Dihydroquercetin (DHQ) (Cat#480-18-2, purity above 98 percent) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Erastin (Cat#E7881) and rapamycin (Cat#53123-88-9) were purchased from Sigma Aldrich (Billerica, MA, United States). The SiO₂ (Cat#S5631) particles (approximately 80% diameter: 1–5 μM) were purchased from Sigma-Aldrich and were filtered by sedimentation in accordance with the law of Stokes, subjected to acidic hydrolysis, and baked throughout the night (200°C, 16 h). The SiO₂ particles were prepared for use in both in vivo and in vitro experiments. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (#BS350B) as well as trypan blue exclusion assay kit (#BL707A) were obtained from Biosharp (Anhui, China). Antibodies against GPX4 (ab125066), ferritin heavy chain 1 (FTH1) (ab65080), NCOA4 (ab86707), and microtubule-related protein 1A/1B-light chain 3 (LC3) (ab192890) were all purchased from Abcam Technology (Abcam, Cambridge, United Kingdom). Antibodies against fibronectin (#26836), collagen I (#72026), alpha-smooth muscle actin (α-SMA) (#19245), and GAPDH (#5174), were acquired from Cell Signaling Technology Inc. (Danvers, MA, United States). A secondary antibody was obtained from Proteintech Biotechnology (Wuhan, China).

Eight-week-old male C57BL/6 mice were purchased from Hubei University of Medicine (Shiyan, China). The SiO₂-induced mouse pulmonary fibrosis model was performed. In brief, each group of mice was anesthetized with 1% pentobarbital sodium intraperitoneally at 40 mg/kg body weight and their tracheae had been surgically exposed. In addition, SiO₂ suspension (20 mg in 50 ul saline) was instilled in the mice. The vehicle control groups were given an equivalent amount of 0.9% sterile saline. After one week of acclimation, mice were divided randomly into four groups (n = 8 per group). Control group, SiO₂ group, SiO₂ and low dose of DHQ group (DHQ-L, 10 mg/kg) as well as large dose of DHQ group (DHQ-H, 50 mg/kg). One week later after SiO₂ administration, DHQ (10 mg/kg or 50 mg/kg) was orally administered to mice at dosages of 10 mg/kg or 50 mg/kg once daily consecutively for 14 days. Meanwhile, the control and the SiO₂ groups received an equal volum of 0.9% sterilized saline. On the day 21, mice were euthanized by intraperitoneal injection of an overdose of pentobarbital sodium. Blood samples from each group were collected for subsequent analysis. Lung tissues were excised and weighed, and the lung weight/body weight (LW/BW, mg/g) ratio was defined as the lung weight divided by the body weight. The left lower lobe lung samples were fixed in 10% formalin for histopathological analysis, and the remaining lung samples were stored at −80°C. All experiments were approved by the Institutional Animal Care and Use Committee of Hubei University of Medicine (Shiyan, China), and performed in accordance with the Provision and General Recommendation of the Chinese Experimental Animals Administration Legislation.

After the mice were sacrificed, lung tissues were collected quickly and fixed with 4% paraformaldehyde, dehydrated with alcohol gradient and then paraffin-embedded. Then the samples were cut into 5 μM sections and were stained for hematoxylin and eosin (H&E) or improved special masson trichrome. The pathological sections of lung tissue were observed and photographed under light microscope.

Sections were stained with hemotoxylin and eosin for the inflammation score evaluation and grading were classified according to the following criteria: none (0), no alveolitis; mild (1+), affected area<20%; moderate (2+), affected area ∼20–50%; severe (3+), affected area >50%. Fibrosis score was evaluated using masson’s trichrome stain sections. The severity of fibrosis in these sections was graded according to the following criteria: none (0), no evidence of fibrosis; mild (1+); lesion range of fibrosis<20% in the whole lung; moderate (2+), lesion range of fibrosis ∼20–50% in the whole lung; severe (3+), lesion range of fibrosis >50% in the whole lung.

The hydroxyproline (HYP) contents in the lung tissues were determined using an HYP kit (#KGT030-2, KeyGen Biotech, Nanjing, China), according to the manufacturer’s instructions. Frozen lungs (∼100 mg) were cut into pieces and then homogenized on ice. Lung tissues were hydrolyzed in 1 ml lysis buffer at 100°C for 20 min. The mixture was incubated at 60°C for 15 min, cooled at room temperature and centrifuged at 8,000 g for 10 min. The absorbance was measured at 550 nm and the expression were calculated as µg hyp per mg wet lung weight.

