Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

Summary

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited therapeutic options and a poor prognosis. Here, we identify the nuclear receptor NR4A1 as a pivotal mediator of estrogen’s antifibrotic effects through its regulation of TGF-β signaling. Clinical analysis revealed increased expression of NR4A1 and estrogen receptor α (ERα) in IPF lung tissues, with NR4A1 phosphorylation correlating with disease progression. Mechanistic investigations demonstrated that sustained TGF-β signaling induces NR4A1 phosphorylation, impairing its negative feedback regulation of fibrosis. Estrogen, acting via ERα, restored NR4A1 activity by suppressing phosphorylation, thereby reducing fibrosis both in vitro and in vivo. Overexpression of NR4A1 or inhibition of its phosphorylation attenuated fibrotic responses, whereas NR4A1 deficiency abrogated estrogen’s protective effects. These findings elucidate a previously unrecognized mechanism of estrogen-mediated antifibrotic activity and highlight NR4A1 as a promising therapeutic target for IPF.

Graphical abstract

Highlights

• •NR4A1 upregulation and phosphorylation correlate with IPF progression
• •Estrogen inhibits NR4A1 phosphorylation via ERα to restore its antifibrotic function
• •NR4A1 overexpression reduces TGF-β-induced fibrosis in vitro and in vivo
• •Estrogen’s antifibrotic effect is lost in NR4A1-deficient mice

Cell biology; Molecular biology

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology, characterized by irreversible interstitial fibrosis of the lung parenchyma and a gradual decline in pulmonary function.¹ Despite recent advances in disease management, no definitive cure is currently available. In recent years, the incidence of IPF has increased substantially, with 2011 U.S. prevalence estimates ranging between 10 and 60 cases per 100,000 individuals, and reaching up to 494 cases per 100,000 in adults aged≥65, over twice the rate reported a decade earlier.^2,3,4 Epidemiological studies reveal marked geographic and sex-based disparities in IPF incidence, with higher rates observed in males, who also tend to experience worse clinical outcomes compared to females.^5,6 Furthermore, IPF incidence has been shown to increase among postmenopausal women, suggesting that estrogen levels may play a potential role in disease progression.⁷

Findings from animal studies further support this hypothesis. Female mice consistently exhibit significantly lower fibrosis scores, reduced collagen deposition, and attenuated fibrosis severity compared to male mice following intratracheal administration of hydrochloric acid (HCl) or nitrogen mustard (NM).^8,9 These findings suggest that estrogen may exert antifibrotic potential in fibrotic diseases. Similar sex-related differences have been observed in other fibrotic conditions, such as chronic viral hepatitis, in which males are more likely than females to develop liver fibrosis or cirrhosis,¹⁰ further indicating a protective role of 17-β-estradiol (E2) in fibrotic diseases.

A central pathogenic mechanism in IPF involves the impaired regenerative capacity of lung epithelial cells following injury.¹¹ Transforming growth factor β (TGF-β) plays a pivotal role in this pathological process,¹² promoting epithelial cell apoptosis,¹³ epithelial-to-mesenchymal transition (EMT),¹⁴ and cellular migration,¹⁵ while simultaneously suppressing cell proliferation.¹⁶ Aberrant overexpression of TGF-β is widely recognized as a key driver of fibrosis, and pharmacological or genetic inhibition of its signaling pathway has been shown to alleviate pulmonary fibrosis in animal models.^17,18

Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been shown to regulate profibrotic signaling pathways and contribute to tissue repair.¹⁹ Activation of NR4A1 has been reported to suppress TGF-β expression and inhibit TGF-β1-induced intestinal fibroblast proliferation and downregulate the expression of fibrosis-related genes.²⁰

In this study, we investigated NR4A1 as a critical mediator of estrogen-mediated regulation of TGF-β signaling and fibrotic progression. Our experimental findings revealed that sustained TGF-β stimulation induces the phosphorylation of NR4A1, whereas estrogen treatment upregulates NR4A1 expression, restoring the balance between total NR4A1 and its phosphorylated form (p-NR4A1). This re-balancing enhances NR4A1-mediated negative feedback on TGF-β signaling, thereby attenuating fibrotic responses. In summary, we identified a novel mechanism by which estrogen mitigates pulmonary fibrosis via NR4A1 activation, highlighting a potential therapeutic strategy for the treatment of IPF.

Results

Abnormal expression of nuclear receptor subfamily 4 group A member 1, p-nuclear receptor subfamily 4 group A member 1, and estrogen receptor in the lung tissue of patients with idiopathic pulmonary fibrosis

To provide clinical context, baseline characteristics of patients with IPF and controls are summarized in Table 1.

Table 1.

Clinical characteristics of patients with IPF and control Subjects

Characteristics	IPF Patients (n = 23)	Controls (n = 11)	p-value
Sex, n (%)
Male	16 (69.6%)	7 (63.6%)	0.74
Female	7 (30.4%)	4 (36.4%)	–
Age (years), mean ± SD	61.7 ± 8.4	59.3 ± 7.9	0.36
Ancestry/Race/Ethnicity
East Asian	23 (100%)	11 (100%)	–
Smoking history, n (%)
Current smoker	4 (17.4%)	2 (18.2%)	–
Former smoker	10 (43.5%)	5 (45.5%)	–
Never smoker	9 (39.1%)	4 (36.4%)	–
FVC (% predicted), mean ± SD	68.4 ± 11.2	95.6 ± 6.8	<0.001
DLCO (% predicted), mean ± SD	52.7 ± 9.6	88.3 ± 7.5	<0.001
Female patients only	–	–	–
Postmenopausal	6/7 (85.7%)	3/4 (75.0%)	–
Premenopausal/perimenopausal	1/7 (14.3%)	1/4 (25.0%)	–
Hormone therapy within 3 months	0 (0%)	0 (0%)	–

Values are presented as mean ± SD or n (%). No significant differences in sex distribution were observed. Postmenopausal status and exclusion of recent hormone therapy are reported for clarity.

qPCR analyses showed that NR4A1 and ERα mRNA levels were significantly higher in IPF lung tissue than in controls, whereas ERβ mRNA was unchanged (Figures 1A–1C). Immunoblotting corroborated these findings, revealing increased NR4A1 and ERα protein together with a marked elevation of p-NR4A1 in IPF samples (Figures 1D and 1E). Correlation analyses demonstrated positive associations between NR4A1 and Erα, but not with ERβ (Figures 1F and 1G). Likewise, p-NR4A1 correlated positively with ERα, with no significant correlation observed with ERβ (Figures 1H and 1I). Collectively, IPF lung tissue exhibits increased NR4A1 and Erα expression accompanied by enhanced NR4A1 phosphorylation.

