SIRT7 Regulates Lipopolysaccharide-Induced Inflammatory Injury by Suppressing the NF- κ B Signaling Pathway

Abstract

Mastitis has severely affected the cattle industry worldwide and has resulted in decreased dairy production and cattle reproduction. Although prevention and treatment methods have been implemented for decades, cattle mastitis is still an intractable disease. Sirtuin 7 (SIRT7) is an NAD⁺-dependent deacetylase that is involved in various biological processes, including ribosomal RNA synthesis and protein synthesis, DNA damage response, metabolism, and tumorigenesis. However, whether SIRT7 participates in inflammation remains unknown. Our results revealed that SIRT7 is downregulated in tissue samples from mastitic cattle. Therefore, we isolated dairy cow mammary epithelial cells (DCMECs) from breast tissues and developed an in vitro model of lipopolysaccharide- (LPS-) induced inflammation to examine SIRT7 function and its potential role in inflammation. We showed that SIRT7 was significantly downregulated in LPS-treated DCMECs. SIRT7 knockdown significantly increased the LPS-stimulated production of inflammatory mediators, like reactive oxygen and nitric oxide, and upregulated TAB1 and TLR4. In addition, SIRT7 knockdown significantly increased the phosphorylation of TAK1 and NF-κBp65 in LPS-treated DCMECs. Moreover, SIRT7 knockdown promoted the translocation of NF-κBp-p65 to the cell nucleus and then increased the secretion of inflammatory cytokines (IL-1β and IL-6). In contrast, SIRT7 overexpression had the opposite effects when compared to SIRT7 knockdown in LPS-treated DCMECs. In addition, SIRT7 overexpression attenuated LPS-induced DCMEC apoptosis. Taken together, our results indicate that SIRT7 can suppress LPS-induced inflammation and apoptosis via the NF-κB signaling pathway. Therefore, SIRT7 may be considered as a potential pharmacological target for clinical mastitis therapy.

Figure 1

SIRT7 expression in tissues from mastitic cattle and LPS-treated DCMECs. (a) SIRT7 expression was reduced in five different samples from mastitic cattle. (b) DCMECs were cultured in vitro and identified using antibodies specific to cytokeratin-18 via immunofluorescence staining. The white arrows indicate the specific localization of cytokeratin-18. Blue: DNA; green: cytokeratin. (c, d) qRT-PCR was carried out to evaluate SIRT7 expression in DCMECs treated with varying concentrations of LPS (1 μg/mL LPS in (c); 10 μg/mL LPS in (d)) at different time points. (e) SIRT7 expression in DCMECs at different time points was evaluated via western blotting after LPS (1 μg/mL LPS) treatment of DCMECs. (f) Quantification of SIRT7 protein levels in DCMECs using ImageJ after LPS (1 μg/mL LPS) treatment of DCMECs. Data are expressed as mean ± S.E.M.^∗p < 0.05 and ^∗∗p < 0.01.

Figure 2

SIRT7 regulated NO and ROS production in LPS-treated DCMECs. (a, b) DCMECs were transfected with three different SIRT7-targeted siRNAs. Knockdown efficiency was assessed by qRT-PCR and western blotting. (c) pcDNA3.0-SIRT7 was used for SIRT7 overexpression. Overexpression efficiency was assessed via western blotting. (d) NO was detected with DAF-FM DA. Scale bar: 100 μm; green: DAF-FM DA; blue: DNA. (e) ROS was labeled with DCFH-DA. Scale bar: 100 μm; green: DCFH-DA; blue: DNA. (f, g) Percentages of NO- and ROS-positive cells were calculated from the counts in five randomly selected visual fields. CTR cells are not transfected; NC (negative control) means the cells were transfected with nontargeting control siRNA; Ctrl (Control) means the cells were transfected with empty vector pcDNA3.0; SIRT7 cells were transfected with pcDNA 3.0-SIRT7. Data are expressed as mean ± S.E.M.^∗p < 0.05 and ^∗∗∗p < 0.001.

Figure 3

SIRT7 is involved in NF-κB signaling. (a) Relative mRNA expression levels of TLR4 and TAB1 after SIRT7 knockdown or overexpression in LPS-treated DCMECs were measured via qRT-PCR. (b, c) TLR4 and TAB1 levels and the phosphorylation levels of TAK1 and NF-κBp65 after the SIRT7 knockdown or overexpression in LPS-treated DCMECs were measured via western blotting. Data are expressed as mean ± S.E.M.^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 4

SIRT7 regulates nucleocytoplasmic translocation of NF-κBp-p65. (a, b) DCMECs were transfected with siSIRT7 or SIRT7 plasmid for 24 h and treated with LPS for 6 h. Immunofluorescence staining was conducted with an anti-NF-κBp-p65 antibody. Red: NF-κBp-p65; blue: DNA; scale bar: 50 μm. (c) The protein levels of NF-κBp-p65 in the nucleus and NF-κBp65 in the cytoplasm were analyzed via western blotting after the cells were transfected with siSIRT7 or SIRT7 plasmid.

Figure 5

SIRT7 regulates proinflammatory cytokines in LPS-treated DCMECs. (a, b) After transfection for 48 h, DCMECs were treated with LPS for 6 h. The levels of TNF-α, IL-1β, and IL-6 mRNA were determined by qRT-PCR. (c) The protein levels of IL-1β and IL-6 were determined via western blotting. (d) The levels of IL-1β and IL-6 in the cell medium were detected by ELISA. Data are expressed as mean ± S.E.M.^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 6

SIRT7 alleviates LPS-induced apoptosis in DCMECs. (a, b) DCMECs were transfected with siSIRT7 or pcDNA3.0-SIRT7 for 48 h and then treated with LPS for 6 h. The percentages of apoptotic cells were examined by flow cytometry. (c) The levels of Bax and Bcl-2 mRNA were determined via qRT-PCR. (d) The protein levels of Bax, Bcl-2, and cleaved-caspase-3 were determined by western blotting. Quantification of the relative protein levels are shown under the bands (protein/GAPDH). Data are expressed as mean ± S.E.M.^∗p < 0.05.

Figure 7

Possible signaling transduction pathways induced by SIRT7 in LPS-treated DCMECs. SIRT7 may attenuate LPS-induced upregulation of TLR4, TAB1, and p-TAK1, deactivate the NF-κB pathway, and downregulate inflammatory cytokines (IL-1β, IL-6, and TNF-α), ROS, and NO. Subsequently, SIRT7 suppresses apoptosis-related gene expression. As a result, SIRT7 may inhibit the proinflammatory response and initiation of apoptosis in LPS-treated DCMECs.