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. 2025 Feb 7;15:4567. doi: 10.1038/s41598-025-88869-6

3,5,6,7,8,3’,4’- Heptamethoxyflavonoid inhibits TGF-β1-induced epithelial–mesenchymal transition by regulating oxidative stress and autophagy through MEK/ERK/PI3K/AKT/mTOR signaling pathway

Yiting Wang 1,2,#, Zhiheng Ma 1,2,#, Weiwen Peng 1,2, Qinglian Yu 1,2, Wenjie Liang 1,2, Liu Cao 1,2, Zhuqiang Wang 1,2,✉,#
PMCID: PMC11802913  PMID: 39915543

Abstract

Epithelial–mesenchymal transition (EMT) is a crucial pathological process in the pathogenesis of fibrosis. 3,5,6,7,8,3′,4′-hepmethoxyflavone (HMF), the main active ingredient extracted from the Chinese herb Breynia fruticosa (L.) Hook. f., has been shown to have beneficial effects on regulating apoptosis and inhibiting collagen deposition. However, it remains unclear whether and how HMF alleviates transforming growth factor-β1 (TGF-β1)-induced EMT. The objective of this study was to investigate the impact of HMF on TGF-β1-induced EMT in human alveolar Type II epithelial cells (A549) and its underlying mechanism. In vitro culture of TGF-β1-induced EMT in A549 cells revealed that HMF reduced cell viability and migration, inhibited collagen deposition, decreased expression levels of mesenchymal cell markers and fibrosis markers α-SMA, MMP2, TIMP1, β-catenin, and Snail. Meanwhile, the expression level of E-cadherin increased as an epithelial cell marker. Additionally, we discussed the effects of HMF on oxidative stress and autophagy. Various experiments confirmed that HMF regulated the expression levels of Nrf2, keap-1, HO-1, ROS, MDA, SOD, GSH, and played a role in reducing oxidative stress. At the same time, HMF significantly activated autophagy by increasing expressions of Beclin-1 and LC3B as well as enhancing autophagosome content. The addition 3-MA, an autophagy inhibitor attenuated these beneficial effects. Furthermore, HMF significantly inhibited phosphorylation levels of MEK, ERK, PI3K, AKT, and mTOR through various pathways. In conclusion, HMF effectively inhibits TGF-β1-induced EMT in A549 cells by targeting the MEK/ERK/PI3K/AKT/mTOR signaling pathway. Moreover, it exhibits a close correlation with the suppression of oxidative stress and induction of autophagy.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-88869-6.

Keywords: 3,5,6,7,8,3′,4′-Heptamethoxyflavonoid; Pulmonary fibrosis; Oxidative stress; Autophagy; Epithelial–mesenchymal transition

Subject terms: Molecular biology, Diseases

Introduction

The epithelial–mesenchymal transition (EMT) is a crucial pathological process in organ fibrosis, characterized by the conversion of epithelial cells to mesenchymal cells through the activation of multiple signaling pathways. This results in alterations in cell morphology, suppression of epithelial characteristics, and acquisition of properties such as increased cell motility and immune function1. Hypoxia, reactive oxygen species (ROS), and transforming growth factor-β1 (TGF-β1) can induce EMT in various types of epithelial cells, including alveolar epithelial cells and renal tubular epithelial cells2,3. Among them, TGF-β1 is significantly upregulated in patient-derived tissues such as pulmonary fibrosis, liver fibrosis, and renal fibrosis. It is primarily secreted by epithelial cells, fibroblasts, and macrophages. TGF-β1 is considered the most potent inducer of EMT during organ fibrosis progression. Treatment with TGF-β1 leads to a transition from an epithelial morphology to a mesenchymal cell phenotype (myofibroblasts) in alveolar epithelial cells (such as AECs or A549 cells) and liver stellate cells (HSCs)4,5. EMT is characterized by downregulation of adhesion molecules like E-cadherin and upregulation of mesenchymal markers such as α-SMA and Snail. While this process plays a role in development and tissue repair under normal circumstances it becomes dysregulated during organ fibrosis where sustained EMT occurs leading to extracellular matrix deposition that ultimately increases fiber formation6. Therefore targeting EMT represents a potential therapeutic approach for treating organ fibrosis.

