1. Academic Validation
  2. Sirtuin 3 relieves inflammatory responses elicited by lipopolysaccharide via the PGC1α-NFκB pathway in bovine mammary epithelial cells

Sirtuin 3 relieves inflammatory responses elicited by lipopolysaccharide via the PGC1α-NFκB pathway in bovine mammary epithelial cells

  • J Dairy Sci. 2022 Dec 6;S0022-0302(22)00707-X. doi: 10.3168/jds.2022-22114.
Lei Liu 1 Baogen Wang 1 Wei Yang 2 Qianming Jiang 3 Juan J Loor 3 Lu Ouyang 1 Huilun Tang 1 Renxu Chang 4 Tao Peng 1 Chuang Xu 5
Affiliations

Affiliations

  • 1 College of Veterinary Medicine, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Hunan Agricultural University, Changsha, 410128, China.
  • 2 College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China.
  • 3 Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801.
  • 4 College of Veterinary Medicine, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Hunan Agricultural University, Changsha, 410128, China; College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China.
  • 5 College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China. Electronic address: xuchuang7175@163.com.
Abstract

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 μg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 μM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of Infection (MOI) for 6 h followed by a 12 h incubation with 150 μg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 μg/mL) for 24 h and then challenged with 150 μg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 μM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 μg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 μg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 μg/mL LPS; concentrations of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 Infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1β, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.

Keywords

PGC1α; Sirtuin 3; dairy cattle; nuclear factor-κB.

Figures
Products