Introduction

Breastfeeding is a crucial nutritional practice for infants. Antibodies in breast milk enhance neonatal immunity and reduce disease incidence1,2. However, the annual incidence of mammary fibrosis is rising, and severe cases may progress to breast cancer. Currently, research into mammary fibrosis is still in its early stages. Further investigation into the pathogenesis of mammary fibrosis and the search for potential prevention strategies are key areas for future research..

Epithelial-mesenchymal transition (EMT) generates abundant extracellular matrix (ECM), which contains collagen, glycoproteins, and other components, and is a primary contributor to fibrosis3. Consequently, EMT is considered a key driver of fibrotic diseases. Transforming growth factor β1 (TGF-β1) is a potent cytokine with pro-fibrotic effects4,5, it induces ECM production and promotes EMT, thereby accelerating fibrotic progression. Thus, TGF-β1 was selected to establish a murine model of mammary fibrosis in this study.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of the antioxidant response. It neutralizes reactive oxygen species (ROS) by binding to antioxidant response elements (ARE), thereby restoring cellular redox balance6. Damaged mitochondria are a major source of ROS, which acts as a key signaling molecule in various biological processes. Excessive ROS accumulation causes oxidative damage, exacerbates mitochondrial dysfunction, and can ultimately lead to cell death7. Therefore, Nrf2 is vital for maintaining mitochondrial homeostasis and structural integrity8. Furthermore, an increasing number of studies have confirmed that Nrf2 plays a crucial role in the mechanism of inhibiting fibrosis9. Zhang et al. demonstrated that Nrf2 inhibits EMT via its antioxidant pathway, alleviating bleomycin-induced pulmonary fibrosis10. Kavian et al. found that Nrf2 inhibits skin fibrosis in systemic sclerosis by regulating antioxidant and ROS-related signaling molecules11. These findings suggest that the Nrf2-mediated antioxidant pathway may be significantly involved in mammary fibrosis pathogenesis.

It is well known that mitochondrial autophagy plays a key role in mitochondrial quality control. Damaged mitochondria and their harmful components can be cleared by mitochondrial autophagy. In mammals, this elimination is primarily mediated by the PTEN-induced putative kinase 1 (Pink1) and Parkin RBR E3 ubiquitin protein ligase (Parkin) pathway. Bhatia et al. reported that macrophage mitophagy, regulated by the Pink1/Mfn2/Parkin pathway, alleviates ECM accumulation in renal fibrosisss SS12. Additionally, studies indicate that Nrf2 protects cardiomyocytes by promoting the clearance of damaged mitochondria via mitophag13.

Whether mitochondrial damage occurs during mammary fibrosis and whether mitigating such damage could be a potential therapeutic strategy remains unreported. Therefore, this study aims to investigate the relationship between Nrf2, mammary fibrosis, and mitochondrial function, to provide experimental evidence and a theoretical foundation for the prevention and treatment of mammary fibrosis.

Material and methods

Animal

Wild-type C57BL/6 mice (WT) were obtained from Liaoning Changsheng Biotechnology, and Nrf2 gene knockout C57BL/6 mice (Nrf2−/−) were acquired from The Jackson Laboratory (Bar Harbor, ME, USA). All animal procedures were approved by the Animal Ethics and Welfare Committee of Jilin University (IACUC; approval number SY202310002) and conducted in accordance with the GB14925 standard. Female and male mice aged 8 weeks were cage mated. Mice were housed under specific pathogen-free conditions at 23–25 °C with a 12 h light/dark cycle and had free access to food and water.

Model establishment

WT and Nrf2−/− mice were randomly divided into two groups post-delivery: a control group and a TGF-β1-induced mammary fibrosis group. Mice were anesthetized with pentobarbital, and the fourth pair of nipples were disinfected with 75% ethanol. The nipples were trimmed to approximately 1 mm using sterilized scissors. Subsequently, 50 μL of TGF-β1 (100 ng/mL) was slowly injected into the mammary papillae. Injections were administered every two days for a total of four injections. Mammary tissues were collected for detection.

