降低使其容易受到氧化应激，从而导致多种疾病的发生，例如奶牛乳腺炎。奶牛的代谢应激导致产前脂质动员，炎症，氧化应激和随后的代谢应激，影响奶牛的产奶量和健康，给奶牛业造成巨大的经济损失4,5。

Oxidative stress reflects an imbalance between free radical production and the body’s ability to detoxify via antioxidants. Under physiological conditions, the antioxidant systems maintain the oxidant-antioxidant balance by rectifying the altering levels of oxidants. In over-conditioned cows, an inflammatory state is caused by the oxidative stress frequently induced by excessive lipolysis that enhances reactive oxygen species (ROS) production via the β-oxidation process.

氧化应激反映了自由基产生与身体通过抗氧化剂解毒能力之间的不平衡。在生理条件下，抗氧化系统通过纠正氧化剂水平的变化来维持氧化剂-抗氧化剂的平衡。在条件过度的奶牛中，炎症状态是由过度脂解引起的氧化应激引起的，过度脂解通过β-氧化过程增强了活性氧（ROS）的产生。

Excessive ROS accumulation enhances the expression of pro-inflammatory mediators, including cytokines6. Therefore, several researchers have focused on reducing the metabolic stress in the mammary glands of dairy cows in order to improve the antioxidants or the anti-inflammatory capacity in the mammary glands and improve milk quality.Fatty acids play an important role in regulating oxidative stress and inflamm.

过量的ROS积累增强了促炎介质（包括细胞因子）的表达6。因此，一些研究人员致力于减少奶牛乳腺中的代谢应激，以提高乳腺中的抗氧化剂或抗炎能力，改善牛奶质量。脂肪酸在调节氧化应激和炎症中起重要作用。

Control group: Cells were not treated with either c9, t11-CLA or H2O2.

对照组：细胞未用c9，t11 CLA或H2O2处理。

H2O2 group: Cells were treated with 600 µmol/L H2O2 for 8 h.

H2O2组：用600μmol/L H2O2处理细胞8小时。

c9, t11-CLA (50) + H2O2 group: Cells were pretreated with 50 µmol/L c9, t11-CLA for 24 h, washed twice with PBS, and treated with 600 µmol/L H2O2 for 8 h.

c9，t11-CLA（50）+H2O2组：用50μmol/L c9，t11-CLA预处理细胞24小时，用PBS洗涤两次，并用600μmol/L H2O2处理8小时。

c9, t11-CLA (100) + H2O2 group: Cells were pretreated with 100 µmol/L c9, t11-CLA for 24 h, washed twice with PBS, and treated with 600 µmol/L H2O2 for 8 h.

c9，t11-CLA（100）+H2O2组：用100μmol/L c9，t11-CLA预处理细胞24小时，用PBS洗涤两次，并用600μmol/L H2O2处理8小时。

Determination of antioxidant enzymes and cytokine levels by ELISABMECs were seeded in each well of 6-well microplates at a density of 1 × 104 cells/well. The MDA content and the activities of antioxidant enzymes (T-AOC, SOD, GPx, and CAT) and pro- and anti-inflammatory interleukins (IL-6, IL-8, IL-1β, and TNF-α) secreted by BMECs were detected respectively using bovine enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.) according to the manufacturer’s instructions.Quantitative real-time polymerase chain reaction (qRT-PCR)The mRNA abundances of the genes encoding bovine pro- and anti-inflammatory interleukins (IL-1 β, IL-6, and IL-8) and antioxidant enzymes (CAT, SOD, and GPx1) in BMECs were detected using qRT-PCR.Briefly, the BMECs were seeded in 6-well microplates at the concentration of 1 × 104 cells/well in DMEM/F12 medium for 48 h and subjected to the four treatments stated above.

