College of Veterinary Medicine, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Hunan Agricultural University, Changsha, 410128, China.
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China.
J Dairy Sci. 2023 Feb;106(2):1315-1329. doi: 10.3168/jds.2022-22114. Epub 2022 Dec 7.
Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 μg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 μM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 μg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 μg/mL) for 24 h and then challenged with 150 μg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 μM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 μg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 μg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 μg/mL LPS; concentrations of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1β, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.
由于乳腺炎导致牛乳腺内皮细胞（BMEC）过度炎症，导致疾病进展和最终淘汰牛。Sirtuin 3（SIRT3）是一种线粒体脱乙酰酶，通过使核因子-κB（NFκB）信号失活，下调暴露于高浓度非酯化脂肪酸的 BMEC 中的促炎细胞因子。在非反刍动物中，SIRT3 受 PGC1α 的控制，PGC1α 是一种转录共因子。具体目标是研究（1）SIRT3 对脂多糖（LPS）刺激的牛乳腺上皮细胞（牛乳腺肺泡细胞-T，MAC-T）模型炎症反应的影响，和（2）PGC1α 通过 SIRT3 衰减 NFκB 信号的作用。为了实现这些目标，首先，将 MAC-T 细胞在三复孔中分别与 0、50、100、150 或 200 μg/mL LPS（源自大肠杆菌 O55：B5）孵育 12 小时，同时或不使用 NFκB 抑制剂铵吡啶二硫代氨基甲酸盐（APDC，10 μM）孵育 2 小时。其次，使用腺病毒表达（Ad-SIRT3）以不同的感染复数（MOI）将 SIRT3 过表达 6 小时，然后用 150 μg/mL LPS 孵育 12 小时。第三，用 PGC1α 激动剂 ZLN005（10 μg/mL）处理细胞 24 小时，然后用 150 μg/mL LPS 刺激 12 小时。第四，细胞最初用 PGC1α 抑制剂 SR-18292（100 μM）处理 6 小时，然后用或不用 50 MOI Ad-SIRT3 培养 6 小时，并在 150 μg/mL LPS 刺激 12 小时。使用单因素方差分析和随后的 Bonferroni 校正分析数据。使用线性和二次对比来确定 LPS 的剂量反应。LPS 剂量对细胞活力有线性和二次影响。孵育 150 和 200 μg/mL LPS 12 小时，细胞活力分别降至 78.6%和 34.9%。与对照组相比，用 150 μg/mL LPS 处理后，IL1B、IL6 和 TNFA 的表达分别上调了 5.2、5.9 和 2.7 倍；细胞培养基中 IL-1β、IL-6 和肿瘤坏死因子-α（TNF-α）的浓度也增加了。与 LPS 组相比，LPS+APDC 增加了细胞活力并逆转了 IL1B、IL6 和 TNFA 表达的上调。然而，随着 LPS 剂量的增加，SIRT3 的 mRNA 和蛋白丰度呈线性下降。Ad-SIRT3 感染（50 MOI）降低了 IL1B、IL6 和 TNFA 的表达及其在细胞培养基中的浓度，并降低了 pNFκB P65/NFκB P65 比值和 NFκB P65 的核丰度。PGC1α 激动剂增加了 SIRT3 的表达，同时降低了细胞因子的表达、pNFκB P65/NFκB P65 比值，并阻止了 NFκB P65 的核转位。与激动剂相反，PGC1α 抑制剂具有相反的作用，并提高了细胞培养基中 IL-1β、IL-6 和 TNF-α 的浓度。总体而言，数据表明，SIRT3 活性可以通过改变 PGC1α-NFκB 途径来减轻乳腺炎引起的乳腺细胞炎症反应。因此，在体内靶向 SIRT3 可能有助于预防或减轻乳腺炎的负面影响。
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