Cell culture and treatment

Murine mammary epithelial cells (mMECs) were purchased from the U.S. Model Culture Repository and cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Clark Bioscience, Richmond, VA, USA) at 37 °C in a 5% CO2. Cells were pretreated with RA (10 μM; Yuanye Bio-Technology, China) or tBHQ (10 μM; Yuanye Bio-Technology, China) for 1 h prior to the addition of TGF-β1 (5 ng/mL) for 48 h. Cell samples were then collected for subsequent experiments.

Histopathological examination

Fresh mice mammary tissue was completely immersed in a 4% formaldehyde solution and fixed for 24 h. After dehydration, transparency, wax infiltration, embedding, slicing, and baking, the pathological tissue sections were prepared for H&E staining and Masson Trichrome staining, using reagents from Beijing Solarbio Science & Technology, Beijing, China.

Immunofluorescence

Paraffin-embedded sections were dewaxed, rehydrated, and subjected to antigen retrieval and blocking. Sections were incubated with a primary antibody against α-SMA (1:100; Affinity, AF1032, China) at 4 °C overnight. After washing with PBS, sections were incubated with a fluorescently labeled secondary antibody (1:1000) for 1 h at room temperature. Nuclei were counterstained with DAPI, and images were captured using a fluorescence microscope. For cell immunofluorescence, cells were fixed with 4% formaldehyde for 15 min after treatment, followed by the same staining procedure as for tissue sections.

Quantitative RT-PCR

Total RNA was extracted from mammary tissues or mMECs using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA using a PrimeScript RT kit (TaKaRa, Japan). Quantitative PCR was performed using SYBR Green PCR Master Mix (Roche, South San Francisco, CA, USA) under the following conditions: 95 °C for 4 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 90 s. Primer sequences are listed in Table 1

Table 1 Primer sequences of α-SMA , Collagen1, MMP , Vimentin and β-actin.
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Mito-tracker

After 48 h of treatment, cells were incubated with 100 nM Mito-Tracker Red CMXRos (Beyotime Institute of Biotechnology, China) for 30 min at 37 °C. The staining solution was replaced with fresh culture medium. Observed under a fluorescence microscope.

ROS detection

Intracellular ROS levels were measured using a ROS assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions.

Small interfering RNA (siRNA) treatment

5 μL Lipofectamine™ 2000 Transfection Reagent (Invitrogen, CA, USA) and 250 μL optimum (Gibco, NY, USA) were mixed together, at the same time, 5 μL of siRNA(Jima Gene Co., Ltd,China) was mixed with 250 μL of optimum for 5 min, then they were mixed for 30 min and then added to a petri dish containing 4 mL of medium for transfection for 48 h. siNrf2, the siNegative control (siNC) sequences information was shown in Table 2.

Table 2 Sequences of siNrf2, siNegative control (siNC).
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Western blot

Proteins were extracted from mammary tissues or mMECs using lysis buffer. Protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology, China). Proteins were denatured in 5 × SDS loading buffer by boiling for 5 min and separated by SDS-PAGE. After transfer, membranes were blocked and incubated overnight at 4 °C with primary antibodies against Hsp60 (15282-1-AP; Proteintech, China), Mfn2 (12186-1-AP; Proteintech, China), P62 (18,420-1-AP; Proteintech, China), PHB2 (12295-1-AP; Proteintech, China), TGF-β1 (21898-1-AP; Proteintech, China), Parkin (14060-1-AP; Proteintech, China), p-Parkin (bs-19882R; Bioss, China), LC3 (14600-1-AP; Proteintech, China), Smad (AF6367; Affinity, China), p-Smad (AF3367; Affinity, China), Vimentin (AF7013; Affinity, China), α-SMA (AF1032; Affinity, China), E-cadherin (A3044; Abclonal, China), Collagen1 (A16891; Abclonal, China), Pink1 (BC100-494; Novus Biologicals, USA), and β-actin (66009-1-Ig; Proteintech, China). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Santa Cruz, CA, USA) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, China).

