Fatty Acid Oxidation-Glycolysis Metabolic Transition Affects ECM Homeostasis in Silica-Induced Pulmonary Fibrosis

Abstract

Silicosis is a fatal occupational pulmonary disease that is characterized by irreversible replacement of lung parenchyma by aberrant Exracellular matrix (ECM). Metabolic reprogramming is a crucial mechanism for fibrosis. However, how the metabolic rewiring shifts the ECM homeostasis toward overaccumulation remains unclear. Herein, a phenotype with reduction in fatty acid oxidation (FAO) but enhanced glycolysis in myofibroblasts is shown. Perturbation of the glycolytic and FAO pathways, respectively, reveals distinct roles in the metabolic distribution of ECM deposition and degradation. Suppressed glycolysis leads to a decrease in insoluble ECM, primarily due to the inhibition of ECM-modifying enzyme activity and a decrease in glycine synthesis. Notably, promoted FAO facilitates the intracellular degradation pathway of ECM. In addition, the findings revealed that hypoxia-inducible factor-1 alpha (HIF-1α) serves as a crucial metabolic regulator in the transition from FAO to glycolysis, thereby playing a significant role in ECM deposition in silica-induced pulmonary fibrosis. Further, the promotion of FAO, inhibition of glycolysis and HIF-1α reduce ECM production and promote ECM degradation, ultimately impeding the progression of fibrosis and providing therapeutic relief for established pulmonary fibrosis in vivo. These findings unveil the metabolic rewire underpinning the deposition of ECM in silica-induced lung fibrosis and identify novel targets for promoting regression of pulmonary fibrosis.

Keywords: extracellular matrix; fatty acid oxidation; glycolysis; hypoxia‐inducible factor‐1α; silicosis.

Figure 1

The development of pulmonary fibrosis in mice is accompanied by a restructuring of cellular metabolism. A) Pathological changes in mouse lung tissues were presented by H&E staining (n = 6 for each group); dashed line scale bar = 1000 µm, solid line scale bar = 100 µm. B)Hydroxyproline content of the lung tissues was used to examine the degree of collagen deposition. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01.C) The protein levels of fibronectin, collagen I, and elastin in each group were examined by the western blot. The results of the experiment were repeated at least 3 times. D) Histological analysis was conducted on mouse lung tissue using Masson staining (first image from left), Hydroxyproline immunohistochemical staining (second image from left), Sirius red staining (second image from right), and polarized light imaging (first image from right); scale bar = 100 µm. E) The heatmap of differential metabolites about glycolysis and FAO identified by metabolomics.F) Protein levels of key molecules involved in glycolysis and fatty acid oxidation in TGF‐β1‐treated MRC‐5 cells were quantified using western blot analysis. Each experiment was performed in triplicate to ensure reproducibility of results.

Figure 2

The deposition of ECM by activated fibroblasts is accomplished by a metabolic perturbation of glycolysis and FAO. A) qRT‐PCR showed that the increased in ACTA2 (the encoding gene for α‐SMA) and collagen I in MRC‐5 cells with treated with different concentrations of TGF‐β1. The data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. B) Western blot analysis for protein levels of core components of ECM in TGF‐β1‐treated MRC‐5 cells. The results of the experiment were repeated at least 3 times. C) Representative confocal microscopy images of immunofluorescence staining for collagen I (green) in MRC‐5 cells. Nuclei were stained with DAPI (blue). D) Mitochondrial content was measured by Mito tracker red after treatment with TGF‐β1 for 48 h, Actin tracker (green) labelling of cellular microfilaments. Nuclei were stained with DAPI (blue). E) Western blot analysis for protein levels of phosphorylated AMPK and AMPK in TGF‐β1‐treated MRC‐5 cells. The results of the experiment were repeated at least 3 times. F) Relative mRNA levels of crucial regulators of glycolysis and FAO in MRC‐5 cells treated by various concentration of TGF‐β1 by qRT‐PCR. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. G) Representative western blot images of crucial regulators of glycolysis and FAO in MRC‐5 cells treated by various concentration of TGF‐β1. The results of the experiment were repeated at least 3 times. H) Glycolysis stress test in MRC‐5 cells with or without TGF‐β1. Glucose (10 mM), oligomycin (1µM), and 2‐DG (2‐Deoxy‐Glucose, 100 mM). I) Fatty acid oxidation in MRC‐5 cells with or without TGF‐β1. Etomoxir (4 µM), oligomycin (2 µM), FCCP (1 µM), antimycin A (0.5 µM) and rotenone (0.5 µM).

