Metformin activated AMPK signaling contributes to the alleviation of LPS-induced inflammatory responses in bovine mammary epithelial cells

BackgroundLipopolysaccharides (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 3mmol/L Metf. for 12h as Metf. group (Metf.) or 2 mu g/mL LPS for 6h as LPS group (LPS). Cells pretreated with 3mmol/L metformin for 12h followed by washing and 2 mu g/mL LPS exposure for 6h were served as ML group (ML). PBS was added to cells as the control group (Con.).ResultsPre-incubation with Metf. inhibited LPS-induced expression of pro-inflammatory genes (TNF, IL1B, IL6, CXCL8, MYD88 and TLR4) and proteins (IL-1 beta, TNF-alpha, NLRP3, Caspase1, ASC) and was accompanied by increased activation of AMPK signaling. Compared with the LPS group, phosphorylation of p65 and I kappa B alpha in the ML group were decreased and accumulation of NF-kappa B in the nucleus was significantly reduced by pretreatment with metformin. Metformin protects the cells from the increase of LPS-induced binding activity of NF-kappa B 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.ConclusionAltogether, our results indicated that pretreatment with metformin dampens LPS-induced inflammatory responses mediated in part by AMPK/NF-kappa B/NLRP3 signaling and modification of histone H3K14 deacetylation and metabolic changes.

Automated summary

This study showed that pretreatment with metformin can inhibit LPS-induced inflammatory responses by activating AMPK signaling pathway and reducing the accumulation of NF-kappa B in the nucleus. It also affects the metabolic changes in cells by regulating the expression of genes and proteins related to lipogenesis and carbohydrate metabolism.