Data analysis

The experimental data were analyzed by means of one-way ANOVA and Turkey test, and presented in the form of Mean ± SD, with each experiment conducted a minimum of 3 times. Statistical analyses and graph creation were performed using GraphPad Prism 7 (San Diego, CA, USA). Combine pictures were performed using Adobe Illustrator CS6 software .The relative density of protein bands was counted using ImageJ software, which also merged fluorescence images and added scale bars.

Results

Nrf2−/− exacerbated pathological damage in mammary fibrosis

To investigate the role of Nrf2 in mammary fibrosis, a mouse model was established by injecting TGF-β1 into the mammary tissue of wild-type (WT) and Nrf2−/− mice, followed by H&E and Masson staining. H&E staining revealed more severe damage to the acinar structure in the Nrf2−/−-TGF-β1 group compared to the WT-TGF-β1 group (Fig. 1A). Masson staining indicated increased collagen deposition in the mammary tissue of Nrf2−/−-TGF-β1 mice (Fig. 1B). Pathological scoring confirmed that mammary tissue damage was more severe in the Nrf2⁻/⁻-TGF-β1 group (Fig. 1C). These results demonstrate that Nrf2 deficiency exacerbates the development of mammary fibrosis in mice.

Fig. 1
figure 1

Nrf2−/− exacerbated pathological damage in mammary fibrosis.100 ng/mL of TGF-β1 was injected into the nipples of mice every two days for a total of four times. (A) H&E staining of mammary tissue. (B) Masson staining of mammary tissue, 200 μm. (C) Mammary injury score.The data were presented as Mean ± SD. ** represents P < 0.01, * represents P < 0.05.

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Nrf2 decreased the increase of molecular indexes of mammary fibrosis

To examine the effect of TGF-β1 on molecular indicators of fibrosis, the changes of fibrotic protein and mRNA were detected in this experiment. First of all, it was clear that the mice used in this experiment were Nrf2−/− mice (Fig. 2A-B). qRT-PCR analysis showed that mRNA levels of COL1, α-SMA and Vim were significantly higher in the Nrf2−/−-TGF-β1 group than in the WT-TGF-β1 group (Fig. 2C). Western blot analysis revealed a significant decrease in E-cadherin protein levels and a significant increase in α-SMA and Vimentin levels in the Nrf2−/−-TGF-β1 group compared to the WT-TGF-β1 group (Fig. 2D–G). Immunofluorescence staining for α-SMA on paraffin-embedded sections showed stronger fluorescence intensity in the Nrf2⁻/⁻-TGF-β1 group (Fig. 2H-I). These findings indicate that Nrf2 knockout promotes the upregulation of molecular markers in TGF-β1-induced mammary fibrosis.

Fig. 2
figure 2

Nrf2 decreased the increase of molecular indexes of mammary fibrosis. (A) Nrf2−/− mice genotype identification. (B) Protein bands and analysis of Nrf2 in mice mammary tissue. (C) Expression of COL1, α-SMA, Vim mRNA in mammary tissue. (D-G) Protein bands and analysis of E-cad, α-SMA, Vim and β-actin in mice mammary tissue. (H-I) Results of immunofluorescence staining and analysis of mammary tissue with α-SMA, 200 μm. The data were presented as Mean ± SD. There were 6 mice in each group (n = 6). Three independent repeatable experiments were performed. ** represents P < 0.01, * represents P < 0.05.

