Sirtuin 3 inhibits nuclear factor-KB signaling activated by a fatty acid challenge in bovine mammary epithelial cells
Sirtuin 3 inhibits nuclear factor-KB signaling activated by a fatty acid challenge in bovine mammary epithelial cells
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DOI：
10.3168/jds.2021-20536
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发表时间：
2021-11-17
影响因子：
3.5
通讯作者：
Li, Xinwei
中科院分区：
文献类型：
--
作者：
Liu, Lei;Lu, Hong;Li, Xinwei
乳腺炎的易感性在围产期（过渡期）最高，通常伴有其他合并症，如酮症。虽然病原微生物感染和产犊前后的免疫功能障碍在乳腺炎的发生中起着关键作用，但其他代谢因素也有影响。Sirtuin 3（SIRT 3）是一种调节能量和氧化还原稳态的线粒体脱乙酰酶，可拮抗非酯化脂肪酸（NEFA）的脂毒作用。因此，我们假设在过渡期观察到的循环NEFA浓度的增加引起炎症反应，该炎症反应可以通过SIRT 3的激活来逆转。在这里，我们旨在研究（1）促炎性NF-κ B信号传导和SIRT 3丰度在酮病奶牛和健康对照的乳腺组织中，和（2）SIRT 3对NF-κ B活化的影响在牛乳腺上皮细胞（BMEC）用高水平的NEFA处理。乳腺活检样本来自先前的研究，其中包括15头健康奶牛和15头酮病奶牛。用胶原酶III消化法从3头健康荷斯坦奶牛中分离原代BMEC。将纯化的BMEC与或不与SIRT 3过表达腺病毒一起孵育48小时，然后用0、0.6、1.2或2.4 mM NEFA处理24小时。酮病奶牛的乳腺组织与SIRT 3沿着较低的蛋白丰度以及较高的NF-κ B P65磷酸化水平（p-NF-κ B P65）、p-NF-κ B P65：NF -κ B P65比率以及IL 1 B和IL 6的mRNA丰度相关。在BMEC中，外源性NEFA剂量依赖性地降低SIRT 3的蛋白丰度，但增加p-NF-Kappa B P65、p-NF-Kappa B P65：NF -Kappa B P65比率以及IL 1 B和IL 6的mRNA丰度。与绿色荧光蛋白腺病毒载体+ NEFA相比，SIRT 3在NEFA处理的BMEC中过表达下调p-NF-κ B P65和IL 1 B和IL 6的mRNA丰度。免疫荧光结果显示，SIRT 3的过表达抑制了NF-κ B B P65的核转位。总之，我们的数据表明，酮病与乳腺中SIRT 3丰度的降低和NF-κ B信号传导的激活相关。体外数据提供的证据表明，高NEFA浓度抑制SIRT 3，这有助于增强NF-κ B信号传导，包括核转位和促炎反应。这些数据表明，SIRT 3作为一种靶点，有助于减轻因暴露于高浓度NEFA而导致的乳腺局部炎症，具有很好的作用。
Susceptibility to mastitis is highest during the peri-partal (transition) period and is often concomitant with other comorbidities such as ketosis. Although infection with pathogenic microorganisms and immune-dysfunc-tion around calving clearly play key roles in mastitis development, other metabolic factors also contribute. Sirtuin 3 (SIRT3), a mitochondrial deacetylase regu-lating energy and redox homeostasis, antagonizes the lipotoxic effects of nonesterified fatty acids (NEFA). Thus, we hypothesized that increases in circulating NEFA concentrations, as observed in the transition period, provokes inflammatory responses that can be reversed via activation of SIRT3. Here we aimed to study (1) proinflammatory NF-Kappa B signaling and SIRT3 abundance in mammary tissue of ketotic cows and healthy controls, and (2) the effect of SIRT3 on NF-Kappa B activation in bovine mammary epithelial cells (BMEC) treated with high levels of NEFA. The mam-mary gland biopsy samples were from a previous study, which included 15 healthy cows and 15 ketotic cows. Primary BMEC were isolated from 3 healthy Holstein cows with collagenase III digestion. Purified BMEC were incubated with or without SIRT3 overexpression adenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 24 h. Mammary tissue of ketotic cows was associated with lower protein abundance of SIRT3 along with greater NF-Kappa B P65 phosphorylation levels (p-NF-Kappa B P65), p-NF-Kappa B P65: NF -Kappa B P65 ratio, and mRNA abundance of IL1B and IL6. In BMEC, exog-enous NEFA dose-dependently reduced protein abun-dance of SIRT3, but increased p-NF-Kappa B P65, p-NF-Kappa B P65: NF -Kappa B P65 ratio, and mRNA abundance of IL1B and IL6. Compared with green fluorescent protein adenovirus vector + NEFA, overexpression of SIRT3 in NEFA-treated BMEC downregulated p-NF-Kappa B P65 and mRNA abundance of IL1B and IL6. Immuno-fluorescence indicated that overexpression of SIRT3 inhibited nuclear translocation of NF-Kappa B P65. Overall, our data demonstrated that ketosis is associated with a reduction in SIRT3 abundance and activation of NF-Kappa B signaling in the mammary gland. In vitro data pro-vided evidence that high NEFA concentrations inhibit SIRT3, which contributes to enhanced NF-Kappa B signaling including nuclear translocation and a pro-inflammatory response. The data suggest a promising role of SIRT3 as a target for helping alleviate localized inflammation of the mammary gland resulting from exposure to high concentrations of NEFA.