Figure 1.

Abnormal expression of NR4A1, p-NR4A1, and ER in lung tissues of patients with IPF

(A–C) qPCR analysis of NR4A1, ERα, and ERβ mRNA expression in lung tissues from healthy controls (Ctrl, n = 11) and patients with IPF (n = 23).

(D) Representative immunoblotting images showing protein levels of NR4A1, p-NR4A1, ERα, and ERβ in lung tissues from Ctrl and IPF groups. β-Actin served as a loading control.

(E) Quantification of immunoblotting results for NR4A1, p-NR4A1, p-NR4A1/NR4A1 ratio, ERα, and ERβ.

(F–I) Correlation analysis between NR4A1 and ERα (F), NR4A1 and ERβ (G), p-NR4A1 and ERα (H), and p-NR4A1 and ERβ (I) in IPF lung tissues. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; Student’s t test for group comparisons, Pearson’s correlation for association analysis.

Nuclear receptor subfamily 4 group A member 1 silencing exacerbates transforming growth factor β-induced fibrosis, and increased nuclear receptor subfamily 4 group A member 1 expression attenuates fibrosis

To determine the contribution of NR4A1 to fibrotic responses, we used siRNA-mediated knockdown to silence NR4A1 expression in embryonic lung fibroblast cell lines MRC5 and HFL1 (Figures 2A and 2B). Knockdown efficiency was confirmed by qPCR and immunoblotting, which showed a significant reduction in NR4A1 mRNA and protein.NR4A1-deficient cells exhibited an exaggerated fibrotic response to TGF-β. Specifically, relative to control cells, NR4A1-silenced cells showed significantly higher mRNA levels of fibrosis markers, including α-SMA, SMAD2, fibronectin, and COL1A1, after TGF-β stimulation (Figures 2C and 2D). Immunoblotting further confirmed concordant increases in the corresponding proteins (Figures 2E and 2F). These results indicate that loss of NR4A1 promotes TGF-β-induced fibrosis, supporting a role for NR4A1 as a negative regulator of profibrotic gene expression.

Figure 2.

Silencing of NR4A1 in cells exacerbates TGF-β-induced fibrosis and increases the expression of α-SMA, SMAD2, fibronectin, and COL1A1

(A and B) qPCR and Western Blot analyses confirm reduced mRNA and protein expression levels of NR4A1 in MRC5 and HFL1 cells following siRNA-mediated NR4A1 silencing.

(C and D) qPCR analysis of α-SMA, SMAD2, Fibronectin, and COL1A1 expression in NR4A1-silenced cells after TGF-β stimulation, showing elevated levels indicative of enhanced fibrosis.

(E and F) Western blot analysis further confirms increased expression of α-SMA, SMAD2, fibronectin, and COL1A1 in NR4A1-silenced cells upon TGF-β stimulation, demonstrating that NR4A1 downregulation aggravates fibrotic responses. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

To test whether NR4A1 overexpression mitigates fibrosis, we generated NR4A1-overexpressing MRC5 and HFL1 cells using a tetracycline-inducible system (Figures 3A and 3B). Overexpression was verified by qPCR and immunoblotting, which showed robust increases in NR4A1 mRNA and protein. In NR4A1-overexpressing fibroblasts, TGF-β-induced fibrotic responses were markedly reduced: mRNA levels of α-SMA, SMAD2, fibronectin, and COL1A1 were decreased (Figures 3C–3J), and immunoblotting confirmed corresponding reductions in protein abundance (Figures 3K and 3L). These findings further support that NR4A1 overexpression suppresses TGF-β-induced fibrosis.

Figure 3.

Increasing NR4A1 expression in cells attenuates TGF-β-induced fibrosis and decreases α-SMA, SMAD2, Fibronectin, and COL1A1 expression

(A and B) NR4A1 overexpression in MRC5 and HFL1 cells induced by tetracycline, confirmed by increased NR4A1 mRNA and protein levels through qPCR and Western blot analyses.

(C–J) qPCR analysis shows reduced expression levels of fibrosis markers α-SMA, SMAD2, Fibronectin, and COL1A1 in NR4A1-overexpressing cells following TGF-β stimulation, indicating diminished fibrotic responses.

(K and L) Western blot analysis further confirms decreased protein levels of α-SMA, SMAD2, Fibronectin, and COL1A1 in TGF-β-stimulated NR4A1-overexpressing cells, demonstrating the antifibrotic effect of NR4A1 upregulation. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

Sustained transforming growth factor β stimulation prompts the phosphorylation of nuclear receptor subfamily 4 group A member 1 and enhances fibrosis

To determine whether TGF-β regulates NR4A1 phosphorylation, MRC5 and HFL1 fibroblasts were treated with increasing concentrations of TGF-β (0–20 ng/mL). Immunoblotting revealed a clear dose-dependent increase in p-NR4A1, with the highest level observed at 20 ng/mL (Figure 4A). In a time course at 20 ng/mL of TGF-β, p-NR4A1 progressively accumulated, became significantly elevated by 72h, and peaked at 96 h (Figure 4B). Thus, TGF-β induces NR4A1 phosphorylation in both dose- and time-dependent manners.

Figure 4.

TGF-β induces NR4A1 phosphorylation and alters its subcellular localization, thereby modulating fibrotic responses in lung fibroblasts

(A) Immunoblotting analysis of p-NR4A1 and NR4A1 in MRC5 and HFL1 cells treated with increasing concentrations of TGF-β. Quantification shows a dose-dependent increase in p-NR4A1, with maximal induction at 20 ng/mL.

(B) Time-course analysis of p-NR4A1 expression in MRC5 and HFL1 cells following stimulation with 20 ng/mL TGF-β. Phosphorylation increased progressively, became significant at 72 h, and peaked at 96 h.