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Activation of Nrf2 inhibited the increase of fibrosis related molecular index in mMECs

EMT refers to the transformation of epithelial cells into mesenchymal phenotypic cells and plays a key role in the fibrosis process. To further validate the effect of Nrf2, an in vitro model of mammary fibrosis was established using TGF-β1-stimulated murine mammary epithelial cells (mMECs), treated with the Nrf2 activator tBHQ. Compared to the TGF-β1 group, the TGF-β1 + tBHQ group showed reduced α-SMA fluorescence intensity and protein levels (Fig. 3A, F), increased E-cadherin protein levels (Fig. 3C), and decreased Collagen 1 and Vimentin levels (Fig. 3D, E). qRT-PCR showed that mRNA levels of α-SMA, VIM, Collagen 1 and MMP1 were significantly lower in the TGF-β1 + tBHQ group (Fig. 3B). These results suggest that Nrf2 activation inhibits the upregulation of fibrosis-related molecular markers.

Fig. 3
figure 3

Activation of Nrf2 inhibited mammary fibrosis related molecular index increase in mMECs. tBHQ (10 μM) was added 1 h before TGF-β1 (5 ng/mL) to stimulate mMECs for 48 h. (A) mMECs α-SMA immunofluorescence staining, 200 μM. (B) Expression of COL1, α-SMA, Vim and MMP1 mRNA in mMECs. (C-F) Protein bands and analysis of E-cad, Collagen1, α-SMA, Vim and β-actin in mMECs. The data were presented as Mean ± SD. Three independent repeatability experiments were performed. ** represents P < 0.01, * represents P < 0.05.

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Inhibition of Nrf2 induced mammary fibrosis related molecular index increase in mMECs

The Nrf2 inhibitor RA was added to TGF-β1-induced mMECs. The TGF-β1 + RA group exhibited stronger α-SMA fluorescence intensity and higher protein levels (Fig. 4 A, E), decreased E-cadherin levels, and increased Collagen 1 and Vimentin levels compared to the TGF-β1 group (Fig. 4C–F). qRT-PCR showed higher mRNA levels of α-SMA, VIM, Collagen 1 and MMP1 in the TGF-β1 + RA group (Fig. 4B). To further investigate the role of Nrf2 inhibition in EMT, Nrf2 expression was knocked down using siRNA. In the TGF-β1 + siNrf2 group, E-cadherin levels decreased, while α-SMA and Vimentin levels increased, contrasting with the effects of tBHQ (Fig. 4G–J). qRT-PCR confirmed increased mRNA levels of these markers in the TGF-β1 + siNrf2 group (Supp. Figure 1). These results indicate that Nrf2 inhibition promotes the expression of fibrosis-related molecular markers in TGF-β1-induced mMECs.

Fig. 4
figure 4

Inhibition of Nrf2 induced mammary fibrosis related molecular index increase in mMECs. RA (10 μM) was added 1 h before TGF-β1 (5 ng/mL) to stimulate mMECs for 48 h. mMECs were transiently transfected with small interfering RNA (siRNA) against Nrf2 or control siRNA for 48 h. (A) mMECs α-SMA immunofluorescence staining, 200 μm. (B) Expression of COL1, α-SMA, Vim and MMP1 mRNA in mMECs. (C-F) Protein bands and analysis of E-cad, Collagen1, α-SMA, Vim and β-actin in mMECs. (G-J)Protein bands and analysis of E-cad, α-SMA, Vim and β-actin in mMECs. The data were presented as Mean ± SD. Three independent repeatability experiments were performed. ** represents P < 0.01, * represents P < 0.05.

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Nrf2 inhibited the activation of TGF/Smad signaling pathway

Given the central role of TGF-β/Smad signaling in fibrosis, its activation was assessed in vivo. The pathway was significantly more activated in the Nrf2−/−-TGF-β1 group than in the WT-TGF-β1 group (Fig. 5A–C). In vitro, RA significantly activated the TGF-β/Smad pathway (Fig. 5D–F), while tBHQ inhibited it (Fig. 5G–I). siRNA-mediated knockdown of Nrf2 also activated the pathway, an effect reversed by tBHQ (Fig. 5J–L). These results demonstrate that Nrf2 mediates mammary fibrosis progression through the TGF-β/Smad signaling pathway.

