Abstract
As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.
Highlights
As a common metabolic disease, the incidence of sub-acute ruminal acidosis (SARA) is greater than20% in the primary and middle lactation of dairy cows, which causes significant economic damage to dairy cattle cultivation [1]
cytokeratin 18 (CK18) was counterstained with fluorescein isothiocyanate (FITC), and nuclei were counterstained with propidium iodide (PI)
Our results demonstrated that treatment of dairy cow mammary epithelial cells (DCMECs) with LPS alone resulted in a remarkable increase in tumor necrosis factor-α (TNF-α) and IL-6 production compared to control group (p < 0.01)
Summary
As a common metabolic disease, the incidence of sub-acute ruminal acidosis (SARA) is greater than20% in the primary and middle lactation of dairy cows, which causes significant economic damage to dairy cattle cultivation [1]. LPS, as a major component of the outer membrane of Gram-negative bacteria, is usually viewed as a highly efficient proinflammatory response factor that activates the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and results in the release of a large number of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and so on [3,4,5,6]. Many studies have shown that proinflammatory cytokines in dairy cows can lead to chronic mastitis [7], changes in nutritional metabolism and cells apoptosis which can influence the milk composition and milk yield [8,9,10,11]. Several intracellular signaling-regulated molecules have been identified [12,13]
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