The levels of cytokines including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in serum and lung tissue homogenate were quantified by ELISA using the corresponding assay kit (#CSB-E08054m, #CSB-E04741m, #CSB-E04726m; Cusabio Biotech, Wuhan, China) following the manufacturer’s protocol. The optical density was measured at 450 nm for each cytokine. The minimum detection limit of IL-1β, TNF-α, and TGF-β was 7.8 pg/ml, 3.9 pg/ml and 0.2 ng/ml, respectively. The levels of the cytokines were obtained with a standard curve.

Human bronchial epithelial (HBE) cells and human fetal lung fibroblast MRC-5 cells were purchased from Procell (Wuhan, China) and cultured in DMEM supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin mixture. When reaching about 80% confluence, cells were passaged with 0.25% trypsin digestion, and plated in 12-well plates and cultured overnight. Subsequently, the medium was changed to fresh culture medium with 10% fetal bovine serum, and subjected to different treatments. A humidified 5% CO2 atmosphere was used to maintain all cell cultures at 37°C.

After overnight culture, HBE cells were incubated with SiO₂ (50 ug per mL medium) or DHQ (40 μM) alone and co-treated with a mixture of SiO₂, respectively. In the co-treatment, DHQ needs to act on HBE cells 1 h in advance and then mixed with SiO₂. HBE cells in normal culture were used as the negative control. 24 h later, the obtained supernatant was collected and filtered through a 0.22 μM disposable filter. Meanwhile, the HBE cells were washed twice with phosphate-buffered saline (PBS) and protein was extracted for further experiments. The collected conditioned media were mixed with an equal amount of complete culture medium and were used to treat MRC-5 cells for 48 h. Then, the total protein was extracted and cell climbing tablets were made for later experiments.

MTT assay was conducted to assess cell viability according to the manufacturer’s instructions. HBE cells (1×10⁵ cells/well) were cultured in 96-well plates with serum-free DMEM medium for 24 h at 37°C in a 5% CO₂ incubator. DHQ were diluted in PBS and then purified using a 0.22-µM pore size filter (Millex-GS; EMD Millipore). Then, the cells were incubated with 190 µl different concentrations of DHQ (0, 5, 10, 20, 40 or 80 µM). Following 24 h, the cells were incubated with 0.5% MTT at 37°C for 4 h. Subsequently, the supernatant was discarded and 150 µl dimethyl sulfoxide was added to each well for 10 min, and the plates were shaken on a horizontal shaker for 10 min. The absorbance of each sample was measured at 490 nm using a microplate reader.

HBE cells were seeded in the 12 well culture plate at 1 × 10⁵ cells/well. When the cells of each well reached approximately 70% confluence, cells were exposed with SiO₂ alone or in combination with DHQ (20 μM, 40μM, or 80 μM) for 48 h at 37°C with 5% CO₂. Following treatment, HBE cells were trypsinized and collected by centrifugation at 700 × g for 5 min at room temperature. Cell suspension (100 μl) was mixed with equal volumes of 0.4% trypan blue for 5 min at room temperature. The viable cell were counted using a hemocytometer under an inverted microscope. Trypan blue stained the dead cells blue while the viable cells remained clear.

Transmission electron microscopy (TEM) was used to examine the mitochondrial features of HBE cells (H-7000, Hitachi, Japan). For intracellular structure observation, The cells were collected and fixed with 2.5% glutaraldehyde overnight, and the morphology of the HBE cells was observed by TEM. Quantification analysis of transmission electron microscopy was determined by the image J software. Any straight line on the image can be defined by two pixels on each end, pixel 1 and pixel 2. Therefore, coordinates of pixels were annotated p1=(x1, y1) and p2=(x2, y2) for pixels 1 and 2. The mitochondria length was measured by applying the pythagorean theorem to the coordinate pixels. Thus, where Lp1, p2 denotes the euclidean distance between pixel 1 and pixel 2. Length was given by (Equation: L(p1+p2) = √(x1−x2) ² +(y1−y2) ² ).

The released iron, 4-HNE, and MDA level of HBE cell lysates were measured with iron, 4-HNE, and MDA assay kit (#ab83366, #ab118970, ab238538; Abcam) respectively following the directions of the manufacturer. The levels of ROS and GSH were assessed in HBE cells by the ROS assay kit (Beyotime, #S0033) and Glutathione assay kit (Beyotime, #S0053), and the guidelines stipulated by the manufacturer.

