Xu Tianle, Wu Xinyue, Lu Xubin, Liang Yusheng, Mao Yongjiang, Loor Juan J, Yang Zhangping
College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, People's Republic of China.
Joint International Research Laboratory of Agriculture and Agri-product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, 225009, People's Republic of China.
BMC Vet Res. 2021 Mar 1;17(1):97. doi: 10.1186/s12917-021-02797-x.
Lipopolysaccharides (LPS) derived from gram-negative bacterial are often regarded as primary inducer of bovine mammary inflammation. This study evaluated the biological response of metformin activated AMPK signaling on LPS-induced inflammatory responses and metabolic changes in primary bovine mammary epithelial cells (pbMEC). The pbMEC were exposed to either 3 mmol/L Metf. for 12 h as Metf. group (Metf.) or 2 μg/mL LPS for 6 h as LPS group (LPS). Cells pretreated with 3 mmol/L metformin for 12 h followed by washing and 2 μg/mL LPS exposure for 6 h were served as ML group (ML). PBS was added to cells as the control group (Con.).
Pre-incubation with Metf. inhibited LPS-induced expression of pro-inflammatory genes (TNF, IL1B, IL6, CXCL8, MYD88 and TLR4) and proteins (IL-1β, TNF-α, NLRP3, Caspase1, ASC) and was accompanied by increased activation of AMPK signaling. Compared with the LPS group, phosphorylation of p65 and IκBα in the ML group were decreased and accumulation of NF-κB in the nucleus was significantly reduced by pretreatment with metformin. Metformin protects the cells from the increase of LPS-induced binding activity of NF-κB on both TNFA and IL1B promoters. Compared with the LPS group, genes (G6PC, PCK2) and proteins (SREBP1, SCD1) related to lipogenesis and carbohydrate metabolism were downregulated while catabolic ones (PPARA, ACSL1, Glut1, HK1) were upregulated in the ML group. Furthermore, increased acetylation of H3K14 by LPS challenge was reversed by pretreatment with metformin.
Altogether, our results indicated that pretreatment with metformin dampens LPS-induced inflammatory responses mediated in part by AMPK/NF-κB/NLRP3 signaling and modification of histone H3K14 deacetylation and metabolic changes.
革兰氏阴性菌衍生的脂多糖（LPS）通常被视为牛乳腺炎症的主要诱导剂。本研究评估了二甲双胍激活的AMPK信号对LPS诱导的原代牛乳腺上皮细胞（pbMEC）炎症反应和代谢变化的生物学反应。将pbMEC暴露于3 mmol/L二甲双胍12小时作为二甲双胍组（Metf.），或暴露于2 μg/mL LPS 6小时作为LPS组（LPS）。用3 mmol/L二甲双胍预处理12小时，然后洗涤并暴露于2 μg/mL LPS 6小时的细胞作为ML组（ML）。向细胞中加入PBS作为对照组（Con.）。
用二甲双胍预孵育可抑制LPS诱导的促炎基因（TNF、IL1B、IL6、CXCL8、MYD88和TLR4）和蛋白质（IL-1β、TNF-α、NLRP3、Caspase1、ASC）的表达，并伴随着AMPK信号激活增加。与LPS组相比，ML组中p65和IκBα的磷酸化降低，二甲双胍预处理显著减少了NF-κB在细胞核中的积累。二甲双胍保护细胞免受LPS诱导的NF-κB对TNFA和IL1B启动子结合活性的增加。与LPS组相比，ML组中与脂肪生成和碳水化合物代谢相关的基因（G6PC、PCK2）和蛋白质（SREBP1、SCD1）下调，而分解代谢相关的基因（PPARA、ACSL1、Glut1、HK1）上调。此外，LPS刺激导致的H3K14乙酰化增加被二甲双胍预处理逆转。
总之，我们的结果表明，二甲双胍预处理可减轻LPS诱导的炎症反应，部分是通过AMPK/NF-κB/NLRP3信号传导以及组蛋白H3K14去乙酰化修饰和代谢变化介导的。
