Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: AS was associated with decreased serum levels of HDCA and impaired Treg accumulation within vascular plaques. (A) Serum HDCA levels were reduced in AS patients relative to healthy individuals. The observed overlap reflects expected biological variability in human populations. (B) A negative correlation was observed between serum HDCA concentration and disease activity scores in AS patients. (C) Histopathological analysis of arterial samples revealed that higher HDCA levels correlated with reduced arterial intima thickness and plaque formation (magnification, 2 × ; scale bar, 1 mm). (D) Confocal microscopy showed increased infiltration of Foxp3 + Tregs (red) in AS patients with higher HDCA levels, whereas the percentages of CD11c + cells (red) did not significantly differ between groups. CD25 + cells (green, in panel A) and MHC-II + cells (green, in panel B) are presented for additional immunofluorescent phenotyping. Cell nuclei were stained with DAPI (blue). Representative images are shown (magnification, 10 × ; scale bar, 100 μm). (E) Serum HDCA concentrations are significantly decreased in AS mouse models. (F) Oil Red O staining of arterial sections showing reduced lipid deposition following HDCA treatment. (G) Assessment of hemodynamic parameters, including systolic and diastolic blood pressure (SBP and DBP) and heart rate. (H) Oil Red O and immunohistochemical analysis of lesions area and IFN-γ expression in arterial section (magnification, 2 × ; scale bar, 1.25 mm). (I) Flow cytometry of PKH26-labeled donor Tregs revealed that HDCA treatment selectively increased Treg accumulation in atherosclerotic plaques, with no effect in spleen tissue. (J) Quantification of Foxp3 + Tregs (red), CD45 + leukocytes (red), CD68 + macrophages (red), and GR1 + monocytes/granulocytes (red) in atherosclerotic plaques following HDCA treatment; approximately 500 cells were counted per group. CD25 + cells (green), CD3 + T cells (green), F4/80 + macrophages (green), and Ly6G + neutrophils (green) were visualized by immunofluorescence co-staining. Cell nuclei were labeled with DAPI (blue). (K – L) ELISA quantification of serum IL-6 (K) and TNF-α (L) levels in AS mice with or without HDCA treatment. (M) IL-35 levels in the supernatant of Treg cells were measured by ELISA after 48 h of culture with or without HDCA treatment. (N) Representative H&E staining of arterial sections from AS mice to evaluate atherosclerotic lesion area following HDCA or anti-CD25 treatment. Data are presented as mean ± SD (n = 5 biological replicates). Statistical significance was determined using two-tailed Student's t -test or one-way ANOVA. ns: not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Concentration Assay, Activity Assay, Confocal Microscopy, Staining, Immunohistochemical staining, Expressing, Flow Cytometry, Labeling, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: HDCA modulates Treg migration and atherosclerotic plaque composition via FXR signaling. ApoE−/− mice (C57BL/6J background, male, 8 weeks old) were fed a high-fat diet for 28 days to induce AS and subsequently received adoptive transfer of control or FXR-knockout (FXR KO) Treg cells generated by CRISPR/Cas9-mediated lentiviral transduction, with HDCA (30 μM) or vehicle treatment as indicated. (A) Representative Western blot analysis of FXR, PD-1, SHP-2, p-Raptor, RAC and IL-10R expression in isolated Treg cells from each group. (B) Oil Red O staining was performed to assess lipid accumulation in the aorta. (C) Masson's trichrome staining of aortic sections from FXR KO mice reveals comparable plaque area and collagen deposition in both HDCA-treated and untreated groups (magnification, 5 × ; scale bar, 1 mm). (D) Representative H&E images of aortic sections show the difference between untreated and HDCA-treated mice in the FXR KO groups (magnification, 5 × ; scale bar, 500 μm). Relative bar graphs show quantification of lesion area and lesion/media area ratio. (E) Confocal immunofluorescence was used to evaluate Foxp3+ Treg infiltration within atherosclerotic plaques (scale bar, 25 μm). (F) Immunohistochemistry images show Treg accumulation in the plaque area across all groups (magnification, 40 × ; scale bar, 100 μm). (G) Flow cytometry analysis of Treg proportions in aortic plaques and spleen from control and FXR KO mice, with or without HDCA treatment. (H) Western blot analysis of matrix remodeling-related proteins, including calpain 1 and matrix metalloproteinase 2, and the anti-inflammatory factor IL-10. Data are presented as mean ± SD (n = 5 biological replicates). Data with four groups were analyzed by one-way ANOVA with Tukey's post hoc test. Comparisons between two groups were performed using the non-parametric Mann-Whitney U test. ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Migration, Adoptive Transfer Assay, Control, Knock-Out, Generated, CRISPR, Transduction, Western Blot, Expressing, Isolation, Staining, Immunofluorescence, Immunohistochemistry, Flow Cytometry, MANN-WHITNEY