Oxidative stress refers to the excessive production of highly reactive molecules, such as reactive oxygen species (ROS), in the body that surpasses its capacity for clearance, resulting in an imbalance between the oxidative and antioxidant systems. The regulation of EMT process involves oxidative stress-induced activation of TGF-β1 expression, with HO-1 acting as a mediator within the TGF-β signaling pathway during EMT7. Studies have demonstrated elevated levels of ROS, superoxide dismutase (SOD), and glutathione (GSH) in tissues associated with various organ fibrosis patients, indicating an imbalance between antioxidants and oxidants8. Crucial antioxidant enzymes like glutathione and superoxide dismutase play a vital role in defending against oxidative damage. Disruption of oxidative homeostasis can lead to abnormal manifestations including inflammation and metabolic disorders, ultimately culminating in tissue and organ fibrosis. The increase in malondialdehyde (MDA) level directly reflects the extent of damage caused by oxygen free radicals9. Furthermore, under normal circumstances, nuclear factor erythroid 2-related factor 2 (Nrf2) localizes to the cytoplasm through interaction with Kelch-like ECH-associated protein 1 (Keap-1). However, exposure to stressful signals induces Nrf2 dissociation from Keap-1 followed by translocation into the nucleus where it upregulates numerous antioxidant genes10. These findings indicate the crucial role of Nrf2 in activating the antioxidant defense system.

Autophagy is a highly conserved cellular process that maintains cellular homeostasis by degrading internal components. Dysregulation of autophagy has been implicated in the pathogenesis of various diseases, including myocardial fibrosis, liver fibrosis, emphysema, and pulmonary fibrosis11–13. Previous studies have demonstrated inhibited autophagy activity in lung tissue with pulmonary fibrosis, characterized by down-regulation of Beclin-1 and LC3B proteins and increased formation of autophagosomes14. Autophagy in hepatic stellate cells (HSCs) can promote the progression of liver fibrosis. Studies have shown that knockout of Atg7 to inhibit HSC autophagy can reduce stromal accumulation and fibrosis15. Beclin-1 and LC3B serve as key markers for assessing autophagic processes16. Beclin-1, a constituent of the class III PI3K complex, plays a pivotal role in initiating autophagosome formation and has been identified as the foremost gene mediating autophagy in mammals. Additionally, it exerts anti-tumor and anti-infective functions. Upregulation of the Beclin1 gene indicates activation of cellular autophagy17. LC3B exists in two forms: cytoplasmic (LC3B-I) and membranous (LC3B-II) isoforms. During autophagy, LC3B-I undergoes transformation into the LC3B-II isoform to facilitate the formation of autophagosome membranes. The expression level of LC3B-II positively correlates with levels of autophagic activity18.

The distinctive characteristic of traditional Chinese medicine and its active components lies in their ability to target multiple pathways, thereby providing comprehensive therapeutic effects for the management of chronic diseases. Traditional Chinese medicine has garnered increasing attention for its preventive and therapeutic effects on fibrosis. 3,5,6,7,8,3′,4′-heptemthoxyflavone (HMF), a lipophilic polymethoxyflavone isolated from Breynia fruticosa (L.) Hook. f19. , exhibits diverse pharmacological activities including anti-inflammatory, anti-apoptotic, and lipid-lowering effects20,21. Importantly, HMF has been shown to inhibit collagenase activity in UV-induced human dermal fibroblast cells (HDFn) as well as modulate the MAPK and TGF-β/Smad signaling pathways to regulate collagen content22. The pathological hallmark of organ fibrosis is an imbalance in extracellular matrix caused by abnormal collagen deposition23. Therefore, we hypothesized that HMF could inhibit TGF-β1-induced EMT and thus prevent excessive extracellular matrix deposition.

However, the specific involvement of HMF in TGF-β1-induced EMT in alveolar epithelial cells remains to be elucidated. The objective of this study was to investigate the direct impact of HMF on TGF-β1-induced EMT in A549 cells and explore potential underlying molecular mechanisms.

Materials and methods

Reagents

HMF used in this study was of 99.4% purity and was obtained from Cuiyirun, Sichuan, China. TGF-β1 was obtained from PEPROTECH, NJ, USA, and 3-Methylamphetamine (3-MA) was sourced from MedChemExpress, TX, USA.

Cell culture and treatment

A549 cell line was purchased from Fuheng Biotechnology Co., Ltd., Shanghai, China. The A549 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Procell, Wuhan, China), 100 U/mL penicillin, and 100 µg/mL streptomycin (GenXion, Guangzhou, China). The cells were cultured at 37 °C in a humid atmosphere with 5% CO2.