(C and D) Subcellular fractionation of MRC5 and HFL1 cells expressing wild-type NR4A1, NR4A1^∧S351A, or NR4A1^∧S351D under TGF-β stimulation. The S351D mutant was predominantly cytoplasmic, whereas the S351A mutant remained nuclear, indicating the phosphorylation-dependent redistribution of NR4A1.

(E and F) Immunoblotting analysis of fibrotic markers, including α-SMA, SMAD2, fibronectin, and COL1A1 in MRC5 and HFL1 cells expressing wild-type NR4A1, NR4A1^∧S351A, or NR4A1^∧S351D. Quantification demonstrates that NR4A1^∧S351D enhances, whereas NR4A1^∧S351A attenuates, TGF-β-induced expression of fibrotic proteins. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA with post hoc tests was used for multiple group comparisons.

To assess the functional consequences of NR4A1 phosphorylation, site-directed mutants were generated. The NR4A1^S351A mutant was designed to prevent phosphorylation, whereas NR4A1^S351D is phosphomimetic. Subcellular fractionation showed that, in cells expressing wild-type NR4A 1, TGF-β stimulation drove a marked redistribution of phosphorylated NR4A1 from the nucleus to the cytoplasm. Compared with wild-type NR4A1, NR4A1^S351D mutant displayed predominant cytoplasmic localization, consistent with phosphorylation-driven nuclear export, whereas NR4A1^S351A remained nuclear even after TGF-β stimulation (Figures 4C and 4D).

Functional analyses further demonstrated that NR4A1^S351D strongly enhanced the induction of fibrotic markers, including α-SMA, SMAD2, fibronectin, and COL1A1, while NR4A1^S351A significantly attenuated their expression under the same conditions (Figures 4E and 4F). SMAD2 was analyzed as a representative downstream effector of TGF-β signaling because it showed stable regulation across experimental conditions.

Together, these findings show that sustained TGF-β stimulation drives NR4A1 phosphorylation in a dose- and time-dependent manner. Importantly, the phosphorylation status of NR4A1 dictates its subcellular localization, which in turn determines the magnitude of fibrotic responses in lung fibroblasts.

Persistent phosphorylation of nuclear receptor subfamily 4 group A member 1 exacerbates pulmonary fibrosis in the idiopathic pulmonary fibrosis model mice

To validate the functional role of NR4A1 phosphorylation in vivo, we used an AAV virus to overexpress either phosphomimetic NR4A1^S351D (pNR4A1⁺) or non-phosphorylatable NR4A1^S351A (pNR4A1^–) in the mouse lung, followed by intratracheal BLM to induce pulmonary fibrosis. Histology showed that HE staining revealed extensive fibrosis in pNR4A1⁺ lungs, with significantly higher Ashcroft scores than controls. By contrast, pNR4A1⁻ mice exhibited attenuated fibrosis with significantly lower fibrosis scores (Figure 5A). Masson’s trichrome staining corroborated these findings, showing substantial collagen deposition in pNR4A1⁺ lungs, minimal deposition in controls, and markedly reduced collagen in pNR4A1⁻ lungs (Figure 5B).

Figure 5.

TGF-β induced more severe lung fibrosis in NR4A1+ mice compared with control mice

(A) Representative images of HE staining in mouse lung tissue (scale bars, 100 μm). Ashcroft scores were significantly increased in pNR4A1⁺ mice, while significantly decreased in pNR4A1^- mice.

(B) Representative images of Masson staining in mouse lung tissue (scale bars, 100 μm), showing increased collagen deposition in pNR4A1⁺ mice and decreased collagen in pNR4A1^- mice.

(C) Representative immunohistochemical images of α-SMA, SMAD2, fibronectin, and COL1A1 in mouse lung tissue (scale bars, 100 μm).

(D) Quantification of immunohistochemical staining for α-SMA, SMAD2, fibronectin, and COL1A1.

(E) Representative Western blot bands for α-SMA, SMAD2, fibronectin, and COL1A1 in mouse lung tissue.

(F–I) Densitometric analysis of α-SMA, SMAD2, Fibronectin, and COL1A1 Western Blot bands in mouse lung tissue. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

Immunohistochemistry and immunoblotting demonstrated that sustained NR4A 1 phosphorylation promotes the expression of fibrosis-related proteins. In pNR4A1+ lungs, α-SMA, SMAD2, fibronectin, and COL1A1 were markedly elevated, whereas these proteins were notably lower in pNR4A1− lungs (Figures 5C–5I). Collectively, the in vivo data show that the sustained phosphorylation of NR4A1 exacerbates BLM-induced lung fibrosis, whereas the non-phosphorylated form significantly mitigates fibrosis severity.

Estrogen promotes nuclear receptor subfamily 4 group A member 1 expression and attenuates fibrosis

Epidemiologic studies show pronounced sex differences in IPF, with a lower incidence in females, particularly premenopausal women, than in males, suggesting a protective role for estrogen. We therefore hypothesized that 17-β-estradiol (E2) alleviates fibrotic responses by upregulating NR4A1. To test this, fibroblasts were exposed to E2, and NR4A1 expression was monitored. In a time course at a fixed E2 concentration, NR4A1 increased over time (Figures 6A and 6B). In a dose-response experiment, NR4A1 rose progressively with increasing E2 (Figures 6C and 6D), indicating time- and dose-dependent induction. To assess whether E2 modulates fibrosis, E2 was added to TGF-β-treated fibroblasts. E2 significantly attenuated TGF-β-induced fibrotic features, reducing mRNA levels of fibrosis markers (Figures 6E and 6F). Immunoblotting corroborated these effects, showing lower protein levels of α-SMA, SMAD2, fibronectin, and COL1A1 with E2 treatment (Figures 6G and 6H). Thus, sustained E2 stimulation upregulates NR4A1 and suppresses TGF-β-induced fibrosis.

Figure 6.

Estrogen promotes NR4A1 expression and inhibits fibrosis progression in IPF

(A and B) Q-PCR and Western blot results show that NR4A1 expression increases gradually with prolonged E2 stimulation.

