Bazedoxifene Attenuates Isoproterenol-Induced Cardiac Fibroblast Activation and Fibrotic Phenotype via Modulation of the IL-6/STAT3 Signaling Axis

Figure 1. Identification of cardiac fibroblasts and determination of the optimal working concentration of bazedoxifene. (A) Representative immunofluorescence images of vimentin staining in neonatal Sprague–Dawley (SD) rat cardiac fibroblasts (CFs). Red fluorescence denotes vimentin-positive cells; blue DAPI staining labels nuclei. Scale bar = 100 μm. (B) Cell viability of CFs following 24 h exposure to bazedoxifene (BAZ, 1 nM–100 μM) or vehicle control (Veh, containing an equivalent volume of DMSO), evaluated using the CCK-8 assay. Data are expressed as mean ± SD (n = 6 independent experiments). (C) IL-6 secretion in culture supernatants of CFs was quantified by ELISA. Experimental groups comprised: CTL (normal control), Veh (DMSO vehicle), ISO (w/o DMSO) (ISO stimulation without DMSO), ISO model (ISO model with DMSO), 1–10 μM BAZ + ISO, and positive control PRO (8 μM propranolol + ISO). Data are presented as mean ± SD (n = 6 independent experiments). *** p < 0.001.

Figure 2. Bazedoxifene-mediated suppression of ISO-induced proliferation in cardiac fibroblasts was linked to G1-to-S phase cell cycle arrest, which was reversed by IL-6 overexpression. (A) Flow cytometric analysis was used to evaluate DNA content distribution across cardiac fibroblast (CF) groups. The experimental groups were defined as follows: CTL (normal control); Veh (equal-volume DMSO solvent control); ISO (w/o DMSO) and ISO model (10 μM ISO stimulation; the latter included an equal volume of DMSO to account for potential solvent effects); 5 μM BAZ + ISO; 5 μM BAZ + Lv-NC + ISO (negative lentiviral control); and 5 μM BAZ + Lv-IL-6 + ISO (IL-6 overexpression rescue). (B) Stacked bar chart illustrating the percentage of cells in each cell cycle phase (G0/G1, S, G2/M) for all groups. Data are expressed as mean ± SD (n = 3 independent experiments). ΔΔΔ p < 0.001, ΔΔ p < 0.01 vs. the CTL group; *** p < 0.001, * p < 0.05 vs. the Veh group; ### p < 0.001, # p < 0.05 vs. the ISO model group; aaa p < 0.001, a p < 0.05 vs. the 5 μM BAZ + ISO group.

Figure 3. BAZ suppressed ISO-induced collagen synthesis and TGF-β1 secretion in cardiac fibroblasts (CFs); this effect was partially counteracted by IL-6 overexpression. (A) Hyp levels in culture supernatants of CFs from each experimental group. (B) TGF-β1 concentrations in culture supernatants of CFs from each experimental group. Data are expressed as mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4. BAZ inhibited ISO-induced cardiac fibroblast migration, an effect that was partially reversed by IL-6 overexpression. (A) Representative Transwell migration micrographs. Original magnification: 200×. After crystal violet staining, cells that traversed the microporous membrane and adhered to the lower chamber were stained purple, indicating migratory cells. (B) Quantitative analysis of the number of cells that migrated through the membrane for each group. Data are presented as mean ± SD (n = 3 independent experiments). ** p < 0.01, *** p < 0.001.

Figure 5. Bazedoxifene inhibits ISO-induced activation of cardiac fibroblasts and collagen synthesis, an effect that is partially reversed by IL-6 overexpression. (A) Western blot analysis showing protein expression bands of α-SMA, Collagen I, and Collagen III in cardiac fibroblasts from the respective groups. (B–D) Quantitative analyses of protein expression levels: α-SMA (B), Collagen I (C), and Collagen III (D). Densitometric values were normalized to β-actin and expressed as relative expression levels. Data are presented as mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6. BAZ suppressed ISO-induced IL-6 expression and secretion in cardiac fibroblasts, an effect that was reversed by IL-6 overexpression. (A) Relative IL-6 mRNA expression levels in cardiac fibroblasts from each group were assessed by qRT-PCR. Data are expressed as mean ± SD (n = 3 independent experiments). (B) IL-6 protein concentrations in cell culture supernatants were quantified by ELISA. Data are expressed as mean ± SD (n = 4 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7. BAZ suppresses IL-6 expression, thereby inhibiting ISO-induced STAT3 phosphorylation in cardiac fibroblasts. (A) Representative Western blot images showing the protein expression levels of IL-6, phosphorylated STAT3 (p-STAT3), and total STAT3 in cardiac fibroblasts across all experimental groups. (B–D) Quantitative analyses of IL-6 (B), p-STAT3 (C), and total STAT3 (D) expression, with grayscale intensities normalized to β-actin and presented as relative expression values. (E) Ratio of p-STAT3 to total STAT3, indicating the activation state of STAT3 phosphorylation. Data are expressed as mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8. Schematic illustration of the proposed mechanism by which bazedoxifene attenuates ISO-induced cardiac fibroblast activation and fibrotic phenotype. In this figure, arrows indicate directionality of signaling and regulatory effects: solid arrows represent direct activation or promotion, whereas dashed arrows denote indirect or downstream effects; red arrows indicate upregulation or increased activity, and blue or green arrows indicate inhibitory effects (as annotated). Under ISO stimulation (left), cardiac fibroblasts upregulate IL-6 expression and secretion, which binds to IL-6Rα and the co-receptor gp130, activating downstream JAK2/STAT3 signaling. Phosphorylated STAT3 (p-STAT3, Tyr705) dimerizes and translocates to the nucleus, where it drives the transcription of pro-fibrotic target genes, leading to CF proliferation (G0/G1 to S phase transition), migration, myofibroblast transdifferentiation (α-SMA upregulation), and excessive collagen synthesis (Collagen I/III and hydroxyproline). Concurrently, IL-6/STAT3 signaling enhances TGF-β1 production, while TGF-β1 further amplifies IL-6 expression, establishing a positive feedback loop that sustains the fibrotic phenotype. BAZ…