TGF-β1 was used to induce EMT in epithelial cells24. HMF intervention methods: The cells were assigned into four groups: control group, TGF-β1 (10 ng/mL) induced PF group, and TGF-β1 (10 ng/mL) induced PF groups with 12.5 and 25 µM HMF. In the control group, the cells were exchanged for fresh culture medium. In the HMF intervention groups, the cells were cultured in the presence of 10 ng/mL TGF-β1 and 12.5 and 25 µM HMF for 48 h.

3-MA intervention: The cells were pretreated with 60 µM 3-MA for 2 h, and then cultured with 10 ng/mL TGF-β1 and 25 µM HMF for 48 h.

Drug preparation and administration

The HMF was prepared as 12.5 and 25 mM stock solutions in DMSO, filtered, sterilized, and stored at −20 °C. TGF-β1 was dissolved to a concentration of 100 µg/mL in PBS with 5% BSA and stored at −80 °C. Both stock solutions were diluted to working concentrations in RPMI-1640 medium and added to cell culture dishes for assays.

Cell viability

A549 cells were seeded at a density of 6000 cells/well in 96-well plates and incubated overnight. They were then exposed to 10 ng/mL TGF-β1 along with varying concentrations of HMF (0, 12.5, 25, 50, 100, 200, and 400 µM) for a duration of 48 h. Cell viability was determined by adding CCK-8 reagent (10 µL/well) (Betotime, Shanghai, China) and incubating for an additional 2 h, after which the optical density was measured at 450 nm using a microplate reader (VICTOR Nivo, PerkinElmer, MA, USA). The cell viability was calculated by the following formula: cell viability = (A of the control group-A of the experimental group)/(A of the control group-A of the blank group) × 100%.

Cellular morphology analysis

A549 cells were seeded at a density of 1 × 105 cells/well in 6-well plates and incubated for 24 h. After replacing the medium, the HMF intervention groups were treated with TGF-β1 and HMF at concentrations of 12.5 or 25 µM. Following a 48-h incubation period, cell morphology was observed and imaged using an inverted microscope (40×) (ECLIPSE Ti2, Nikon, Japan).

Wound-healing assay

Equal numbers of A549 cells were seeded in 6-well plates. When the cell confluence reached 80%, two scratch wounds were created in each well using a sterile P-200 pipette tip. The wells were then washed three times with precooled PBS to remove the detached cells. Subsequently, the cells were treated with HMF and TGF-β1 according to the experimental design. Photomicrographs were taken at 0, 24, and 48 h after generating the wounds. The wound area was determined using Image J software (https://imagej.net). The ratio of wound closure was calculated using the following formula: ratio of wound closure (%) = (Initial scratch area − final scratch area)/Initial scratch area × 100.

Immunofluorescence analysis

A549 cells were seeded at a density of 1 × 104 cells/well in 24-well plates and treated with HMF and TGF-β1 after attachment. After incubating for 48 h, the cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100. Following blocking with goat serum, the cells were incubated overnight at 4 °C with primary antibodies. Then they were incubated for 1 h at room temperature with secondary antibody. The cells were stained with DAPI (1:100, Betotime Shanghai China), washed, and sealed using an anti-fluorescence quenching solution (Betotime Shanghai China). Images were captured at a magnification of 200× and the relative fluorescence intensity was quantified using ImageJ according to the formula: sum of fluorescence intensity in the region (IntDen)/Area. Supplementary Table 1 contained a list of the used antibodies.

MDC staining

A549 cells were seeded at a density of 1 × 105 cells/well in 6-well plates and treated with HMF and TGF-β1 after adhesion. After 48 h, the cells were stained using the MDC reagent kit (G0170; Solarbio, Beijing, China), washed, and observed under a fluorescence microscope at a magnification of 100× for imaging.

ROS detection

The A549 cells were seeded at a density of 2 × 105 cells/well in 6-well plates and treated with HMF and TGF-β1. After 48 h, the levels of ROS were determined using a ROS assay kit (S0033S; Betotime, Shanghai, China). The cells were incubated with 10 µM DCFH-DA diluted in serum-free culture medium, washed, trypsinized, and collected. After centrifugation, they were resuspended and analyzed for average fluorescence intensity using a flow cytometer (CytoFLEX, Beckman Coulter, CA, USA).

Determination of of MDA, GDH, and SOD contents

The A549 cells were seeded at a density of 2 × 105 cells/well in 6-well plates, treated with HMF and TGF-β1, and incubated for 48 h. Ripa lysis solution was then added, and the cells were lysed on ice. Supernatants were collected, and the levels of MDA (S0131; Betotime, Shanghai, China), GSH (S0057S; Betotime, Shanghai, China), and SOD (A001-3; Nanjing Jiancheng, Nanjing, China) were measured using specific reagent kits.

