Joint International Research Laboratory of Agriculture and Agri-product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, 225009, P. R. China.
College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, P. R. China.
J Anim Sci. 2021 Jul 1;99(7). doi: 10.1093/jas/skab153.
The occurrence of bovine ketosis involves the accumulation of β-hydroxybutyric acid (BHBA), which contributes to the initiation and acceleration of hepatic metabolic stress and inflammation. Metformin has other beneficial effects apart from its medical intervention for diabetes, such as prevention of laminitis and hyper-triglyceridemic. AMPK maintains energy homeostasis and is the intracellular target of metformin action. This study aims to uncover the role of metformin in modulating BHBA-induced inflammatory responses through the activation of AMPK signaling. The hepatocytes were isolated from the liver tissue of mid-lactation multiparous Holstein cows (~160 d postpartum). Treatments were conducted as follows: treated with PBS for 18 h (control); pretreated with PBS for 12 h followed by treatment of 1.2 mM BHBA for 6 h (BHBA); pretreated with 1.5 mM or 3 mM metformin for 12 h followed by the BHBA treatment (1.2 mM) for 6 h (M(1.5)+B; M(3)+B). The inhibitor of AMPK, Compound C, at a concentration of 10 μM, was applied to substantiate the AMPK-dependent responses. RT-qPCR were applied for the mRNA expression while Western-blots and immunofluorescence were conducted for the target proteins expression. Among dose-dependent assays for BHBA, the concentration of BHBA at 1.2 mM activated NF-κB signaling by upregulating the expression of phosphorylated NF-κB and pro-inflammatory cytokines compared with the control cells (P < 0.05). Along with the upregulation of phosphorylated AMPKα and ACCα, metformin at 1.5 and 3 mM inactivated NF-κB signaling components (p65 and IκBα) and the inflammatory genes (TNFA, IL6, IL1B and COX-2) which were activated by BHBA. Additionally, BHBA inhibited cells staining intensity in EdU assay were increased by pretreatment with metformin. The activation of AMPK resulted in the increased gene and protein expression of SIRT1, along with the deacetylation of H3K9 and H3K14. However, the AMPK inhibitor compound C blocked this effect. Compared with BHBA treated cells, the protein expression of COX-2 and IL-1β were decreased by the pretreatment with metformin, and the inhibitory effect of metformin was released by compound C. The bound of NF-κB onto IL1B promoter displayed higher in BHBA group and this was suppressed by pretreatment with metformin (P < 0.05). Altogether, metformin attenuates the BHBA-induced inflammation through the inactivation of NF-κB as a target for AMPK/SIRT1 signaling in bovine hepatocytes.
奶牛酮病的发生涉及β-羟丁酸（BHBA）的积累，这有助于启动和加速肝代谢应激和炎症。二甲双胍除了对糖尿病的医学干预外，还有其他有益作用，如预防蹄叶炎和高甘油三酯血症。AMPK 维持能量平衡，是二甲双胍作用的细胞内靶点。本研究旨在通过激活 AMPK 信号来揭示二甲双胍在调节 BHBA 诱导的炎症反应中的作用。肝细胞从泌乳中期多胎荷斯坦奶牛的肝组织中分离出来（产后约 160 天）。处理如下：用 PBS 处理 18 小时（对照）；用 PBS 预处理 12 小时，然后用 1.2 mM BHBA 处理 6 小时（BHBA）；用 1.5 mM 或 3 mM 二甲双胍预处理 12 小时，然后用 1.2 mM BHBA 处理 6 小时（M（1.5）+B；M（3）+B）。用浓度为 10 μM 的 AMPK 抑制剂 Compound C 来证实 AMPK 依赖性反应。应用 RT-qPCR 进行 mRNA 表达，应用 Western-blot 和免疫荧光进行靶蛋白表达。在 BHBA 的剂量依赖性试验中，与对照细胞相比，浓度为 1.2 mM 的 BHBA 通过上调磷酸化 NF-κB 和促炎细胞因子的表达激活 NF-κB 信号（P < 0.05）。随着磷酸化 AMPKα和 ACCα的上调，1.5 和 3 mM 的二甲双胍失活了由 BHBA 激活的 NF-κB 信号成分（p65 和 IκBα）和炎症基因（TNFA、IL6、IL1B 和 COX-2）。此外，BHBA 抑制了 EdU 测定中细胞染色强度，而用二甲双胍预处理则增加了这种强度。AMPK 的激活导致 SIRT1 的基因和蛋白表达增加，同时 H3K9 和 H3K14 的去乙酰化。然而，AMPK 抑制剂 Compound C 阻断了这种作用。与 BHBA 处理的细胞相比，用二甲双胍预处理降低了 COX-2 和 IL-1β的蛋白表达，而 Compound C 释放了二甲双胍的抑制作用。NF-κB 与 IL1B 启动子的结合在 BHBA 组中显示出更高的结合，而这种结合被二甲双胍预处理所抑制（P < 0.05）。总之，二甲双胍通过 AMPK/SIRT1 信号通路失活 NF-κB 作为靶标来减轻奶牛肝细胞中 BHBA 诱导的炎症。