Figure 3

Inhibiting glycolysis or enhancing FAO affects ECM deposition in activated fibroblasts. A) Western blot analysis for total protein levels of core components of ECM in MRC‐5 cells co‐treated with TGF‐β1 and 3PO or pioglitazone. The results of the experiment were repeated at least 3 times. B) Representative image showing collagen I (green) in MRC‐5 cells. Cell membranes were stained with DiI (red) and nuclei were stained with DAPI (blue). C) Western blot analysis for the levels of ECM molecules in extracellular and soluble proteins. Each experiment was performed in triplicate to ensure reproducibility of results. D) The content of soluble collagen in cell culture media detected by Sircol soluble collagen assay kit. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. E) Lysyl oxidase activity in MRC‐5 cells co‐treated with TGF‐β1 and 3PO or pioglitazone. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. F) The effects of the TGF‐β1 and 3PO on relative abundance of glycine in MRC‐5 cells. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. G) Colocalization of lysosome and collagen I in MRC‐5 cells. MRC‐5 cells were subjected to immunofluorescence analysis with lyso tracker (red) and anti‐ collagen I (green). Nuclei are stained with DAPI (blue). H) Representative images of DQ‐collagen internalized by MRC‐5 cells. Nuclei are stained with DAPI (blue).

Figure 4

Inhibiting glycolysis or enhancing FAO impedes the progression of fibrosis in vivo. A) Schematic outline of the 3PO or pioglitazone intervention model in mice with silica‐induced lung fibrosis. B) Histological analysis was conducted on mouse lung tissue using H&E staining (first image from left), Masson staining (second image from left), Hydroxyproline immunohistochemical staining (third image from left), Sirius red staining (second image from right), and polarized light imaging (first image from right); scale bar = 100 µm. C) The protein levels of fibronectin, collagen I, and elastin in each group were examined by the western blot. The results of the experiment were repeated at least 3 times. D) Representative fluorescence micrographs of fibrotic lung areas were stained with fluorescently labelled collagen hybridisation peptide (Cy3‐CHP). Nuclei are stained with DAPI (blue). Selected micrographs are representative of images collected from each group.

Figure 5

The administration of 3PO or pioglitazone has been demonstrated to provide therapeutic relief for established pulmonary fibrosis in vivo. A) Schematic outline of the 3PO or pioglitazone treatment model in mice with silica‐induced lung fibrosis. B) Histological analysis was conducted on mouse lung tissue using H&E staining (first image from left), Masson staining (second image from left), Hydroxyproline immunohistochemical staining (third image from left), Sirius red staining (second image from right), and polarized light imaging (first image from right), scale bar = 100 µm. C) The protein levels of fibronectin, collagen I, and elastin in each group were examined by the western blot. The results of the experiment were repeated at least 3 times. D) Representative fluorescence micrographs of fibrotic lung areas were stained with fluorescently labelled collagen hybridisation peptide (Cy3‐CHP). Nuclei are stained with DAPI (blue). Selected micrographs are representative of images collected from each group.

Figure 6

HIF‐1α regulated the transformation of FAO‐glycolysis in fibroblast. A) Representative image showing HIF‐1α (red) stained with anti‐HIF‐1α antibody in mouse lung tissue. Nuclei were stained with DAPI (blue). B) Relative expression of HIF‐1α and α‐SMA mRNA in MRC‐5 cells at different time‐points after 5ng/mL TGF‐β1 treatment by qRT‐PCR. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. C) The protein levels of fibronectin, collagen I, HIF‐1α, and elastin in MRC‐5 cells at different time‐points after 5ng/mL TGF‐β1 treatment were examined by the western blot. The results of the experiment were repeated at least 3 times. D) Relative expression of HIF‐1α and PPAR‐γ mRNA in MRC‐5 cells transfected with siHIF‐1α by qRT‐PCR. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. E) Mitochondrial content was measured by Mito tracker red after transfected with siHIF‐1α, Actin tracker (green) labelling of cellular microfilaments. Nuclei were stained with DAPI (blue). F) Western blot analysis for protein levels of phosphorylated AMPK and AMPK in MRC‐5 cells. The results of the experiment were repeated at least 3 times. G) and H) Real‐time measurements to determine the effect of knockdown HIF‐1α on ECAR and OCR in MRC‐5 cells.