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: PD-1 blockade promotes Treg migration via mTORC1-driven glycolytic pathways . (A) Representative en face images of Oil Red O-stained aortas from control and PD-1 blockade groups. (B) Representative images of arterial lesions stained with H&E (scale bar, 100 μm) and Oil Red O (scale bar, 200 μm), with quantification of the necrotic core area and Oil Red O-positive areas. (C) Flow cytometry detection of the enrichment of Foxp3+ Tregs in plaque areas following adoptive transfer of Tregs transduced with shRaptor or control vector, with or without PD-1 blockade. (D) Representative H&E staining of aortic sections showing the percentage of atherosclerotic plaque area relative to the total arterial area in each group (scale bar, 100 μm; magnification, 10 × ). (E) Kaplan-Meier survival analysis of skin grafts in recipients treated with control Tregs (pLKO.1), shRaptor Tregs, PD-1 blockade, or shRaptor plus PD-1 blockade. (F–I) Metabolic analysis of donor Tregs after shRaptor or PD-1 blockade, showing effects on glycolysis (ECAR) and mitochondrial respiration (OCR). (J – K) Western blot and flow cytometry analysis of CPT1a expression levels in Treg isolated from plaques to evaluate alterations in fatty acid oxidation pathways following PD-1 blockade. (L) Western blot analysis was used to assess pERK, ERK, pS6K, S6K, and RAC levels in Treg cells transduced with control shRNA (pLKO.1) or Raptor shRNA (shRaptor) following PD-1 blockade at 0, 15, and 30 min. Statistical analyses were performed using one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons, and the log-rank test for survival analysis. Data are presented as mean ± SD (n = 3-5 biological replicates). Statistical significance is indicated as follows: ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Migration, Staining, Control, Flow Cytometry, Adoptive Transfer Assay, Transduction, Plasmid Preparation, Western Blot, Expressing, Isolation, shRNA

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: ZNF671 governs HDCA-driven STAT5 activation and metabolic adaptation in Treg cells. Ex vivo Tregs were transduced with shZNF671 or vector treated w/wo HDCA prior to iv-injection in recipient AS recipients. Tregs isolated from atherosclerotic plaque based on Foxp3 expression. (A) Flow cytometric analysis and quantification ZNF671 expression in Foxp3+ Tregs isolated from plaques in Vector and FXR KO groups ± HDCA. (B) Western blot analysis of protein expression levels of ZNF671, MAPK6, and SIAH1 in each group. (C) Quantification of atherosclerotic plaque area by percentage of Oil Red O staining-positive area in aorta from different groups. (D) Quantification of atherosclerotic lesion area in indicated groups. (E – F) ELISA was used to quantify the levels of anti-inflammatory cytokines IL-10 and IL-35 in culture supernatants of Treg cells from the indicated groups. (G) Migration of PKH26-labeled Treg cells was quantified by flow cytometry 24 h post-transfer. (H–I) Seahorse assay showed that OCR was significantly reduced in the ZNF671 KO group and this reduction was further exacerbated by HDCA treatment. (J) Immunofluorescence staining showed that ZNF671 KO enhanced pSTAT5 signaling, and this effect was significantly amplified by HDCA treatment. Quantification of pSTAT5 levels is shown in the bar graphs as mean ± SD from 500 cells per coverslip. scale bar, 25 μm. (K) Flow cytometry plots and quantification the percentage of pSTAT5+Foxp3+ Treg cells across all groups. Data are presented as the mean ± SD (n = 3-5 biological replicates). Statistical analysis was performed using one-way ANOVA followed by post-hoc tests. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: We used the Oil Red O staining kit (C0157S, Beyotime).

Techniques: Activation Assay, Ex Vivo, Transduction, Plasmid Preparation, IV Injection, Isolation, Expressing, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Migration, Labeling, Flow Cytometry, Immunofluorescence, Amplification