Western blot

Cells were lysed with RIPA solution on ice for 30 min, then centrifuged at 12,000 rpm at 4 °C for 15 min using a high-speed refrigerated centrifuge (5910 Ri, Eppendorf, HAMBURG, GER). Total protein was extracted and quantified using the BCA kit (Betotime, Shanghai, China). Equal amounts of protein were loaded onto 8% or 12% SDS-PAGE gels, electrophoresed, and transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked and incubated with various primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at room temperature for 1 h. Bands were visualized with ECL reagent (Affinty Ohio USA) and quantified using ImageJ software. Supplementary Table 1 contained a list of the used antibodies.

Statistical analyses

The experiments were conducted in three independent replicates. GraphPad Prism version 9.4.1 software (https://www.graphpad.com/updates) was used for graph preparation, and SPSS version 26.0 software (http://www.spss.com.cn) was used for data analysis. All data are expressed as medians with interquartile ranges. The Kruskal–Wallis test was used to compare overall differences, and the Mann–Whitney U test was used to compare pairwise differences between groups. A P-value of less than 0.05 was considered statistically significant.

Results

HMF effectively inhibits the migration and activation of A549 cells

The chemical structure of HMF is presented in Fig. 1A. Cell toxicity and viability were assessed using the CCK8 assay to determine the optimal treatment concentration. The results showed no significant differences in cell viability among the groups treated with various concentrations of HMF (12.5, 25, and 50 µM) compared to the control group. However, significant differences were observed between the groups treated with 100, 200, and 400 µM HMF and the control group (P < 0.05). From a concentration of 12.5 µM HMF onwards, cell activity was significantly reduced compared to the TGF-β1 group (P < 0.05) (Fig. 1B, C). Consequently, subsequent experiments were conducted using concentrations of 12.5 µM and 25 µM HMF. TGF-β1 promotes EMT during the progression of pulmonary fibrosis. The TGF-β1 group exhibited significant cell thinning and elongation compared to the control group, indicating changes in fibroblast morphology and a transition towards mesenchymal cell morphology. In contrast, cells in the HMF treatment groups gradually transitioned towards a spindle shape resembling that of the control group when compared to the TGF-β1 group (Fig. 1D). The migration of alveolar epithelial cells is closely associated with EMT and ECM accumulation. The findings revealed that after 24 and 48 h of intervention, cells from the TGF-β1 group migrated towards the center of the wound with a substantial reduction in wound width compared to the control group. However, the HMF treatment groups exhibited significantly slower cell movement speed, widening of the wound gap, and decreased cell migration rate compared to those in the TGF-β1 group (P < 0.05) (Fig. 1E, F).

Fig. 1.

Fig. 1

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HMF inhibits the activity and migration of A549 cells. A549 cells were treated with TGF-β1 (10 ng/mL) for 48 h in the presence or absence of HMF. (A) The chemical structural formula of HMF. (B) The cck8 method was used to detect the effects of different concentrations of HMF on cell viability. (C) The cck8 method for detecting the effect of different concentrations of HMF on TGF-β1-induced cell viability. (D) The effect of HMF on cell morphology. (E) Detection of HMF (12.5, 25 µM) through wound healing analysis of the impact on cell migration. (F) Image J software was used to quantify the area of the wound (the area between two lines). Scale: 500 μm. Kruskal–Wallis test, #control vs. TGF-β1, P < 0.05; *TGF-β1 vs. HMF, P < 0.05.

HMF regulates the expression of EMT and ECM-related proteins in A549 cells

Western blot analysis was performed to assess the levels of E-cadherin, β-catenin, and Snail in A549 cells to explore whether HMF can inhibit the EMT process in these cells. The results demonstrated that upon TGF-β1 induction, HMF significantly downregulated the expression of β-catenin and Snail but upregulated the expression of E-cadherin in A549 cells (P < 0.05) (Fig. 2A–D). It is worth noting that ECM deposition plays a crucial role in fibrosis occurrence and development. In this study, compared with the control group, the expression levels of MMP2, collagen I, and α-SMA proteins were significantly up-regulated in the TGF-β1 group (P < 0.05), while they were significantly down-regulated in the HMF groups compared with the TGF-β1 group (P < 0.05). The expression level of TIMP1 protein did not show significant change. Additionally, after HMF intervention, there was a significant downregulation in MMP2/TIMP1 expression level which could balance MMPs/TIMPs ratio and inhibit extracellular matrix deposition (P < 0.05) (Fig. 2E–H). The original blots are presented in Supplementary Fig. 1. Immunofluorescence staining showed a significant increase in collagen-I and α-SMA expression in A549 cells induced by TGF-β1. However, HMF treatment effectively reduced average immunofluorescence intensity in A549 cells after TGF-β1 induction (Fig. 2I–K).

