Estrogen-dependent activation of TRX2 reverses oxidative stress and metabolic dysfunction associated with steatotic disease

Figures

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  Fig. 1. Phenotypic and functional characterization of HLC derived from hESC. a Representative bright field images of the differentiation phases that lead WA01 and WA09 hESC to differentiate into HLC. Scale bar, 40 µm. b hESC, HLC and LPO-treated HLC were subjected to qRT-PCR analysis. The relative quantity is shown using ∆Ct. Data represent means ± SEM. One-way ANOVA, Tukey-corrected. **p < 0.01; ***p < 0.001. Each dot represents a biological replicate, n = 3. c Total protein lysates from WA01 and WA09 hESC, derived HLC and LPO-treated HLC were subjected to western blot analysis with the antibodies indicated. The Histone H3 (H3) is used as a protein loading control normalizer.

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  Fig. 2. Lactate, pyruvate, and octanoic acid-induced steatosis in HLC and immortalized cells. Untreated and LPO-exposed a WA01 and WA09 HLC, b AML12, and HepG2 cells were subjected to confocal analysis as indicated in the figure. Representative confocal images of BODIPY493/503 stained cells are shown (orange/yellow: LD; blue: DAPI, nuclei. Scale bar, 25 µm). Quantification of LD number/cell and their average size (arbitrary unit) were reported. The confocal analysis of LPO-treated AML12 and HepG2 cells has been used as reference in Fig. 5b. c Untreated and LPO-exposed WA01, WA09, AML12, and HepG2 cells were subjected to qRT-PCR analysis. The relative quantity normalized on untreated is shown using ∆∆Ct for AML12 and HepG2 cells, whereas the relative quantity is shown using ∆Ct for WA01 and WA09 cells. Data represent means ± SEM. (a—n° lipid droplets; b; c—AML12 and HepG2) Student’s t-test, (a—average size) Mann–Whitney, (c—HLC) Kruskal–Wallis, Dunnett-corrected, *p < 0.05, ***p < 0.001, ****p < 0.0001. Each dot represents a biological replicate, n ≥ 3.

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  Fig. 3. LPO treatment increased oxidative stress in HLC and immortalized cells. a Untreated and LPO-exposed (48 h) WA01 and WA09 cells were subjected to confocal analysis and subsequent quantification. Representative images of MitoSOX and CellROX-stained cells are shown (Red: MitoSOX; Green: CellROX; blue: DAPI, nuclei. Scale bar, 25 µm). b, c AML12 and HepG2 cells were treated with LPO for 48 h and subjected to confocal and fluorescent analyses. b Representative pictures of CellROX-stained cells are shown (Green: CellROX. Scale bar, 25 µm), n ≥ 3. Quantification of CellROX is reported. c ROS levels were measured using CellROX and MitoSOX fluorescent probes in AML12 and HepG2 cells treated with LPO (48 h). Untreated cells were used as comparator. d LPO-exposed AML12 cells was subjected to TMRE staining and subsequent quantification of mitochondrial morphology parameters: aspect ratio and form factor (Red: TMRE. Scale bar, 25 µm). The confocal analysis of LPO-treated AML12 cells has been used as reference in Fig. 4h. e Representative pictures of MitoTracker Green-stained AML12 cells are shown (Green: MitoTracker Green; blue: DAPI, nuclei. Scale bar, 25 µm). Quantification of MitoTracker…

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  Fig. 4. Estrogens reduced oxidative stress and altered mitochondrial morphology in LPO-treated hepatocytes. a Untreated and LPO-exposed WA01 and WA09 HLC were subjected to western blot analysis with the antibodies indicated. ER+ MCF7 breast cancer cell line is used as a positive control (CTR+) for estrogen receptor (ER) expression. The Histone H3 (H3) is used as a protein loading control normalizer. b LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of 17β-estradiol (E2) for 48 h and subjected to qRT-PCR using the assays described in the figure. The relative quantity is shown using ∆Ct. c LPO-treated AML12 and HepG2 cells were cultured with or without 50 nM of E2 for 48 h and subjected to ERE-luciferase reporter assay. d LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of E2 for 48 h and subjected to confocal analysis. Representative confocal images of MitoSOX-stained cells are shown (red: MitoSOX; blue: DAPI, nuclei. Scale bar, 25 µm). e LPO-treated AML12 cells were cultured with or without 50 nM of E2 for 48 h and subjected to confocal analysis. Representative images of CellROX-stained cells are shown (Green: CellROX. Scale bar, 25 µm). Quantification of…