(C and D) Q-PCR and immunoblotting results demonstrate that NR4A1 expression increases with increasing E2 concentrations.

(E and F) Under TGF-β stimulation, mRNA levels of α-SMA, SMAD2, fibronectin, and COL1A1 were significantly increased, but E2 treatment significantly reduced the expression of these mRNAs.

(G and H) immunoblotting results indicate that E2 alleviates TGF-β-induced fibrosis by decreasing the protein expression of α-SMA, SMAD2, fibronectin, and COL1A1. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

E2 increases nuclear receptor subfamily 4 group A member 1 expression through estrogen receptor α and attenuates lung fibrosis

To define the pathway by which E2 regulates NR4A1, we examined the role of ERα. Of the two estrogen receptors (ERα and ERβ), prior studies report altered ERα expression in IPF lung, implicating ERα in E2-induced NR4A1 expression. Consistent with this, E2 treatment of MRC5 and HFL1 fibroblasts significantly upregulated NR4A1, whereas E2 failed to increase NR4A1 in ERα-knockout cells (Figures 7A and 7B), indicating that ERα is required for E2-induced NR4A1 expression. We next tested whether ERα mediates the antifibrotic effects of E2. In TGF-β-treated MRC5 and HFL1 cells with intact ERα, E2 markedly attenuated fibrotic responses, reducing α-SMA, SMAD2, fibronectin, and COL1A1 as assessed by immunofluorescence and immunoblotting (Figures 7C–7J). By contrast, in ERα-deficient cells, E2 did not mitigate TGF-β-induced fibrosis. These data support a model in which E2 upregulates NR4A1 via ERα, thereby suppressing TGF-β-driven fibrotic programs; loss of ERα abrogates both NR4A1 induction and the antifibrotic effect.

Figure 7.

Silencing ERα signaling abolishes the E2-induced regulation of NR4A1 expression

(A) q-PCR and immunoblotting results show that in MRC5 cells, silencing ERα prevents E2 from stimulating an increase in NR4A1 mRNA levels.

(B) q-PCR and immunoblotting results show that in HFL1 cells, silencing ERα prevents E2 from stimulating an increase in NR4A1 protein expression.

(C–F) Representative images and quantification of immunofluorescence for fibrosis-related factors α-SMA, SMAD2, fibronectin, and COL1A1 in cells (n = 6, scale bars, 100 μm).

(G–J) immunoblotting results indicate that in ERα siRNA-silenced cell lines, E2 cannot reduce protein levels of α-SMA, SMAD2, fibronectin, and COL1A1, and fails to inhibit TGF-β-induced fibrosis. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

E2 inhibits lung fibrosis in the pulmonary fibrosis model mice by stimulating nuclear receptor subfamily 4 group A member 1 signaling

To test whether E2 attenuates pulmonary fibrosis via NR4A1, we established BLM-induced PF in C57BL/6 wild-type mice and NR4A1^−/− mice () and administered E2. The PF model was successfully established in both genotypes. In wild-type mice, E2 markedly reduced histopathologic lung injury (Figure 8B), decreased collagen fiber deposition (Figure 8C), lowered α-SMA, SMAD2, fibronectin, and COL1A1 expression (Figures 8D–8H). By contrast, NR4A1^−/− mice developed more severe BLM-induced fibrosis, as indicated by higher fibrosis-marker expression, and E2 failed to significantly ameliorate lung fibrosis (Figures 8A–8H). These data indicate that the antifibrotic effect of E2 is mediated through NR4A1; loss of NR4A1 diminishes E2’s ability to limit fibrosis.

Figure 8.

E2 alleviates pulmonary fibrosis in mice, while NR4A1 deficiency exacerbates the condition

(A) Schematic illustration of estrogen capsule preparation.

(B) Representative images of HE staining of mouse lung tissue (n = 6 per group, scale bars, 100 μm) and Ashcroft fibrosis scores.

(C) Representative images of Masson staining of mouse lung tissue (n = 6 per group, scale bars, 100 μm) and collagen quantification analysis.

(D) Representative images of immunohistochemical staining for α-SMA, SMAD2, Fibronectin, and COL1A1 in mouse lung tissue (n = 6 per group, scale bars, 100 μm).

(E–H) Quantification analysis of immunohistochemical staining for α-SMA, SMAD2, Fibronectin, and COL1A1 in mouse lung tissue. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

Placement of estrogen extended-release capsules increases nuclear receptor subfamily 4 group A member 1 expression and attenuates pulmonary fibrosis in mice

Prior studies suggest that E2 can ameliorate pulmonary fibrosis. To evaluate therapeutic efficacy at different time points, we tested E2 in a bleomycin (BLM)–induced PF mouse model. In a prophylactic regimen, E2 slow-release capsules were implanted 7 days before BLM (Figure 9A). In a post-exposure regimen, capsules were implanted within 24 h after BLM (Figure 10A). Across both regimens, E2 increased NR4A1 expression in the lung (Figure 9B). Histopathology showed reduced fibrosis in E2-treated mice with lower Ashcroft scores (Figures 9C, 9D, 10B, and 10C). Masson’s trichrome corroborated decreased collagen deposition (Figures 9E and 10D). qPCR and immunoblotting further demonstrated reduced α-SMA, SMAD2, fibronectin (FN), and COL1A1 (Figures 9F–9N and 10E–10M), supporting the antifibrotic activity of E2.

Figure 9.

Subcutaneous placement of estrogen capsules in mice before TGF-β staining attenuates pulmonary fibrosis in mice

(A) Schematic of the procedure for the subcutaneous implantation of estrogen capsules to treat IPF.

(B) qPCR and immunoblotting analysis of NR4A1 expression in mice following estrogen capsule implantation.

(C–N) Estrogen implantation reduces BLM-induced pulmonary fibrosis in mice.

(C) Representative images of HE and Masson staining of mouse lung tissue (n = 6 per group, scale bars, 100 μm).

(D) Fibrosis scoring of lung tissue.

(E) Collagen quantification analysis of lung tissue.

(F–N) qPCR and immunoblotting analysis of α-SMA, SMAD2, Fibronectin (FN), and COL1A1 expression in lung tissue. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

Figure 10.