Fig. 2.

Fig. 2

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HMF inhibits the expression of EMT and fibrosis-related proteins in A549 cells. (A) Protein levels of E-cadherin, β-catenin, and Snail were measured with Western blotting, respectively. (B–D) E-cadherin, β-catenin, and Snail protein levels were quantified by grayscale value, and Image J software was used. (E) Protein levels of collagen-I, α-SMA, MMP2, and TIMP1 were measured with Western blotting, respectively. (F–H) Collagen-I, α-SMA, and MMP2/TIMP1 protein levels were quantified by grayscale value, and Image J software was used. (I) Immunofluorescence assay was used to detect the expression of collagen-I (green) and α-SMA (green). The nucleus is stained with DAPI (blue). Scale: 50 μm. (J–K) Average immunofluorescence intensity of collagen-I and α-SMA. GAPDH or β-Actin as a loading control. Kruskal–Wallis test, #control vs. TGF-β1, P < 0.05; *TGF-β1 vs. HMF, P < 0.05.

HMF alleviates oxidative stress

The levels of antioxidant enzymes, oxidase enzyme and malondialdehyde (MDA) were assessed to determine their involvement in the regulation of EMT process by activating TGF-β1 expression, as previous studies have shown that oxidative stress plays a role in this process. In the TGF-β1 group, there was a significant decrease in GSH and SOD levels (P < 0.05), but a notable increase in MDA level compared to the control group (P < 0.05). Conversely, treatment with HMF resulted in a significant upregulation of GSH and SOD levels (P < 0.05), but a substantial downregulation of MDA level (P < 0.05) compared to the TGF-β1 group (Fig. 3A–C). ROS content was determined using flow cytometry, which revealed that HMF treatment effectively reduced ROS levels in A549 cells (P < 0.05) (Fig. 3D, E). Furthermore, analysis of the expression levels of oxidative stress-related proteins showed that HMF treatment significantly upregulated Nrf2 and HO-1 antioxidant proteins (P < 0.05), but downregulated keap-1 prooxidative protein expression compared to the TGF-β1 group (P < 0.05) (Fig. 3F–I). The original blots are presented in Supplementary Fig. 1. These findings indicate that HMF potentially alleviates EMT progression in A549 cells by inhibiting oxidative stress.

Fig. 3.

Fig. 3

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HMF inhibits oxidative stress. (A) The content of SOD in A549 cells. (B) The content of GSH in A549 cells. (C) The content of MDA in A549 cells. (D,E) Flow cytometry was used to detect the content of ROS in A549 cells. (F) Protein levels of Nrf2, keap-1, and HO-1 were measured with Western blotting, respectively. (G–I) Nrf2, keap-1, and HO-1 protein levels were quantified by grayscale value, and Image J software was used. β-Actin as a loading control. Kruskal–Wallis test, #control vs. TGF-β1, P < 0.05; *TGF-β1 vs. HMF, P < 0.05.

HMF induces autophagy activation

Previous studies have demonstrated an association between autophagy dysfunction and the progression of fibrosis. Therefore, we assessed the expression levels of key autophagy markers, including LC3B and Beclin-1 proteins. The results revealed that HMF treatment significantly upregulated the expression of LC3B and Beclin-1 proteins (P < 0.05) (Fig. 4A–C). The original blots are presented in Supplementary Fig. 1. MDC staining confirmed the activation of autophagy by HMF. Notably, TGF-β1 treatment induced the formation of a small number of autophagosomes in A549 cells, but HMF treatment significantly increased their number (Fig. 4D). These findings suggest that HMF potentially inhibits the EMT process in A549 cells by activating autophagy. We used 3-MA as an autophagy inhibitor that interferes with early stages of autophagosome formation by inhibiting class III PI3K to explore whether HMF inhibits EMT in alveolar epithelial cells by activating autophagy. Notably, pretreatment of the cells with 3-MA reversed the effect of HMF, leading to a significant reduction in immunofluorescence intensity of LC3B protein (P < 0.05) (Fig. 5D, E), downregulation in Beclin-1 protein expression (P < 0.05), and upregulation in α-SMA protein expression (P < 0.05) (Fig. 5A–C). The original blots are presented in Supplementary Fig. 1. Additionally, pretreatment with 3-MA significantly reduced the number of autophagosomes in these cells (Fig. 5F).