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  Fig. 5. Estrogens reduced LD accumulation in LPO-treated hepatocytes. a LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of E2 for 48 h and subjected to confocal analysis. Representative confocal images of BODIPY493/503 stained cells are shown (orange/yellow: LD; blue: DAPI, nuclei. Scale bar, 25 µm). Quantification of BODIPY493/503 spots/cell is reported. b LPO-treated AML12 and HepG2 were cultured with or without 50 nM of E2 for 48 h and subjected to confocal analysis. Representative confocal images of BODIPY493/503 stained cells are shown (orange/yellow: LD; blue: DAPI, nuclei. Scale bar, 25 µm). Quantification of LD number/cell and their average size (arbitrary unit) is reported. The confocal analysis of LPO-treated AML12 cells and HepG2 cells have been used as reference in Fig. 2b and has been processed in parallel with E2-treated cells. c LPO-exposed AML12 cells were treated with 50 nM of E2 for 48 h and 20 µM of ATGL inhibitor (ATGLi) for 24 h. Representative confocal images of BODIPY493/503 stained cells are shown (orange/yellow: LD; blue: DAPI, nuclei. Scale bar, 25 µm). Quantification of LD number/cell and their average size (arbitrary unit) is reported. Data…

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  Fig. 6. TRX2 inhibition reverted estrogen-mediated ROS buffering. a LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM E2 for 48 h and subjected to qRT-PCR and western blotting analysis using the assays described in the figure. The Hsp90 and ACTB are used as protein-loading control normalizers. b ROS levels were measured with CellROX and MitoSOX probes in LPO-treated AML12 and HepG2 cells cultured with or without 50 nM of E2 and/or 250 nM of auranofin (AU) for 48 h. The fluorometric values of the NT and E2-treated AML12 and HepG2 cells are those reported in Fig. 4f, g and have been processed in parallel with AU-treated cells. c LPO-treated WA01 and WA09 cells were cultured with or without 1 nM of E2 and/or 500 nM of auranofin (AU) for 48 h and subjected to confocal analysis. Representative images of MitoSOX-stained cells are shown (Red: MitoSOX; blue: DAPI, nuclei. Scale bar, 25 µm). d Total protein lysates from AML12 and HepG2 cells transfected with 50 nmol/L of siTRX2 or siCTR for 72 h were subjected to western blot analysis, as indicated. The Hsp90 and ACTB are used as protein-loading control normalizers. e Mitochondrial ROS levels were measured using MitoSOX probe in…

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  Fig. 7. TXN2 expression profile in a cohort of female MASLD patients. Gene expression of TXN2 was analyzed with GEO2R and R-program in a cohort of normal or MASLD-bearing patients (GSE163211) divided by age. TXN2 expression levels a in the whole female population, b in the postmenopausal (Age ≥ 50), and c in premenopausal women (Age < 50). d Differences in TXN2 expression between postmenopausal and premenopausal women compared to normal tissue (Normal-Steatosis, Normal-early MASH, and Normal-late MASH stage). Wilcox test.

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  Fig. 8. Graphical abstract of the study. (1) Human embryonic stem cells (hESC) were differentiated into hepatocyte-like cells (HLC) using an 18-day differentiation protocol; (2) HLC and hepatic immortalized cell lines (AML12, HepG2) were then treated with sodium L lactate (L), sodium pyruvate (P) and octanoic acid (O), a protocol of induction called LPO. LPO administration induces distinct steatotic traits, such as the increase of lipid droplets and reactive oxygen species (ROS) levels. (3) Treatment with 17-β estradiol (E2) results in a reduction of the distinctive traits of MASLD through the activation of the mitochondrial antioxidant mechanism of thioredoxin 2 (TRX2), which is inhibited by the administration of auranofin.