通过ELISABMECs测定抗氧化酶和细胞因子水平将其以1×104个细胞/孔的密度接种在6孔微孔板的每个孔中。根据制造商的说明，使用牛酶联免疫吸附测定（ELISA）试剂盒（上海酶联生物技术有限公司）分别检测BMEC分泌的MDA含量和抗氧化酶（T-AOC，SOD，GPx和CAT）以及促炎和抗炎白细胞介素（IL-6，IL-8，IL-1β和TNF-α）的活性。定量实时聚合酶链反应（qRT-PCR）使用qRT-PCR检测BMEC中编码牛促炎和抗炎白细胞介素（IL-1β，IL-6和IL-8）和抗氧化酶（CAT，SOD和GPx1）的基因的mRNA丰度。简而言之，将BMEC以1×104个细胞/孔的浓度接种在DMEM/F12培养基中的6孔微孔板中48小时，并进行上述四种处理。

The total RNA of the cells was extracted using the RNAprep Pure Cell/Bacteria kit (DP430, Tiangen Biotech, China) according to the manufacturer’s instructions and stored at − 80 °C. The RNA purity and concentrations were determined by measuring the 260 nm/280 nm ratio using a BioTek Synergy H4 Hybrid Reader.

根据制造商的说明，使用RNAprep Pure Cell/Bacteria kit（DP430，Tiangen Biotech，China）提取细胞的总RNA，并保存在-80℃。通过使用BioTek Synergy H4杂交阅读器测量260 nm/280 nm的比率来确定RNA的纯度和浓度。

The RNA purity results of 260 nm/280 nm ratio were all between 1.8 and 2.0. The RNA integrity was assessed using 1% agarose gel electrophoresis. Next, 100 ng total RNA was added in the reaction system to reverse transcribed into cDNA by a PrimeScript RT Master Mix Kit (TaKaRa, RR820A, Dalian, China).

260 nm/280 nm比率的RNA纯度结果均在1.8至2.0之间。使用1%琼脂糖凝胶电泳评估RNA完整性。接下来，在反应系统中加入100 ng总RNA，通过PrimeScript RT Master Mix试剂盒（TaKaRa，RR820A，大连，中国）逆转录成cDNA。

RT-qPCR was conducted on a Light Cycler 480 (Roche Holding AG, Basel, Switzerland) with a SYBR Premix Ex TaqTM II kit (TaKaRa, RR047A, Dalian, China) following the manufacturer’s recommendations. The PCR conditions were set as follows: 95 °C for .

按照制造商的建议，使用SYBR Premix Ex TaqTM II试剂盒（TaKaRa，RR047A，大连，中国），在Light Cycler 480（Roche Holding AG，瑞士巴塞尔）上进行RT-qPCR。PCR条件设定如下：95°C。

Data availability

数据可用性

All data generated or analysed during this study are included in this published article and its supplementary information file.

本研究期间生成或分析的所有数据均包含在本文及其补充信息文件中。

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PubMed Google ScholarContributionsH.Z wrote the original main manuscript text and conducted the investigation and computational experiments. Y.Q.W did the investigation, validation, and visualization; C.L.G. analyzed and interpreted the data; and N.D. acquired funding and drafted and critically reviewed the manuscript.

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Reprints and permissionsAbout this articleCite this articleZhang, H., Dan, N., Wang, Yq. et al. Protection effect of cis 9, trans 11-conjugated linoleic acid on oxidative stress and inflammatory damage in bovine mammary epithelial cells.

转载和许可本文引用本文Zhang，H.，Dan，N.，Wang，Yq。顺式9，反式11共轭亚油酸对牛乳腺上皮细胞氧化应激和炎症损伤的保护作用。

Sci Rep 14, 26295 (2024). https://doi.org/10.1038/s41598-024-77711-0Download citationReceived: 25 July 2024Accepted: 24 October 2024Published: 01 November 2024DOI: https://doi.org/10.1038/s41598-024-77711-0Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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KeywordsCis 9, trans 11-conjugated linoleic acidBovine mammary epithelial cellsOxidative stressInflammation

关键词SCIS 9，反式11共轭亚油酸牛乳腺上皮细胞氧化应激炎症