Fig. 4.

Fig. 4

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HMF activates autophagy in A549 cells. (A) Protein levels of Beclin-1 and LC3B II/I were measured with Western blotting, respectively. (B,C) Beclin-1 and LC3B II/I protein levels were quantified by grayscale value, and Image J software was used. (D) Effect of HMF on autophagosome content in MDC staining. The red arrows are typical autophagosomes. Scale: 50 μm. β-Actin as a loading control. Kruskal–Wallis test, #control vs. TGF-β1, P < 0.05; *TGF-β1 vs. HMF, P < 0.05.

Fig. 5.

Fig. 5

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Autophagy inhibitor 3-MA can reverse the anti-pulmonary fibrosis effect of HMF. Pretreat with or without 3-MA for 2 h, then coculture HMF at a concentration of 25 µM with TGF-β1 at a concentration of 10 ng/mL for 48 h. (A) Protein levels of Beclin-1 and α-SMA were measured with Western blotting, respectively. (B,C) Beclin-1 and α-SMA protein levels were quantified by grayscale value, and ImageJ software was used. (D) Immunofluorescence assay was used to detect the expression of LC3B (green). The nucleus is stained with DAPI (blue). Scale: 50 μm. (E) Average immunofluorescence intensity of LC3B. (F) Effect of HMF on autophagosome content in MDC staining. The red arrows are typical autophagosomes. Scale: 100 μm. β-Actin as a loading control. Kruskal–Wallis test, #control vs. TGF-β1, P < 0.05; &HMF vs. HMF + 3-MA, P < 0.05.

HMF effectively inhibits the PI3K/AKT/MEK/ERK/mTOR signaling pathways

Western blot analysis was performed to assess the expression levels of p-PI3K, p-AKT, p-MEK, p-ERK, IKKα, and p-mTOR proteins in order to explore whether HMF activates autophagy and suppresses the EMT process in A549 cells through pathways associated with these proteins. TGF-β1-treated A549 cells exhibited upregulated expression of p-PI3K, p-AKT, p-MEK, p-ERK, IKKα, and p-mTOR proteins compared to the control group. However, treatment of the cells with HMF significantly inhibited the expression of these proteins (P < 0.05) (Fig. 6A–G). The original blots are presented in Supplementary Fig. 1. Immunofluorescence staining showed that the positive expression of p-mTOR in A549 cells induced by TGF-β1 increased significantly. Nevertheless, administration of HMF reduced the average immunofluorescence intensity of p-mTOR (P < 0.05) (Fig. 6H, I). Notably, pretreatment with 3-MA reversed the effect of HMF and substantially increased the immunofluorescence intensity of p-mTOR (Fig. 6J, K). Finally, we created a schematic chart illustrating the potential mechanism underlying the therapeutic role of HMF in TGF-β1-induced EMT in A549 cells as displayed in Fig. 7.

Fig. 6.

Fig. 6

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HMF inhibits PI3K/AKT/MEK/ERK/mTOR signaling pathways. (A) Protein levels of p-mTOR/mTOR, p-PI3K/PI3K, p-AKT/AKT, p-MEK/MEK, p-ERK/ERK, and Ikkα measured with Western blotting, respectively. (B–G) p-mTOR/mTOR, p-PI3K/PI3K, p-AKT/AKT, p-MEK/MEK, p-ERK/ERK, and Ikkα protein levels were quantified by grayscale value, and Image J software was used. (H) Immunofluorescence assay was used to detect the expression of p-mTOR (green). The nucleus is stained with DAPI (blue). (I) Average immunofluorescence intensity of p-mTOR. (J) An immunofluorescence assay was used to detect the expression of p-mTOR (green) following treatment of 3-MA. The nucleus is stained with DAPI (blue). (K) Average immunofluorescence intensity of p-mTOR with the treatment of 3-MA. Scale: 50 μm. β-Actin as a loading control. Kruskal–Wallis test, #control vs. TGF-β1, P < 0.05; *TGF-β1 vs. HMF, P < 0.05; &HMF vs. HMF + 3-MA, P < 0.05.

Fig. 7.

