Quercetin alleviates liver fibrosis via regulating glycolysis of liver sinusoidal endothelial cells and neutrophil infiltration

Abstract

Liver fibrosis, a common characteristic in various chronic liver diseases, is largely influenced by glycolysis. Quercetin (QE), a natural flavonoid known to regulate glycolysis, was studied for its effects on liver fibrosis and its underlying mechanism. In a model of liver fibrosis induced by carbon tetrachloride (CCl4), we aimed to assess pathological features, serum marker levels, and analyze the expression of glycolysis-related enzymes at both mRNA and protein levels, with a focus on changes in liver sinusoidal endothelial cells (LSECs). Our results showed that QE effectively improved liver injury and fibrosis evident by improved pathological features and lowered levels of serum markers, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), total bile acid (TBA), total bilirubin (TBIL), direct bilirubin (DBIL), hyaluronic acid (HA), laminin (LN), and procollagen type III (PCIII). QE also decreased lactate production and downregulated the expression of glycolysis-related enzymes-pyruvate kinase M2 (PKM2), phosphofructokinase platelet (PFKP), and hexokinase II (HK2)-at both the mRNA and protein levels. QE reduced the expression and activity of these enzymes, resulting in reduced glucose consumption, adenosine triphosphate (ATP) production, and lactate generation. Further analysis revealed that QE inhibited the production of chemokine (C-X-C motif) ligand 1 (CXCL1) and suppressed neutrophil recruitment. Overall, QE showed promising therapeutic potential for liver fibrosis by targeting LSEC glycolysis and reducing neutrophil infiltration.

Figure 1.

Efficacy of QE in the treatment of liver injury. (A) Diagram of QE treatment in a CCl4-induced liver fibrosis mouse model; (B) Body weight; (C) Liver index; (D–J) Serum levels of AST, ALT, ALP, GGT, TBA, TBIL, and DBIL in mice subjected to different treatments were determined using serology. For the statistical significance of this figure, bars indicate means ± SEM, n ═ 8 per group, ^###P < 0.001 compared with the control, and ^*P < 0.05, ^**P < 0.01, ***P < 0.001 compared with the model. QE: Quercetin; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; GGT: γ-glutamyl transferase; TBA: Total bile acid; TBIL: Total bilirubin; DBIL: Direct bilirubin.

Figure 2.

Efficacy of QE in the treatment of liver fibrosis. (A) H&E, Masson and Sirius red staining in liver tissues, scale bar: 200 µm; (B) Serum levels of HA, LN, and PC-III in mice subjected to different treatments were determined using serology. For the statistical significance of this figure, bars indicate means ± SEM, and n ≥ 3 per group, ^###P < 0.001 compared with the control, and *P < 0.05, **P < 0.01, ***P <0.001 compared with the model. QE: Quercetin; HA: Hyaluronic acid; LN: Laminin; H&E: Hematoxylin and eosin; SEM: Standard error of the mean.

Figure 3.

QE inhibits glycolysis of CCl4-induced liver fibrosis in mice. (A) Lactic acid levels in mice subjected to different treatments were detected using serological analysis; (B) The RT-qPCR results showed each group’s mRNA expressions of HK2, PFKP, and PKM2 in the liver tissues. β-Actin was used as the loading control; (C) Western blot analysis revealed the protein expressions of HK2, PFKP, and PKM2 in liver tissues from each group. GAPDH was used as the loading control. For the statistical significance of this figure, bars indicate means ± SEM, and n ≥ 3 per group, ^###P < 0.001 compared with the control, and *P < 0.05, **P < 0.01, ***P < 0.001 compared with the model. QE: Quercetin; PKM2: Pyruvate kinase M2; PFKP: Phosphofructokinase platelet; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR: Quantitative real-time PCR.

Figure 4.

Inhibition of QE on LSEC glycolysis. (A) Effect of QE on cell viability was detected using the CCK8 reagent; (B) Measurements of glucose consumption are indicated by GOD activity; (C) Measurements of ATP levels; (D) Measurements of intracellular lactate levels; (E) Western blot analyses of PKM2, PFKP, and HK2 protein expression with quantification; (F) Measurements of intracellular enzyme activities of PKM2, PFKP, and HK2. For the statistical significance of this figure, bars indicate means ± SEM, and n ≥ 3 per group, ^###P < 0.001 compared with the control, and *P < 0.05, **P < 0.01, ***P < 0.001 compared with the model. QE: Quercetin; PKM2: Pyruvate kinase M2; PFKP: Phosphofructokinase, platelet; LSEC: Liver sinusoidal endothelial cell; CCK8: Cell counting kit 8; GOD: Glucose oxidase; ATP: Adenosine triphosphate; HK2: Hexokinase II; SEM: Standard error of the mean.

Figure 5.

Effect of QE on the neutrophil infiltration. (A) Immunohistochemistry analysis of CXCL1 in liver tissues. Scale bars: 100 µm; (B) The level of CXCL1 release in the culture medium was evaluated using an ELISA kit; (C) Immunofluorescence analysis of MPO in liver tissues. Cells were stained for DAPI (blue) and MPO (green). Scale bars: 100 µm; (D) The expression levels of MPO in liver tissue; (E) The expression levels of NE in liver tissue. For the statistical significance of this figure, bars indicate means ± SEM, and n ≥ 3 per group, ^### P < 0.001 compared with the control, and *P < 0.05, **P < 0.01, ***P < 0.001 compared with the model. QE: Quercetin; ELISA: Enzyme-linked immunosorbent assay; MPO: Myeloperoxidase; DAPI: Diamidino-2-phenylindole; IHC: Immunohistochemistry; NE: Neutrophil elastase; SEM: Standard error of the mean.

Figure 6.

Diagram of QE alleviating liver fibrosis via LSEC glycolysis and neutrophil infiltration. QE: Quercetin; LSEC: Liver sinusoidal endothelial cell; HK2: Hexokinase II; PFKP: Phosphofructokinase platelet; PKM2: Pyruvate kinase M2.