Metformin Inhibits Lipoteichoic Acid-Induced Oxidative Stress and Inflammation Through AMPK/NRF2/NF-κB Signaling Pathway in Bovine Mammary Epithelial Cells.
Metformin Inhibits Lipoteichoic Acid-Induced Oxidative Stress and Inflammation Through AMPK/NRF2/NF-κB Signaling Pathway in Bovine Mammary Epithelial Cells.
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二甲双胍通过 AMPK/NRF2/NF-κB 信号通路抑制牛乳腺上皮细胞中脂磷壁酸诱导的氧化应激和炎症
DOI：
10.3389/fvets.2021.661380
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发表时间：
2021
影响因子：
3.2
通讯作者：
Yang Z
中科院分区：
文献类型：
--
作者：
Arbab AAI;Lu X;Abdalla IM;Idris AA;Chen Z;Li M;Mao Y;Xu T;Yang Z
本研究的目的是探讨二甲双胍对脂磷壁酸（LTA）诱导的乳腺炎模型的影响，使用分离的原代牛乳腺上皮细胞（PBMEC）。PBMEC暴露于3 mM二甲双胍12 h作为二甲双胍组（MET）或100 μg/mL LTA 6 h作为LTA组（LTA）。用3 mM二甲双胍预处理12 h，然后洗涤并暴露于100 μg/mL LTA 6 h的细胞作为MET + LTA组。将磷酸盐缓冲盐水加入细胞中作为对照组。流式细胞术（annexin V-异硫氰酸荧光素法）检测不同剂量二甲双胍预处理的PBMEC的细胞凋亡率。我们进行了定量逆转录酶-聚合酶链反应和蛋白质印迹分析，通过测量细胞毒性、mRNA表达和蛋白质表达来评估二甲双胍和LTA的炎症和氧化反应。免疫荧光法用于评价核定位。结果显示，与对照细胞相比，用LTA剂量攻击的细胞中COX 2、IL-1β和IL-6的基因表达显著增加。在炎症性PBMEC中，二甲双胍减弱了LTA诱导的炎症基因核因子κB（NF-κB）p65、肿瘤坏死因子α、环氧合酶2和白细胞介素1β的表达，以及NF-κ Bp 65蛋白的核定位和磷酸化，但增加核因子红细胞2相关因子2（Nrf 2）和Nrf 2靶向抗氧化基因血红素加氧酶-1（HO-1）的转录和Gpx 1的表达以及HO-1蛋白的核定位。重要的是，二甲双胍诱导的Nrf 2活化是AMP活化蛋白激酶（AMPK）依赖性的;由于二甲双胍预处理的PBMEC通过上调磷酸化AMPK水平激活AMPK信号传导，二甲双胍预处理的细胞也逆转了LTA抑制的Nrf 2易位。AMPK和Nrf 2通路之间的这种融合对于二甲双胍在LTA刺激的PBMEC中的抗炎作用至关重要。总之，我们的研究结果表明，二甲双胍通过调节AMPK/Nrf 2/NF-κB信号通路发挥抗炎和氧化应激作用，这突出了AMPK作为治疗牛乳腺炎的潜在治疗策略的作用。
The objective of this research was to explore the effect of metformin on the lipoteichoic acid (LTA)–induced mastitis model using isolated primary bovine mammary epithelial cells (PBMECs). The PBMECs were exposed to either 3 mM metformin for 12 h as a metformin group (MET) or 100 μg/mL LTA for 6 h as LTA group (LTA). Cells pretreated with 3 mM metformin for 12 h followed by washing and 100 μg/mL LTA exposure for 6 h served as the MET + LTA group. Phosphate-buffered saline was added to cells as the control group. PBMECs pretreated with different metformin doses were analyzed by a flow cytometry (annexin V–fluorescein isothiocyanate assay) to detect the cell apoptotic rate. We performed quantitative reverse transcriptase–polymerase chain reaction and Western blot analysis to evaluate the inflammatory and oxidative responses to metformin and LTA by measuring cellular cytotoxicity, mRNA expression, and protein expression. Immunofluorescence was used to evaluate nuclear localization. The results showed that the gene expression of COX2, IL-1β, and IL-6 significantly increased in the cells challenged with LTA doses compared to control cells. In inflammatory PBMECs, metformin attenuated LTA-induced expression of inflammatory genes nuclear factor κB (NF-κB) p65, tumor necrosis factor α, cyclooxygenase 2, and interleukin 1β, as well as the nuclear localization and phosphorylation of NF-κBp65 protein, but increased the transcription of nuclear factor erythroid 2–related factor 2 (Nrf2) and Nrf2-targeted antioxidative genes heme oxygenase-1 (HO-1) and Gpx1, as well as the nuclear localization of HO-1 protein. Importantly, metformin-induced activation of Nrf2 is AMP-activated protein kinase (AMPK)–dependent; as metformin-pretreated PBMECs activated AMPK signaling via the upregulation of phosphorylated AMPK levels, cell pretreatment with metformin also reversed the translocation of Nrf2 that was LTA inhibited. This convergence between AMPK and Nrf2 pathways is essential for the anti-inflammatory effect of metformin in LTA-stimulated PBMECs. Altogether, our results indicate that metformin exerts anti-inflammation and oxidative stress through regulation of AMPK/Nrf2/NF-κB signaling pathway, which highlights the role of AMPK as a potential therapeutic strategy for treatment of bovine mastitis.
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