Fig. 5
figure 5

Nrf2 mediated the development of in mice mammary fibrosis induced by TGF-β1 through the TGF/Smad signaling pathway. (A-C) Protein bands and analysis of p-Smad, Smad, TGF and β-actin in mice mammary tissue. (D-F) Protein bands and analysis of p-Smad, Smad, TGF and β-actin in mMECs of RA group. (G-I) Protein bands and analysis of p-Smad, Smad, TGF and β-actin in mMECs of tBHQ group. (J-L) Protein bands and analysis of p-Smad, Smad, TGF and β-actin in mMECs tissue. The data were presented as Mean ± SD. Three independent repeatability experiments were performed. ** represents P < 0.01, * represents P < 0.05.

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Effects of Nrf2 on mitochondrial damage and autophagy in vitro

To explore the mechanism by which Nrf2 influences mammary fibrosis, mMECs were treated with TGF-β1 and RA. ROS levels were higher in the TGF-β1 + RA group than in the TGF-β1 group (Fig. 6A). Mitochondrial damage, indicated by reduced Mfn2 and Hsp60 expression, was more severe in the TGF-β1 + RA group (Fig. 6B–D). MitoTracker Red staining showed a significant decrease in mitochondrial membrane potential (Fig. 6E). Mitophagy-related proteins PHB2 and LC3 were decreased, P62 was increased, and the Pink1-Parkin pathway was suppressed in the TGF-β1 + RA group (Fig. 6F–K). These results indicate that Nrf2 inhibition exacerbates mitochondrial damage, suppresses mitophagy, and increases ROS production.

Fig. 6
figure 6

Effects of Nrf2 on mitochondrial damage and autophagy in vitro. (A) Determination of ROS content. (B-D) Protein bands and analysis of Mfn2, Hsp60 and β-actin in mMECs. (E) Mito-Tracker Red was used to detect changes in mitochondrial membrane potential, 200 μm. (F-K) Protein bands and analysis of PHB2, LC3, P62, p-Parkin, Parkin, Pink1 and β-actin in mMECs. The data were presented as Mean ± SD. Three independent repeatable experiments were performed. ** represents P < 0.01, * represents P < 0.05.

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Effects of Nrf2 on mitochondrial damage and autophagy in vivo

In vivo, Nrf2−/−-TGF-β1 mice produced more ROS than WT-TGF-β1 mice (Fig. 7A-B). Transmission electron microscopy revealed mitochondrial rupture in WT-TGF-β1 mice and more severe damage, including cristae loss and vacuolization, in Nrf2−/ – -TGF-β1 mice (Fig. 7C). Western blot confirmed reduced Mfn2 and Hsp60 expression (Fig. 7D–F), decreased PHB2 and LC3, increased P62, and suppressed Pink1-Parkin signaling in Nrf2−/−-TGF-β1 mice (Fig. 7G–L). These results were consistent with the in vitro findings.

Fig. 7
figure 7

Effect of Nrf2−/− on mice mammary mitochondrial damage and autophagy induced by TGF-β1. (A, B) Determination of ROS content in mice mammary tissue. (C) The mitochondrial damage in mammary tissue of mice was observed by transmission electron microscopy. (D-F) Protein bands and analysis of Mfn2, Hsp60 and β-actin in mice mammary tissue. (G-L) Protein bands and analysis of PHB2, LC3, P62, p-Parkin, Parkin, Pink1 and β-actin in mice mammary tissue. The data were presented as Mean ± SD. Three independent repeatable experiments were performed. ** represents P < 0.01, * represents P < 0.05.

