Sirtuin 3 inhibits nuclear factor-κB signaling activated by a fatty acid challenge in bovine mammary epithelial cells

摘要

Susceptibility to mastitis is highest during the peripartal(transition) period and is often concomitant withother comorbidities such as ketosis. Although infectionwith pathogenic microorganisms and immune-dysfunctionaround calving clearly play key roles in mastitisdevelopment, other metabolic factors also contribute.Sirtuin 3 (SIRT3), a mitochondrial deacetylase regulatingenergy and redox homeostasis, antagonizes thelipotoxic effects of nonesterified fatty acids (NEFA).Thus, we hypothesized that increases in circulatingNEFA concentrations, as observed in the transitionperiod, provokes inflammatory responses that canbe reversed via activation of SIRT3. Here we aimedto study (1) proinflammatory NF-κB signaling andSIRT3 abundance in mammary tissue of ketotic cowsand healthy controls, and (2) the effect of SIRT3 onNF-κB activation in bovine mammary epithelial cells(BMEC) treated with high levels of NEFA. The mammarygland biopsy samples were from a previous study,which included 15 healthy cows and 15 ketotic cows.Primary BMEC were isolated from 3 healthy Holsteincows with collagenase Ⅲ digestion. Purified BMECwere incubated with or without SIRT3 overexpressionadenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4mM NEFA for 24 h. Mammary tissue of ketotic cowswas associated with lower protein abundance of SIRT3along with greater NF-κB P65 phosphorylation levels(p-NF-κB P65), p-NF-κB P65: NF -κB P65 ratio, andmRNA abundance of IL1B and IL6. In BMEC, exogenousNEFA dose-dependently reduced protein abundanceof SIRT3, but increased p-NF-κB P65, p-NF-κBP65: NF -κB P65 ratio, and mRNA abundance of IL1Band IL6. Compared with green fluorescent proteinadenovirus vector + NEFA, overexpression of SIRT3in NEFA-treated BMEC downregulated p-NF-κB P65and mRNA abundance of IL1B and IL6. Immunofluorescenceindicated that overexpression of SIRT3inhibited nuclear translocation of NF-κB P65. Overall,our data demonstrated that ketosis is associated witha reduction in SIRT3 abundance and activation of NF-κB signaling in the mammary gland. In vitro data providedevidence that high NEFA concentrations inhibitSIRT3, which contributes to enhanced NF-κB signalingincluding nuclear translocation and a pro-inflammatoryresponse. The data suggest a promising role of SIRT3as a target for helping alleviate localized inflammationof the mammary gland resulting from exposure to highconcentrations of NEFA.