Subcutaneous placement of estrogen capsules in mice within 24h after TGF-β staining attenuates pulmonary fibrosis

(A) Schematic illustrating the procedure of estrogen capsule implantation for IPF treatment. (B-M) Estrogen implantation reduces BLM-induced pulmonary fibrosis in mice.

(B) Representative images of HE and Masson staining of mouse lung tissue (n = 6 per group, scale bars, 100 μm).

(C) Fibrosis scoring of lung tissue.

(D) Quantification of collagen in lung tissue.

(E–M) qPCR and immunoblotting analysis of α-SMA, SMAD2, fibronectin (FN), and COL1A1 expression levels in lung tissue. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

To compare the timing of E2 intervention, we quantified p-NR4A1 in the lung under the two regimens. Relative to the BLM model group, E2-treated mice showed significantly lower p-NR4A1 (Figures 11A and 11B), with the lowest levels in the pre-treatment regimen, indicating stronger suppression by pre-exposure E2. Immunohistochemistry further showed reduced α-SMA, SMAD2, fibronectin (FN), and COL1A1 across both regimens, with COL1A1 particularly lower after pre-treatment (Figures 11C–11G). These data suggest that E2 pre-treatment is more effective at limiting pulmonary fibrosis.

Figure 11.

Comparing the therapeutic effects of estrogen capsule placement at the two time points, early placement of estrogen capsules was better

(A) Representative Western blot bands of p-NR4A1 protein in mouse lung tissue under different interventions.

(B) Quantification of p-NR4A1 protein bands by grayscale analysis.

(C) Representative immunohistochemistry images of mouse lung tissue (n = 6 per group, scale bars, 100 μm).

(D–G) Immunohistochemical analysis of p-NR4A1 expression in mouse lung tissue, showing differences in expression levels across different interventions. Each group n = 6; ∗p < 0.05, ∗∗p < 0.01 compared to the two groups.

Discussion

We propose a model in which sustained TGF-β signaling keeps profibrotic pathways persistently active. persistently active. Prolonged TGF-β stimulation increases NR4A1 expression but also induces its phosphorylation, which diminishes NR4A1 activity and blunts its negative feedback on TGF-β signaling, thereby exacerbating fibrosis. E2, acting via ERα, restores NR4A1 inhibitory function, reduces collagen deposition, and slows fibrosis progression (Figure 12). These findings provide theoretical support for the potential application of E2-based therapeutic strategiesin IPF.

Figure 12.

Regulatory role of NR4A1 in TGF-β signaling in normal, IPF, and estrogen treatment conditions

(A) Under normal physiological conditions, brief TGF-β stimulation induces the upregulation of NR4A1. NR4A1 then suppresses epithelial-mesenchymal transition (EMT) and collagen deposition via a negative feedback mechanism.

(B) In the IPF state, sustained TGF-β signaling leads to prolonged EMT and collagen deposition, while promoting NR4A1 phosphorylation, which disables its negative feedback regulation of TGF-β signaling.

(C) Estrogen treatment enhances NR4A1 transcription and inhibits NR4A1 phosphorylation, thereby restoring its negative feedback regulation on TGF-β signaling.

The clinical cohort analyzed in this study consisted mainly of men and postmenopausal women, both with low-estrogen states. This hormonal milieu aligns with reduced NR4A1 activity and greater fibrosis, reinforcing our mechanistic model that impaired E2–ERα-NR4A1 signaling contributes to disease severity. These observations are consistent with epidemiologic data showing higher IPF incidence and poorer outcomes in men than in women and suggest a protective role for estrogen signaling in fibrotic lung disease. Recognizing these sex-specific differences may refine risk stratification and inform future hormone-targeted therapeutic strategies in IPF. NR4A1 functions beyond fibrosis and participates in diverse pathophysiologic processes,²¹ including tumorigenesis,²² inflammation,²³ glucose- and lipid-metabolic homeostasis, adipogenesis, and vascular remodeling.²⁴ As a regulator of innate and adaptive immunity, NR4A1 plays a prominent role in controlling inflammatory responses.^25,26 NR4A1 also modulates fibrogenesis across multiple organs—skin, lung, liver, and kidney¹⁹—and can mitigate fibrosis, for example via the inhibition of cyclophilin B (CypB) activity.²⁷ Collectively, these observations suggest tissue-specific and context-dependent roles for NR4A1 in fibrosis. Here, we generated NR4A1-silenced and NR4A1-overexpressing cell models to interrogate NR4A1’s regulatory function in fibrosis. Under TGF-β stimulation, control cells exhibited epithelial-to-mesenchymal transition (EMT) and collagen deposition, whereas NR4A1-silenced cells showed significantly augmented EMT and collagen deposition. Conversely, NR4A1 overexpression markedly attenuated TGF-β-induced fibrotic responses. These data underscore NR4A1 as a central inhibitor of fibrosis and support the potential of NR4A1 agonists as antifibrotic therapeutics.

TGF-β is widely regarded as the “master regulator” of fibrosis,²⁸ showing overexpression and persistent activation across numerous fibrosis models and diseases.^29,30 As a key driver, TGF-β accelerates fibrotic progression by activating myofibroblasts, inducing epithelial-to-mesenchymal transition (EMT), and promoting extracellular matrix (ECM) accumulation.^31,32 However, because TGF-β participates broadly in cellular metabolic and physiologic processes, direct inhibition can cause substantial adverse effects,^33,34 making it a less viable antifibrotic strategy. A more practical approach is to target specific downstream nodes within the TGF-β pathway—particularly negative-feedback regulators such as NR4A1. Our data show that sustained TGF-β stimulation induces NR4A1 expression but also drives extensive NR4A1 phosphorylation, which impairs its negative-feedback function and exacerbates fibrosis. E2 restores NR4A1 function via ERα, enhancing negative feedback on TGF-β signaling and thereby suppressing fibrosis progression. The physiologic roles of E2 and its signaling in lung disease are well documented,³⁵ particularly in surfactant synthesis and regulation of alveolar morphology.³⁶ E2 is also implicated in lung cancer biology³⁷; estrogen receptors are widely expressed in lung cancer tissues and cell lines.^38,39 Notably, in ER-deficient mice, the protective effects of E2 are markedly diminished, leading to atelectasis and fibrotic changes in lung tissue,⁹ underscoring the relevance of E2 signaling across pulmonary pathologies.