Fig. 7

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HMF reduces TGF-β1-induced EMT in A549 cells by inhibiting oxidative stress and activating autophagy. HMF inhibits the imbalance of MMPs/TIMPs induced by TGF-β1, reducing the content of ROS, inhibiting the production of oxidative stress, and then activating autophagy. HMF inhibits EMT and alleviates abnormal repair of collagen through signaling pathways such as MEK/ERK, PI3K/AKT, etc. This figure was created with BioRender.com.

Discussion

EMT has long been regarded as a mechanism underlying fibrotic diseases2. In this study, we investigated the impact of HMF on EMT in A549 cells. The findings demonstrate that HMF effectively suppresses TGF-β1-induced activation of alveolar epithelial cells and inhibits EMT in vitro. Moreover, the results indicate that HMF exerts its effects through various pathways, including attenuation of oxidative stress, promotion of autophagy activation, and inhibition of phosphorylation levels of proteins involved in PI3K, AKT, MEK, and ERK signaling cascades to impede EMT progression in A549 cells. Traditional Chinese medicine derived from plants has gained significant attention due to its diverse pharmacological activities and minimal side effects. HMF is the primary active compound found in Breynia fruticosa (L.) Hook. f. and classified as a polymethoxylated flavonoid (PMF), exhibiting several potent bioactive properties25. A previous study demonstrated that HMF effectively alleviates hyperlipidemia and lipid levels in mice fed a high-fat diet20. Furthermore, HMF inhibits collagenase activity in UV-induced human dermal fibroblasts HDFn and modulates the MAPK and TGF/Smad signaling pathways to regulate collagen content22. Abnormal collagen deposition is a key pathogenic mechanism underlying fibrosis characterized by the activation of epithelial cells by TGF-β1. In this current study, the results demonstrate that stimulation with TGF-β1 upregulates the expression of fibrosis-related proteins leading to an imbalance between MMPs/TIMPs. However, treatment with HMF downregulated α-SMA and collagen-I expression in TGF-β1-induced A549 cells inhibited collagenase activity and reduced MMP2/TIMP1 expression levels. Consequently, restoring balance between MMPs/TIMPs may exert anti-fibrotic effects.

Oxidative stress arises from an imbalance between the body’s oxidants and antioxidants, leading to cellular stress. Elevated levels of oxidative products trigger the activation of inflammatory cells, resulting in the generation of free radicals such as ROS. Excessive intracellular ROS levels can induce oxidative stress and modulate gene expression, cytoskeletal rearrangement, and cell migration in epithelial cells, which are crucial for TGF-β1-induced EMT26. Furthermore, an imbalance in MMPs/TIMPs contributes to oxidative stress, causing varying degrees of lung damage27,28. Previous findings suggest that oxidative stress plays a role in the pathogenesis of organ fibrosis. Consequently, reducing ROS levels and maintaining redox balance represent promising therapeutic strategies for managing organ fibrosis29. Therefore, we investigated the extent of HMF treatment-induced oxidative stress. Exposure of A549 cells to TGF-β1 resulted in significant levels of oxidative stress characterized by increased MDA and ROS contents, decreased GSH and SOD levels, as well as reduced expression levels of Nrf2 and HO-1 proteins. Conversely, TGF-β1 induction upregulated keap-1 protein expression. Importantly, the results demonstrate that HMF effectively inhibits oxidative stress while simultaneously alleviating the EMT process.

Autophagy is a highly conserved cellular self-protective mechanism that facilitates the elimination of excessive or damaged organelles in organisms. This crucial process maintains a balanced protein metabolism and intracellular environmental homeostasis. Notably, defects in autophagy play a significant role in the pathogenesis and progression of various fibrotic diseases30. Recent research revealed abrogation of the autophagy pathway of pulmonary epithelial cells among organ fibrosis patients, impairing the regulation of collagen degradation and autophagy-associated cell death, ultimately leading to the development of organ fibrosis31,32. These findings demonstrate a strong association between dysregulation of autophagy and the pathogenesis of organ fibrosis, highlighting the need to evaluate autophagy levels during HMF treatment. Our study revealed dysregulation of autophagy in TGF-β1-induced A549 cells, characterized by decreased autophagosomes and the expression of LC3B II/I and Beclin-1. Notably, HMF effectively activated autophagy and inhibited EMT. Furthermore, we observed that the protective effect of HMF was attenuated by the autophagy inhibitor 3-MA. These findings indicate the ability of HMF to activate autophagy and abrogate the EMT process in alveolar epithelial cells.