Figure 7

HIF‐1α inhibition reduced ECM deposition by regulating glycolytic‐FAO metabolic disturbance. A) Western blot analysis for total protein levels of core components of ECM in MRC‐5 cells co‐treated with HIF‐1α siRNA and TGF‐β1. The results of the experiment were repeated at least 3 times. B) Relative expression of core components of ECM in MRC‐5 cells co‐treated with HIF‐1α siRNA and TGF‐β1 by qRT‐PCR. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. C) Representative image showing collagen I (green) in MRC‐5 cells. Cell membranes were stained with DiI (red) and nuclei were stained with DAPI (blue). D) qRT‐PCR analysis for the mRNA expression of some ECM‐modifying enzymes (LOX, LOXL2, P4AH2, PLOD1, PLOD2) in MRC‐5 cells co‐treated with HIF‐1α siRNA and TGF‐β1. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. E) Western blot analysis for the levels of ECM molecules in extracellular and soluble proteins. Each experiment was performed in triplicate to ensure reproducibility of results. F) The content of soluble collagen in cell culture media detected by Sircol soluble collagen assay kit. All data were expressed as the means ± SD of at least 3 independent experiments, ^* p < 0.05 and ^** p < 0.01. G) Colocalization of lysosome and collagen I in MRC‐5 cells. MRC‐5 cells were subjected to immunofluorescence analysis with lyso tracker (red) and anti‐ collagen I (green). Nuclei are stained with DAPI (blue). H) and I) Representative images of DQ‐collagen internalized by MRC‐5 cells. Nuclei are stained with DAPI (blue). LOXL2: lysyl oxidase like 2; P4AH2: prolyl 4‐hydroxylase 2; PLOD1: procollagen‐lysine,2‐oxoglutarate 5‐dioxygenase 1; PLOD2: procollagen‐lysine,2‐oxoglutarate 5‐dioxygenase 2.

Figure 8

Inhibition of HIF‐1α or FAO‐glycolysis metabolic perturbation impedes the progression of fibrosis in vivo. A) Schematic outline of the intervention model in mice with silica‐induced lung fibrosis. B) Histological analysis was conducted on mouse lung tissue using H&E staining (first image from left), Masson staining (second image from left), Hydroxyproline immunohistochemical staining (third image from left), Sirius red staining (second image from right), and polarized light imaging (first image from right); scale bar = 100 µm. C) The protein levels of fibronectin, collagen I, and elastin in each group were examined by the western blot. D) Severity of fibrosis score and distributions of fibrosis grade in mice (n = 6), ^** p < 0.01.

Figure 9

Inhibition of HIF‐1α or improvement of FAO‐glycolysis metabolic perturbation promotes regression of established fibrosis in vivo. A) Schematic outline of the treatment model in mice with silica‐induced lung fibrosis. B) Histological analysis was conducted on mouse lung tissue using H&E staining (first image from left), Masson staining (second image from left), Hydroxyproline immunohistochemical staining (third image from left), Sirius red staining (second image from right), and polarized light imaging (first image from right); scale bar = 100 µm. C) The protein levels of fibronectin, collagen I, and elastin in each group were examined by the western blot. D) Severity of fibrosis score and distributions of fibrosis grade in mice (n = 6), ^* p < 0.05 and ^** p < 0.01.

Figure 10

Schematic diagram of the mechanism of HIF‐1α regulating ECM deposition by regulating the glycolytic‐FAO metabolic pathway in silica‐induced pulmonary fibrosis.

Figure 11

A) Representative images of cells with or without decellularization (left) and immunofluorescence staining for collagen I (green) in cells. Nuclei were stained with DAPI (blue).