To establish whether NR4A1 is required for the antifibrotic effects of E2, we induced idiopathic pulmonary fibrosis (IPF) with bleomycin (BLM) in NR4A1^−/− mice and assessed E2 efficacy. Exogenous E2 failed to reduce lung fibrosis in NR4A1^−/− mice, whereas in wild-type mice, E2 significantly attenuated TGF-β-driven fibrotic features. These results indicate that E2’s antifibrotic activity depends on NR4A1 and support a model in which E2 alleviates lung fibrosis via NR4A1-mediated negative feedback on TGF-β signaling, highlighting NR4A1 as a critical determinant and potential therapeutic target.

In summary, this study establishes NR4A1 as a pivotal regulator of TGF-β-induced fibrosis: sustained TGF-β drives NR4A1 phosphorylation, blunting its negative-feedback function. As an NR4A1 agonist,⁴⁰ E2 increases NR4A1 expression, rebalances total and phosphorylated NR4A1, and strengthens negative feedback on TGF-β signaling, thereby limiting fibrosis progression. These findings support E2 and NR4A1 as promising therapeutic targets for antifibrotic intervention.

Limitations of the study

Although our data implicate NR4A1 phosphorylation in promoting fibrosis, we did not directly test whether loss of ERα alters NR4A1 phosphorylation. Based on current results, we speculate that ERα may both enhance NR4A1 transcription and influence its phosphorylation status. Future studies using ChIP-qPCR and promoter-reporter assays will be required to define direct ERα-dependent transcriptional control of NR4A1 and its impact on phosphorylation. In addition, while we delineated NR4A1’s regulation of total SMAD2 and baseline fibrotic responses, we did not validate p-SMAD2—the activated effector of the TGF-β/SMAD cascade—precluding definitive conclusions about effects on canonical signaling; this is a priority for follow-up. Other limitations include the use of BLM-induced pulmonary fibrosis, which only partially recapitulates human IPF complexity, and a modest clinical sample size that may limit power and generalizability, particularly for sex- and hormone-related analyses. Finally, retrospective sample collection precluded uniform assessment of circulating estrogen. Prospective studies with larger cohorts and dynamic hormone profiling are needed to validate and extend these findings.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Bao Liu (liubao2019@126.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Data

• •Data reported in this article will be shared by the lead contact upon request.

Code

• •This article does not report original code.

Additional information

• •Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

Acknowledgments

Funding: 1. Henan Province Traditional Chinese Medicine Inheritance and Innovation Talent Project (Zhongjing Project) Talent Program (Yu Wei Zhong Yi Han [2021] No. 15); 2. Henan Province Key Traditional Chinese Medicine Discipline Construction Project (Yu Wei Zhong Yi Yao Ke Jiao [2024] BBLL No. 1).

Author contributions

B.L. analyzed and interpreted the levels of NR4A1 and E.R. in a patient’s lung tissue and played a major role in writing the article. H.C. contributed significantly to the writing of the article and organized the experimental data. T.J. was also a major contributor to the writing of the article. X.W. created the flowchart of the process. F.C. and R.Z. assisted B.L. in completing the testing of patient tissue. Z.L. and X.H. proposed ideas, developed the experimental protocol, and revised the article. All authors read and approved the final article.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

The use of AI and AI-assisted technologies was not employed in this article.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
β-Actin	Abcam, England	Cat# 70-ab008-100; RRID: AB_2750915
α-SMA	Abcam, England	Cat#ab7818; RRID: AB_262054
SMAD2	Abcam, England	Abcam Cat# 1736-1; RRID:AB_598187
Fibronectin	Abcam, England	Abcam Cat# ab45688; RRID:AB_732380
COL1A1	Abcam, England	Cat#ab316222; RRID:AB_2081873
NR4A1	Abcam, England	Cat#ab153914; RRID: AB_2155955
p-NR4A1	Thermo Fisher Scientific, USA	Cat#PA5-105155 ; RRID: AB_2816628
Goat Anti-Rabbit IgG H&L (HRP)	Abcam, England	Cat#ab205718; RRID:AB_955447
Goat Anti-Rabbit IgG H&L	Abcam, England	Cat#ab150077; RRID:AB_956012
Goat Anti-Mouse IgG H&L	Abcam, England	Cat#ab150113; RRID:AB_10687623
Bacterial and virus strains
LPP-Mm03063-Lv201-100	GeneCopoeia	Cat#EX-Mm03063-Lv201
LPP-MSH197809-LVRU6GP-500	GeneCopoeia	Cat#EX-NEG-Lv201
LP146-100	GeneCopoeia	Cat# LP146-100
Biological samples
IPF lung tissue	Henan Provincial People’s Hospital	–
Control lung tissue	Henan Provincial People’s Hospital	–
Experimental models: Cell lines
MRC5	Cell Resource Center of Chinese Academy of Sciences	Cat# GNHu41; CSTR:19375.09.3101HUMGNHu41
HFL1	Cell Resource Center of Chinese Academy of Sciences	Cat#GNHu28; CSTR:3101HUMGNHu28
Experimental models: Organisms/strains
Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA)	Cyagen Biosciences	Cat# S-KO-02468
C57BL/6 mice	Beijing Vital River Laboratory Animal Technology Co., Ltd	Cat# C57BL∕6NCrl
Oligonucleotides
Primers for NR4A1, see Table 3	This paper	–
Primers for ERα, see Table 3	This paper	–
Primers for ERβ, see Table 3	This paper	–
Primers for α-SMA, see Table 3	This paper	–
Primers for SMAD2, see Table 3	This paper	–
Software and algorithms
GraphPad Prism	GraphPad Software 9	https://www.graphpad.com/features
ImageJ	National Institutes of Health(NIH)	https://imagej.nih.gov/ij/

Experimental model and, experimental model and study participant details

Human participants

This study enrolled 23 adult patients diagnosed with idiopathic pulmonary fibrosis (IPF) admitted to the Department of Respiratory and Critical Care Medicine between May 2018 and January 2023. All participants were self-reported East Asian (race and ethnicity not used for stratification due to the homogeneous, single-center cohort). The cohort consisted of 16 males and 7 postmenopausal females (age range: 52–72 years). All patients met the diagnostic criteria of the 2018 ATS/ERS/JRS/ALAT guidelines. Control lung tissue samples were obtained from the adjacent non-tumorous lung tissues of patients undergoing lung cancer resection surgery at the same institution. The potential influence of sex and gender was considered in this study. In the human cohort, all female participants were postmenopausal, which may influence hormone-related responses. However, due to the small sample size and the clinical focus on a specific IPF population, the study was not statistically powered to stratify outcomes conclusively by sex.

