Deficiency of S100A9 Alleviates Sepsis-Induced Acute Liver Injury through Regulating AKT-AMPK-Dependent Mitochondrial Energy Metabolism

Abstract

Acute liver injury (ALI) is recognized as a serious complication of sepsis in patients in intensive care units (ICUs). S100A8/A9 is known to promote inflammation and immune responses. However, the role of S100A8/A9 in the regulation of sepsis-induced ALI remains known. Our results indicated that S100A8/A9 expression was significantly upregulated in the livers of septic mice 24 h after cecal ligation and a puncture (CLP) operation. Moreover, S100A9-KO in mice markedly attenuated CLP-induced liver dysfunction and injury, promoting the AMPK/ACC/GLUT4-mediated increases in fatty acid and glucose uptake as well as the improvement in mitochondrial function and ATP production. In contrast, treatment with the AMPK inhibitor Compound C reversed the inhibitory effects of S100A9 KO on CLP-induced liver dysfunction and injury in vivo. Finally, the administration of the S100A9 inhibitor Paquinimod (Paq) to WT mice protected against CLP-induced mortality, liver injury and mitochondrial dysfunction. In summary, our findings demonstrate for the first time that S100A9 plays an important pro-inflammatory role in sepsis-mediated ALI by regulating AKT-AMPK-dependent mitochondrial energy metabolism and highlights that targeting S100A9 may be a promising new approach for the prevention and treatment of sepsis-related liver injury.

Keywords: AMPK; S100A9; acute liver injury; mitochondrial energy metabolism; sepsis.

Figure 1

Upregulation of S100A8/A9 in the liver of CLP-treated mice. Polymicrobial sepsis in wild-type (WT) mice was induced by cecal ligation and puncture (CLP) for 24–72 h. Sham group: 24 h. (A) qPCR assay of S100A8 and S100A9 mRNA levels in the liver at each time point (n = 6). (B) Immunoblotting analysis of S100A8 and S100A9 protein levels in the liver at each time point (left), and quantification of the relative protein level (right, n = 4). (C) Immunohistochemical staining of liver sections with anti-S100A9 antibody (left, brown area), and quantification of the S100A9⁺ area (right, n = 6). (D) Immunofluorescent staining of liver sections with anti-F4/80 (green) or anti-S100A9 (red) antibody to examine localization of S100A9 in F4/80⁺ macrophages. Data are shown as mean ± SEM, and n represents the number of mice in each group.

Figure 2

Knockout of S100A9 in mice suppresses CLP-induced liver dysfunction, hepatocyte damage, apoptosis, inflammation and superoxide production. (A) Schematic diagram of WT or S100A9-knockout (KO) mice subjected to CLP for 24 h. (B) Survival rate of WT and S100A9 KO mice after sham or CLP operation (sham group, n = 5 per group; CLP group, n = 25 per group). (C) The levels of serum ALT and AST in each group (n = 6). (D) H&E staining of liver sections (left, CV: central veins), and quantification of the histological score (right, n = 6). (E) TUNEL (red) and DAPI (blue) staining of liver sections (left), and quantification of the TUNEL⁺ nuclei (right, n = 6). (F) Immunoblotting assay of Bax and Bcl-2 protein levels (left), and the ratio of Bax to Bcl-2 (right, n = 4). (G) Immunohistochemical staining of liver sections using anti-CD68 antibody (left, brown area), and quantification of the CD68⁺ area (right, n = 6). (H) Dihydroethidium (DHE) staining of liver sections to measure superoxide production (left) and quantification of fluorescence intensity (right, n = 6). (I) qPCR assay of IL-1β, IL-6, TNF-α, NOX1 and NOX2 mRNA levels (n = 6). Data are shown as mean ± SEM, and n represents the number of mice in each group.

Figure 3

Knockout of S100A9 ameliorates liver mitochondrial dysfunction through increasing AMPK-medaited glucose and lipid metabolism. (A) Representative images of O₂ concentration change (blue line) and O₂ flux per mass (red line) in groups. (B) Summarized data for the oxygen consumption capacity measured by high-resolution respirometry in CI leak, CI P (complex I OXPHOS), CI + CII P, CI + CII ETS (electron transfer system capacity), and CII ETS (n = 6). (C) ATP production and (D) fluorescence (represent mitochondrial membrane potential) (n = 6). (E) TEM of liver tissue (left) and the percentage of damaged mitochondria (right, n = 3). Asterisk: normal mitochondria; Arrowheads: damaged mitochondria; N: nucleus; magnification is 2000×. (F) Serum and (G) tissue levels of TG and FFA (n = 6). (H) Immunoblotting analysis of S100A9, p-AKT, AKT, p-AMPK, AMPK, ACC, GLUT4 (left) and quantification of the relative protein levels (right, n = 4). Data are shown as mean ± SEM, and n represents the number of mice in each group.