The MEK/ERK signaling pathway is a widely activated MAPK pathway that facilitates extracellular signal transduction through cell membrane receptors, thereby regulating the expression of specific intracellular proteins and controlling various cellular functions such as proliferation and differentiation33. PI3K is an intracellular phosphatidylinositol kinase that mediates the translocation of Akt from the cytoplasm to the plasma membrane, leading to downstream protein phosphorylation and subsequent promotion of abnormal ECM deposition and EMT. AKT, a direct downstream target protein of PI3K, can activate mTOR, thereby regulating cell proliferation and metabolism while promoting fibrosis34. mTOR is a highly conserved kinase and key regulator of autophagy; it exhibits inhibitory effects on autophagy when activated but induces autophagy when inactivated35,36. Activation of the MEK/ERK signaling pathway indirectly triggers the PI3K/AKT/mTOR signaling pathway, resulting in inhibition of autophagy37. Researchers have reported that inhibition of the ERK signaling pathway can lead to downregulation of Snail-1 expression, thereby impeding epithelial–mesenchymal transition (EMT) and the progression of fibrosis38. Furthermore, previous studies have demonstrated that suppression of the PI3K/AKT/mTOR signaling pathway alleviates pulmonary fibrosis in a bleomycin-induced PF model39. Importantly, both the PI3K/AKT/mTOR and MEK/ERK signaling pathways play pivotal roles in the pathogenesis of organ fibrosis. Inhibition of these pathways attenuates fibrosis and represents significant downstream signaling cascades regulated by TGF-β140. Our study revealed that administration of HMF effectively reduced the phosphorylation levels of PI3K, AKT, MEK, ERK, and mTOR in TGF-β1-induced A549 cells. Furthermore, we observed that the autophagy inhibitor 3-MA decreased mTOR phosphorylation levels and inhibited mTOR-associated signaling pathways. These findings strongly suggest that the protective effect of HMF against EMT may be attributed to its ability to activate autophagy by inhibiting the MEK/ERK/PI3K/AKT/mTOR signaling pathways.

TGF-β1 is a pivotal regulatory factor in various organ fibrotic lesions, and its markedly elevated expression level is closely associated with the onset and progression of organ fibrosis. While preliminary studies have explored the effects of HMF on TGF-β1-induced EMT in alveolar epithelial cells, the impact of HMF on EMT in renal and hepatic epithelial cells remains largely unexplored. This study is currently confined to the A549 cell line, which originates from human lung adenocarcinoma and may not fully represent pathways and processes observed in primary cells or in vivo models. Furthermore, our investigation has not yet extended to mouse fibrosis models, thereby limiting a comprehensive understanding of the EMT process and the in vivo mechanisms of HMF action. Additionally, research on the intricate interplay between oxidative stress and autophagy in fibrosis is relatively limited. Oxidative stress is considered a critical factor in promoting fibrosis, while autophagy plays an essential role in maintaining cellular homeostasis and responding to stressors. Therefore, we aim to further investigate the interaction between oxidative stress and autophagy using animal models in future studies, and extend our examination to primary alveolar epithelial cells, as well as explore the EMT process and the role of HMF in mice, to gain a more thorough understanding of the relevant biological processes and provide novel therapeutic strategies for fibrotic lesions.

Conclusion

In conclusion, the findings demonstrate that HMF effectively inhibits the EMT process in alveolar epithelial cells through suppression of the MEK/ERK/PI3K/AKT/mTOR signaling pathways, attenuation of oxidative stress, and induction of autophagy.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (6.6MB, pdf)

Acknowledgements

The authors express their gratitude for the support provided by the National Demonstration Pilot Project for the Inheritance and Development of Traditional Chinese Medicine, as well as for the drawing materials provided by BioRender.

Author contributions

Zhuqiang Wang conceived and guided the research. Yiting Wang and Zhiheng Ma performed the experiment and data analyses and wrote the manuscript. Weiwen Peng, Qinglian Yu, Wenjie Liang and Liu Cao helped perform the analysis with constructive discusions. All the authors read, critically evaluated, gave their feedback, and edited the manuscript.

Funding

This research was supported by funds from the Science and Technology Project of Zhongshan City, Guangdong Province (No. 2020B3007).

Data availability

The datasets used and analyzed in the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yiting Wang and Zhiheng Ma contributed equally.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (6.6MB, pdf)

Data Availability Statement

The datasets used and analyzed in the current study are available from the corresponding author on reasonable request.

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