This study was approved by the Ethics Committee of Henan Provincial People’s Hospital (approval number: HNRH-KY-2018-011) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrollment.

Cell lines

The human embryonic lung fibroblast cell lines MRC5 and HFL1 were obtained from the Cell Resource Center of the Chinese Academy of Sciences. Both cell lines were authenticated using short tandem repeat (STR) profiling, and were confirmed to be free of mycoplasma contamination. Cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO₂. All experiments were performed using fibroblasts between passages 4 and 8. Stable cell lines were generated via lentiviral transduction followed by puromycin selection.

Animals

Animal studies utilized male Nr4a1 knockout (NR4A1^−/−) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.). Mice were housed under specific pathogen-free (SPF) conditions in individually ventilated cages on a 12-h light/dark cycle with ad libitum access to food and water. All animal procedures were reviewed and approved by the Ethics Committee of Henan Provincial People’s Hospital (approval No. HNRH-KY-2018-011).

Method details

Study populations

This study enrolled 23 adult patients diagnosed with idiopathic pulmonary fibrosis (IPF) who were admitted to the Department of Respiratory and Critical Care Medicine between May 2018 and January 2023. All participants were self-reported East Asian. Race and ethnicity were not used as stratification variables in this study due to the relatively homogeneous population recruited at a single center. Among the 23 IPF patients, 16 were male and 7 were female. All female participants were postmenopausal (52–72 years). All patients met the diagnostic criteria defined by the 2018 ATS/ERS/JRS/ALAT guidelines. At the time of sample collection, none of the patients had received antifibrotic therapy (including antifibrotic agents or immunosuppressants), and no participant had received hormone therapy within the preceding 3 months. All enrolled individuals were free of other significant pulmonary diseases or active infections (e.g., pneumonia). Control lung tissue samples were obtained from patients undergoing lung cancer resection surgery at the same institution. Adjacent non-tumorous lung tissues were confirmed histologically to be free of malignant infiltration. All participants underwent comprehensive clinical assessment—including high-resolution CT, pulmonary function testing, and symptom evaluation—to ensure eligibility.

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki, and the research protocol was reviewed and approved by the Ethics Committee of Henan Provincial People’s Hospital (approval number: HNRH-KY-2018-011). Written informed consent was obtained from all participants prior to enrollment, and participation was entirely voluntary.

Following clinical sample collection, each specimen was promptly divided into two portions for molecular and pathological analyses. One portion was snap-frozen in liquid nitrogen for subsequent gene expression profiling, protein detection, and molecular mechanism studies. The other portion was fixed in formalin for histopathological evaluation and fibrosis scoring, ensuring preservation of tissue architecture and minimizing post-sampling variability.

Cell culture and viral transduction

The human embryonic lung fibroblast cell lines MRC5 and HFL1 used in this study were obtained from the Cell Resource Center of Chinese Academy of Sciences. Cells were cultured at 37°C in a humidified incubator with 5% CO₂. The culture medium consisted of high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco, Cat. No. 10099141C, Australia) and 100 U/mL penicillin-streptomycin solution (Gibco, Cat. No. 15140-122, Australia). To ensure experimental consistency and stable cell conditions, all experiments were performed using fibroblasts at passages 4 to 8.

The lentiviral and adeno-associated viral (AAV) vectors used for cell and animal experiments were obtained from GeneCopoeia (Rockville, MD, U.S.A) (Table 2). Lentiviruses were used to transduce MRC5 and HFL1 cells, and stable cell lines were established through puromycin selection. Expression of the target gene was subsequently confirmed by quantitative real-time PCR and Western blotting. AAV was administered to mice via intranasal instillation. After 72 h, one mouse was harvested to verify transduction efficiency by PCR.

Table 2.

Viruses and Plasmids

AVV	plasmids
LPP-Mm03063-Lv201-100	EX-Mm03063-Lv201
LPP-MSH197809-LVRU6GP-500	EX-NEG-Lv201
LP146-100	–

Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (GeneCopoeia, QP020, USA), and RNA concentration and purity were assessed with using a NanoDrop spectrophotometer. RNA was then reverse transcribed into complementary DNA (cDNA) using the GeneCopoeia reverse transcription kit (QP056, USA). Quantitative real-time PCR (qRT-PCR) was performed on a GeneCopoeia real-time PCR system (QP031, China), with each reaction containing 10 μL of 2× SYBR Green PCR Master Mix, 0.4 μL of each specific primer (10 μM), and 1 μL of cDNA template, in a total volume of 20 μL. PCR cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s. Non-template controls and melt curve analysis were included to exclude nonspecific amplification. β-Actin served as the reference gene for normalization, and gene expression levels were calculated using the 2^∧-ΔΔCt method. Primer sequences are listed in Table 3.

Table 3.