Trizol reagent (TAKARA, Japan) was utilized for the purpose of extracting total RNAs from the cell lines, and the PrimeScript™ RT Master Mix kit (TAKARA, Japan) was used to generate cDNAs from the total RNAs. The PCR was carried out utilizing the TB Green® Premix Ex Taq™ II kit (TAKARA, Japan) in accordance with the guidelines stipulated by the manufacturer. Supplementary Table S1 lists the primer sequences that were used in the present study. The conditions of PCR were shown as follows: 95°C for 5 min, then 40 cycles of denaturation at 95°C for 15 s, annealing 60°C for 30 s and extension at 72°C for 30 s. GAPDH served as the internal reference. The 2^−ΔΔCT method was used for calculation of the relative gene level.

The protein levels of collagen I, fibronectin and α-SMA, GPX4, FTH1, NCOA4 and LC3 were detected by western blotting. Total proteins extracted from lung homogenate or cell lysate were lysed in RIPA buffer (Beyotime, #P0013B). Total protein concentration was determined by BCA protein assay kit (Beyotime, #P0012) and 50 µg protein was separated by SDS-PAGE gel, and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, IPVH00010). The membrane was blocked for 2 h at room temperature in tris-buffered saline (TBS) (Sigma-aldrich, A9418), and then incubated with primary antibodies overnight at 4°C followed by detection with the HRP-labeled secondary antibodies and visualized using super ECL detection reagent (Beyotime, #P0018). The protein bands intensity was determined by the image J package and the relative protein levels were normalized to that of GAPDH.

For cell immunofluorescence, MRC-5 and HBE cells were fixed with 4% paraformaldehyde, washed with PBS, permeabilized with 0.2% Triton X-100 for 5 min at room temperature, and blocked with 1% bovine serum albumin for 1 h. MRC-5 and HBE cells were then incubated with primary antibodies against collagen-1, fibronectin, α-SMA, LC3 and GPX4 overnight at 4°C. After washing with PBS, cells were incubated with relative secondary antibody and counterstained with DAPI staining. The fluorescent intensity of target proteins was calculated using software image J.

All statistical analyses were performed by using the GraphPad 8.0.2 statistical software and results were presented as mean ± SD (standard deviation). Comparison among multiple groups were assessed by one-way ANOVA. Differences were defined as significant for p value < 0.05.

Two doses of DHQ-L (10 mg/kg) and DHQ-H (50 mg/kg) were orally administered for 14 consecutive days, respectively, starting 7 days after SiO₂ (0.2 g/kg) administration. The high and low doses of DHQ treatment markedly attenuated SiO₂-induced the weight loss and the increasing LW/BW ratio as well as HYP content in lungs (Figures 1A–C). HE and masson’s staining showed that DHQ ameliorated alveolar walls, inflammatory reaction, and deposition of collagen fibers at day 21 compared to SiO₂ group (Figures 1D–F). Furthermore, the levels of pro-inflammatory cytokines, including IL-1β, TNF-α, and TGF-β, in serum and lung homogenate were elevated in SiO₂ groups, but significantly reduced after DHQ treatment (Figure 1G). In addition, the expression of α-SMA, collagen I and fibronectin was also significantly reduced in DHQ-treated mice compared to SiO₂ group (Figure 1H). Taken together, these findings further demonstrated that DHQ could greatly alleviate SiO₂-induced inflammation and fibrosis degree of lung tissues.

Our study had shown that DHQ attenuated SiO₂-induced inflammation and pulmonary fibrosis in vivo. Subsequently, we conducted experiments in vitro to further corroborate experimental results in vivo. As demonstrated in Figure 2A, DHQ at concentration ranging from 5 to 40 µM had no obvious cytotoxicity to HBE cells. However, viability was below 90% when concentration reached 80 μM. Therefore, 40 μM of DHQ was used in our subsequent experiments in vitro. We used conditioned media from SiO₂ or DHQ-treated HBE cells to verify whether DHQ treatment could inhibit MRC-5 cells activation in vitro. The immunofluorescence staining and western blot analysis of MRC-5 activation makers showed that DHQ significantly reduced the levels of α-SMA, collagen1 and fibronectin (Figures 2B–D). Altogether, conditioned media from DHQ-treated HBE cells inhibited MRC-5 cells activation.