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Nrf2 mediated the development of fibrosis through ROS

To determine if ROS mediates the effect of Nrf2 on fibrosis, the ROS scavenger NAC was added to mMECs. ROS levels were significantly reduced in the TGF-β1 + RA + NAC group compared to the TGF-β1 + RA group (Fig. 8A). NAC treatment attenuated the decrease in E-cadherin and the increase in α-SMA and Vimentin (Fig. 8B–E) and inhibited TGF-β/Smad pathway activation (Fig. 8F–H). In vivo, intraperitoneal NAC injection in Nrf2−/− mice alleviated collagen deposition and ROS accumulation (Fig. 8I, J), mitigated changes in E-cadherin, α-SMA, and Vimentin levels (Fig. 8K–N), and suppressed TGF-β/Smad activation (Fig. 8O–Q). These results confirm that Nrf2 knockout increases ROS production, activating the TGF-β/Smad pathway and exacerbating fibrosis, while ROS scavenging effectively alleviates mammary fibrosis. The in vivo and in vitro results are consistent.

Fig. 8
figure 8

Nrf2 mediated the development of TGF-β1 induced fibrosis through ROS. NAC (3 mM) was added 1 h before TGF-β1 (5 ng/mL) to stimulate mMECs for 48 h. In vivo experiments, 100 mg/kg NAC was administered intraperitoneally for 7 days. (A) Determination of ROS content. (B-H) Protein bands and analysis of E-cad, α-SMA, Vim, p-Smad, Smad, TGF and β-actin in mMECs. (I) Masson staining of Nrf2−/− mice mammary tissue, 200 μm (J) Determination of ROS of Nrf2−/− mice mammary tissue. (K-N) Protein bands and analysis of E-cad, Vim and β-actin in Nrf2−/−mice mammary tissue. (O-Q) Protein bands and analysis of p-Smad, Smad, TGF and β-actin in Nrf2−/− mice mammary tissue. The data were presented as Mean ± SD. Three independent repeatable experiments were performed. ** represents P < 0.01, * represents P < 0.05.

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Discussion

Mammary fibrosis poses significant risks to breastfeeding, endangering maternal health and indirectly impacting infant health and development14. Consequently, identifying therapeutic targets to alleviate mammary fibrosis is crucial. In this study, we found that Nrf2−/− mice stimulated with TGF-β1 exhibited more severe mitochondrial damage, suppressed autophagy, and significantly higher expression of fibrotic proteins and genes. Further investigation revealed that Nrf2 mitigates mammary fibrosis progression in mice by modulating the TGF-β/Smad signaling pathway and ROS levels. Our findings highlight, for the first time, the crucial role of Nrf2-mediated mitophagy in alleviating mammary fibrosis.

TGF-β1 is a primary inducer of epithelial-mesenchymal transition (EMT). It promotes the aggregation of inflammatory cells and fibroblasts, as well as collagen and fibrin synthesis, leading to ECM deposition and degradation15. Liu et al. found that organs or tissues with fibrosis have higher levels of TGF-β1. Stimulating experimental animals with exogenous TGF-β1 can induce the occurrence of fibrosis and excessive deposition of ECM proteins in their tissues and organs16.Furthermore, TGF-β1 has been shown to induce EMT in bovine mammary epithelial cells via the TGF-β1/Smad signaling pathway17.Therefore, we selected TGF-β1 to establish a mouse model of mammary fibrosis.