Primer sequences

	Gene	NM number	Forward primer	Reverse primer
Human	NR4A1	NM_001202234.2	ACTTGGATG GAGACTTAA	GCCTTCAATTA GAATCTTG
	ERα	NM_000125.3	AGAACAACAT CAGCAGTA	TGGCATTATAG AATCATTACG
	ERβ	NM_001214902.1	ATACATACCTT CCTCCTAT	TTCCAAGTTAG TGACATT
Mouse	NR4A1	NM_010444.3	CCCACTATTTG TCTTATCC	CATCTCAACC TCTTCCTT
	α-SMA	NM_007392.3	TGGCATCAATC ACTTCAA	CCTATCTGG TCACCTGTA
	SMAD2	NM_010754.5	TTGTCCTTGC CTCTGTAG	GTTGGTTAGT TATTCTCGTGAT
	Fibronectin	NM_010233.2	ATACGAAGTC AGTGTCTA	ATTCTCCAG AGTAGTGAT
	COL1A1	NM_007742.4	AGGTATGCTT GATCTGTAT	CAGTCCAG TTCTTCATTG

Western blot analysis

Proteins were extracted from cells or tissues using RIPA lysis buffer (P0013B, Beyotime, China) according to the manufacturer’s instructions. Protein samples were separated by SDS-PAGE on 10%–15% polyacrylamide gels, selected according to the molecular weight of the target protein. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Merck Millipore, USA) by electrophoresis at 100V for 1h in standard transfer buffer. Non-specific binding was blocked with 5% skim milk or BSA (Sigma-Aldrich, USA) for 1h at room temperature (RT). Membranes were incubated with specific primary antibodies overnight at 4°C Table 4, washed three times in TBST (TBS with 0.1% Tween 20; 10 min each), and then incubated with secondary antibodies (e.g., Goat Anti-Rabbit IgG H&L; Table 3) for 1 h at RT. Protein bands were visualized by enhanced chemiluminescence (ECL) and imaged using an automated imaging imaging system (iBright FL1000, Thermo Fisher Scientific, USA). Band intensities were quantified in ImageJ (https://imagej.nih.gov/ij/) and normalized to β-actin as the loading control to ensure reproducibility.

Table 4.

Antibody

Antibody	Concentration	Number	Company
β-Actin	1:1000	ab8226	Abcam, England
α-SMA	1:1500	ab7818	Abcam, England
SMAD2	1:1000	ab119907	Abcam, England
Fibronectin	1:1500	ab2314	Abcam, England
COL1A1	1:3000	ab316222	Abcam, England
NR4A1	1:1500	ab153914	Abcam, England
p-NR4A1	1:2000	ITP1019	G-Biosciences, USA
Goat Anti-Rabbit IgG H&L (HRP)	1:10000	ab205718	Abcam, England
Goat Anti-Rabbit IgG H&L	1:10000	ab150077	Abcam, England
Goat Anti-Mouse IgG H&L	1:10000	ab150113	Abcam, England

Immunofluorescence staining

Cells were first fixed in 4% paraformaldehyde (P0099; Beyotime, China) for 10–15 min and then permeabilized with 0.1% Triton X-100 (ST795; Beyotime, China) for 30 min at RT. Next, cells were incubated overnight at 4°C with primary antibodies against α-SMA, SMAD2, fibronectin, and COL1A1 (Table 4). The following day, cells were washed three times with PBS and then incubated for 1 h atRT with the appropriate fluorophore -conjugated secondary antibodys Table 3). Fluorescence images were captured on an inverted fluorescence microscope (ICX41; Leica, Germany), and images were signal intensity was quantifiedin ImageJ.

Animal studies

Nr4a1 knockout (NR4A1^−/−) mice on a C57BL/6 background (Catalog S-KO-02468) were obtained from Cyagen Biosciences. In addition, 8-week-old male C57BL/6 specific pathogen-free (SPF) mice (C57BL/6NCrl) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (certificate No. HPPH-2022XS241). Mice were housed in individually ventilated cages on a 12-h light/dark cycle and were acclimated for 1 week before experiments. Before experimentation, all mice underwent PCR genotyping to confirm the Nr4a1 knockout genotype. All procedures complied with the regulations of the Ethics Committee of Henan Provincial People’s Hospital (approval No. HNRH-KY-2018-011). Pulmonary fibrosis (PF) was induced by intratracheal instillation of bleomycin (BLM) to establish a model that partially recapitulates IPF. Briefly, each mouse received 0.05 mL of a 0.1 U/mL BLM via intratracheal administration. After instillation, the thorax was gently tapped to facilitate uniform distribution. Mice were monitored for 21–28 days, and histological and molecular analyses were performed at prespecified time points.

Pathology tests

Pathology tests Paraffin-embedded tissue sections were processed for histopathology. Sections were deparaffinized, stained with hematoxylin and eosin (H&E) to assess morphology, dehydrated through ethanol, cleared in xylene, and mounted with neutral resin. Slides were examined on an upright microscope (Eclipse Ci; Nikon, Japan) with an attached imaging system (DS-U3; Nikon, Japan). Fibrosis severity was scored using the modified Ashcroft scoring method.

Collagen deposition was evaluated by Masson’s trichrome staining. Sections were sequentially treated with hematoxylin, Ponceau acid fuchsin, and aniline blue, dehydrated, cleared, and sealed with resin. Collagen content was quantified from captured images using ImageJ. Immunohistochemistry (IHC) was performed to detect ERα, ERβ, TGF-β1, NR4A1, p-NR4A1, α-SMA, SMAD2, fibronectin, and COL1A1. After deparaffinization and antigen retrieval, sections were incubated with primary antibodies followed by horseradish peroxidase (HRP)–conjugated secondary antibodies (Goat Anti-Rabbit IgG H&L). Signal was developed with DAB and nuclei were counterstained with hematoxylin. Stained sections were imaged on the Eclipse Ci system, and quantitative image analysis was performed in ImageJ.

Preparation of estrogen extended-release capsules

As previously described,⁴¹ 17β-estradiol slow-release capsules were prepared by dissolving 17β-estradiol in ethanol or DMSO and filling medical-grade silicone tubing (inner diameter, 1.5 mm; outer diameter, 2.0 mm) with the solution. Tubing segments (1–2 cm) were sealed with sterile silicone plugs, cured, and stored in a dark, sterile environment until use for subcutaneous implantation to achieve sustained estrogen release.

Quantification and statistical analysis

Statistical analyses were performed in GraphPad Prism, and data are presented as mean ± standard deviation (SD). Between-group comparisons were conducted using an unpaired t test for non-normal data. One-way ANOVA followed by Dunnett’s post hoc multiple-comparison test was applied for comparisons across multiple groups. Spearman’s rank correlation was used to assess associations among NR4A1, p-NR4A1, ERα, ERβ, and TGF-β. Two-tailed p < 0.05 was considered statistically significant.

Published: January 29, 2026