Recently, ferroptosis was identified as an iron-dependent oxidative cell death that may be responsible for lung disorders (30-34). Our hypothesis in this study was that DHQ had a significant role in the anti-fibrosis effects by regulating ferroptosis. To validate this conjecture, we examined the effects of DHQ on the death levels of HBE cells. Trypan blue staining confirmed that the DHQ significantly reduced cell death in activated HBE cells (Figure 3A). Following that, we investigated whether DHQ mediated inhibition of the ferroptosis in vivo and in vitro by measuring ferroptosis-associated biomarkers. The results of immunofluorescence staining and western blot analysis displayed that the expressing of the ferroptosis marker GPX4 was significantly increased by DHQ treatment compared to SiO₂ group (Figures 3B,C). Given that ferroptosis is mainly dependent on the intracellular iron accumulation, lipid peroxidation, and the depletion of antioxidant enzymes. The levels of Fe²⁺, ROS, MDA, 4-HNE content and GSH were detected to validate the occurrence of ferroptosis. As expected, our data also displayed that DHQ treatment markedly idecreased iron level and lipid peroxidation products, and increased GSH content (Figure 3D). Finally, electron morphology of cell mitochondria further testified the occurrence of ferroptosis. Compared with DHQ-treated HBE cells, SiO₂-treated cells showed smaller, crumpled and broken mitochondria, which is cellular morphological characteristic of ferroptosis (Figure 3E). In conclusion, these data showed that DHQ suppressed activated HBE ferroptosis in vivo and in vitro.

In order to further explore the role of ferroptosis in the anti-fibrotic effect of DHQ, ferroptosis specific inducer erastin was used to activate ferroptosis in activated HBE cells. As a result, HBE cells treated by DHQ exerted promotion of cell viability, whereas erastin showed the effect of cell survival inhibition (Figure 4B). In addition, DHQ treatment remarkably decreased the levels of iron, ROS, MDA and 4-HNE, but markedly increased the levels of GSH and GPX4 compared with SiO₂ group. Interestingly, erastin treatment strongly blocked the inhibition effect of DHQ on ferroptosis (Figures 4A,B). Subsequently, real-time PCR assay and western blot analysis were carried out to determine whether stimulation of ferroptosis could reverse the inhibitory effect of DHQ on MRC-5 cells. Interestingly, the results indicated that treatment with DHQ significantly reduced the expression of α-SMA, collagen I and fibronectin, whereas this effect was significantly reversed by erastin (Figures 4C,D). These data collectively suggested that stimulation of ferroptosis impaired anti-fibrosis effects of DHQ in vitro.

An increasing number of studies have confirmed that the ferritinophagy participates in the occurrence and development of pulmonary diseases (Yoshida et al., 2019; Qin et al., 2021). Therefore, we speculated that DHQ ameliorated pulmonary fibrosis by regulating ferritinophagy of activated HBE cells. Western blotting revealed that the expression of vital autophagy makers LC3 was down-regulated, and the ferritinophagy markers FTH1 and NCOA4 were up-regulated by DHQ treatment (Figure 5A). Moreover, immunofluorescence staining displayed that DHQ inhibited the expression of LC3 (Figure 5B).

To further investigate the role of ferritinophagy in SiO₂-induced pulmonary fibrosis, activated HBE cells were pretreated with rapamycin (Rapa), followed by DHQ treatment for 24 h. Our results demonstrated that DHQ alleviated ferroptosis by promoting cell viability, decreasing Fe²⁺ level and MDA content, and increasing GSH content, which was reversed by Rapa co-treatment (Figure 5C). In addition, results from western blot showed that DHQ treatment significantly increased the level of GPX4 and decreased the expression of ferritinophagy markers LC3, while ferritinophagy inducer Rapa co-treatment triggered the degradation of LC3. Moreover, the results showed that DHQ notably inhibited degradation of FTH1 and NCOA4, but co-treatment with Rapa eliminated this effect (Figure 5D). Furthermore, The results also demonstrated that DHQ treatment significantly reduced the expression of α-SMA, collagen1 and fibronectin, whereas Rapa co-treatment dramatically increased the expression of MRC-5 activation makers (Figure 5E). Overall, these findings revealed that DHQ suppressed activated HBE cells ferroptosis by inhibiting ferritinophagy to alleviate MRC-5 activation in vitro.