The extracellular matrix (ECM), composed of various collagens, proteoglycans, and glycoproteins, serves as the adhesion matrix and structural scaffold for cellular components18. A key indicator of fibrosis is the accumulation of fibrous collagen, particularly Collagen I, which constitutes approximately 30% of the ECM19and is pivotal in pathological processes such as inflammation, tumor growth, and tissue fibrosis20.Our results indicate that under TGF-β1 stimulation, mammary tissue from Nrf2−/− mice showed more severe acinar damage and collagen fiber deposition in Nrf2−/− mice. These results preliminarily confirm that Nrf2 deficiency aggravates TGF-β1-induced mammary fibrosis development. In addition, numerous studies have established a close relationship between the TGF/Smad signaling pathway and ECM deposition/collagen fiber formation21,22. Upon TGF-β1 activation, Smad2 and Smad3 are phosphorylated, form complexes, and translocate to the nucleus to promote TGF/Smad-dependent transcriptional activation of downstream genes23,24, Activation of the TGF-β1-dependent Smad2/3 pathway in rat cardiac fibroblasts, for instance, contributes to ECM protein production and fibrosis progression25. Xu et al. explored the possibility of finding new ways to alleviate liver fibrosis by targeting TGF/Smad signaling26. Therefore, we investigated whether Nrf2 regulates mammary fibrosis progression via TGF/Smad. These findings suggest that Nrf2 mediates the development of TGF-β1-induced mammary fibrosis in mice through the TGF/Smad signaling pathway.

Nrf2 is a master regulator of the cellular antioxidant response and a key player in maintaining mitochondrial homeostasis and structural integrity27,28.Chen et al. found that mitochondrial damage was alleviated through the regulation of Nrf229.Just as we have studied, mitochondrial damage was more severe in Nrf2−/− mice, manifested by significantly reduced Hsp60 and Mfn2 protein expression and a pronounced decrease in mitochondrial membrane potential.In addition, mitophagy plays a vital role in controlling mitochondrial quality and intracellular environment stability. Damaged mitochondria are selectively engulfed by autophagosomes and degraded by lysosomes30,31. Mitophagy exerts protective effects in some fibrotic diseases32, and its inhibition, leading to failed clearance of damaged mitochondria, may explain the negative correlation between Nrf2 and mammary fibrosis. Chang et al. found that Nrf2 can induce mitophagy in acute lung injury models to alleviate injury33. suggesting Nrf2 may mitigate TGF-β1-induced mammary fibrosis by inducing mitophagy. The PINK1-Parkin pathway primarily mediates mitophagy. Prohibitin 2 (PHB2) acts as a mondrial inner membrane receptor recruiting LC3 (during which cytosolic LC3-I is lipidated to membrane-bound LC3-II)34,which then binds to p62 for selective autophagic degradation35. Our results indicate that mitophagy was inhibited following Nrf2 knockout or inhibition. This conclusion was further verified in vitro.

Accumulating evidence identifies damaged mitochondria as a major source of excessive ROS accumulation36,37.Mitochondrial damage induces substantial ROS production; excessive ROS damages cellular proteins, DNA, and other macromolecules, disrupts homeostasis, exacerbates mitochondrial dysfunction, and can ultimately lead to cell death. We found that Nrf2 deficiency significantly increased ROS levels both in vivo and in vitro compared to controls. ROS accumulation is closely linked to fibrosis progression38. In liver fibrosis, ROS activate hepatic stellate cells, increasing excessive ECM deposition and ultimately leading to fibrosis39,40,41. Therefore, we further explored the effects of ROS on fibrosis and the TGF/Smad pathway by adding the ROS scavenger NAC in vivo and in vitro. These results indicate that Nrf2 loss increases ROS production, thereby activating the TGF/Smad signaling pathway and promoting mammary fibrosis in mice. Inhibiting TGF/Smad activation by removing ROS significantly alleviates mammary fibrosis development. This study has some limitations: due to time constraints, we did not overexpress Nrf2 to confirm specificity, and whether the observed effects are specific to mammary tissue or applicable to other organs remains unclear. These aspects warrant further investigation.

In summary, Nrf2 alleviates mammary fibrosis in mice primarily by regulating mitophagy and mitigating mitochondrial damage, thereby reducing ROS accumulation. Additionally, Nrf2 reduces TGF/Smad pathway activation by inhibiting ROS accumulation, preventing fibrosis progression. This study provides experimental evidence supporting Nrf2 as a potential therapeutic target for mammary fibrosis treatment.