Figure 4

Inhibition of AMPK reverses S100A9-KO-mediated protection of CLP-induced liver dysfunction. (A) Diagram of AMPK inhibitor Compound C (CC). (B) Schematic diagram of WT or S100A9-knockout (KO) mice treated with CC and CLP operation for 24 h. (C) The levels of serum ALT and AST in each group (n = 6). (D) H&E staining of liver sections (left), and quantification of the histological score (right, n = 6). (E) TUNEL (red) and DAPI (blue) staining of liver sections (left), and quantification of TUNEL⁺ nuclei (right, n = 6). (F) Immunohistochemical staining of liver sections with anti-CD68 antibody (left), and quantification of the CD68⁺ area (right, n = 6). (G) DHE staining of liver sections to measure superoxide production (left), and quantification of fluorescence intensity (right, n = 6). Data are shown as mean ± SEM, and n represents the number of mice in each group.

Figure 5

Blocking AMPK abrogates the S100A9-KO-mediated improvement of CLP-induced liver mitochondria dysfunction and reduction of glucose and lipid metabolism. (A) Representative images of O₂ concentration change (blue line) and O₂ flux per mass (red line) in groups. (B) Summarized data for the oxygen consumption capacity measured by high-resolution respirometry in CI leak, CI P, CI + CII P, CI + CII ETS, CII ETS (n = 6). (C) ATP production and (D) fluorescence (represent mitochondrial membrane potential) (n = 6). (E) Serum and (F) tissue levels of TG and FFA (n = 6). (G) Immunoblotting analysis of S100A9, p-AMPK, AMPK, ACC, GLUT4 (left) and quantification of the relative protein levels (right, n = 4). Data are shown as mean ± SEM, and n represents the number of mice in each group.

Figure 6

Administration of S100A9 inhibitor Paquinimod suppresses CLP-induced liver dysfunction, damage, apoptosis, inflammation and superoxide production. (A) Diagram of S100A9 inhibitor Paquinimod (Paq). (B) Schematic diagram of wild-type (WT) treated with S100A9 inhibitor (Paq) and CLP operation for 24 h. (C) The levels of serum ALT and AST in each group treated with Paq at dosages of 5, 10 and 20 mg/kg (n = 6). (D) The S100A9 protein level of each group treated with vehicle or Paq at dose of 10 mg/kg. (E) Survival rate in vehicle- or Paq-administered mice after 24 h of sham or CLP operation (sham group, n = 5 per group; CLP group, n = 25 per group). (F) H&E staining of liver sections (left, CV: central veins), and quantification of the histological score (right, n = 6). (G) TUNEL (red) and DAPI (blue) staining of liver sections (left), and quantification of TUNEL⁺ nuclei (right, n = 6). (H) Immunohistochemical staining of liver sections with anti-CD68 antibody (left, brown area), and quantification of the CD68⁺ area (right, n = 6). (I) DHE staining of liver sections to measure superoxide production (left) and quantify fluorescence intensity (right, n = 6). (J) Immunoblotting analysis of Bax and Bcl-2 protein levels (left), and quantification of Bax to Bcl-2 ratio (right, n = 4). (K) qPCR analysis of IL-1β, IL-6, TNF-α, NOX1 and NOX2 mRNA levels (n = 6). Data are shown as mean ± SEM, and n represents the number of mice in each group.

Figure 7

Administration of S100A9 inhibitor ameliorates liver mitochondrial dysfunction through increasing AMPK-mediated glucose and lipid metabolism. (A) Representative images of O₂ concentration change (blue line) and O₂ flux per mass (red line) in groups. (B) Summarized data for the oxygen consumption capacity measured by high-resolution respirometry in CI leak, CI P, CI + CII P, CI + CII ETS, CII ETS (n = 6). (C) ATP production and (D) fluorescence (represent mitochondrial membrane potential) (n = 6). (E) Immunoblotting analysis of p-AKT, AKT, p-AMPK, AMPK, ACC, GLUT4 (left) and quantification of the relative protein levels (right, n = 4). Data are shown as mean ± SEM, and n represents the number of mice in each group.