This study employs animal and cell models to investigate whether NRF2 inhibitor ML385 attenuates silica-induced malignant progression of lung adenocarcinoma through the ROS/NRF2-autophagy axis pathway.
Research Article
This study employs animal and cell models to investigate whether NRF2 inhibitor ML385 attenuates silica-induced malignant progression of lung adenocarcinoma through the ROS/NRF2-autophagy axis pathway.
Silica exposure is associated with an increased risk of lung adenocarcinoma, but its molecular mechanism remains unclear. This study aims to establish a repeatable experimental protocol to explore how the nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor ML385 inhibits silica-induced malignant progression of lung adenocarcinoma by regulating the ROS/NRF2-autophagy axis pathway. Firstly, a silicosis model was established by intranasal perfusion of silica suspension in C57BL/6 mice. The degree of pulmonary fibrosis was evaluated by Masson staining, the infiltration of immune cells was analyzed by immunofluorescence, and the expressions of NRF2, cyclin-dependent kinase 1 (CDK1), and voltage-dependent anion channel 1 (VDAC1) in lung tissue were detected by immunohistochemistry. Subsequently, in in vitro experiments, the RAW264.7 macrophage cell line was treated with silica. Autophagic flux and oxidative stress levels were evaluated by LC3-lysosome co-localization and ROS probes, and intervention was carried out with the NRF2 inhibitor ML385. Finally, the effects of ML38 on migration, invasion, and apoptosis of lung adenocarcinoma cells were analyzed by scratch assay, cells passing through pores of a specific size assay, and flow cytometry. The results showed that silica could induce pulmonary fibrosis, immune cell infiltration, and upregulation of NRF2, CDK1, and VDAC1 in mice (p < 0.001), and inhibit the autophagic flux of macrophages and reduce ROS levels. ML385 can reverse these effects (p < 0.05). This protocol provides a complete experimental process from in vivo model construction to in vitro mechanism research, offering an operational technical path for studying the molecular mechanism of silica-related lung adenocarcinoma and therapeutic strategies targeting NRF2.
Lung adenocarcinoma is the leading cause of cancer-related deaths worldwide, with an estimated 2 million new cases and 1.76 million deaths each year1. In recent years, the incidence of lung adenocarcinoma among people who have never smoked has risen, which may be related to environmental factors such as air pollution2. Silica is a common environmental pollutant and has been identified as a potential pathogenic factor for human lung adenocarcinoma3. Silica can cause persistent chronic inflammation, leading to infiltration of macrophages, lymphocytes, and neutrophils, and the release of reactive oxygen species and inflammatory factors4. This long-term inflammatory microenvironment promotes the occurrence of lung adenocarcinoma5,6. Although the association between silica exposure and lung adenocarcinoma has been confirmed, its specific molecular mechanism has not been fully elucidated.
The transcription factor NRF2, encoded by the NFE2L2 gene, plays a crucial role as a master regulator in maintaining cellular redox homeostasis in response to oxidative stress7,8. Under normal circumstances, NRF2 is labeled and degraded by the KEAP1-CUL3 ubiquitin ligase complex. Under the stimulation of oxidative stress or electrophilic substances, the activity of KEAP1 is inhibited, allowing NRF2 to accumulate stably and enter the nucleus, thereby exerting the defense and regulatory role of the core9. Nevertheless, excessive NRF2 activation in the tumor microenvironment may enhance glycolysis and the survival of tumor cells by stopping ROS accumulation and boosting apoptotic resistance10,11. Studies have proven that silica exposure can result in abnormal activation of the NRF2 signaling pathway12,13. Therefore, targeting NRF2 may be an effective strategy for intervening in the malignant progression of lung adenocarcinoma induced by silica14.
This study aims to clarify whether the NRF2 inhibitor ML385 inhibits silica-induced malignant progression of lung adenocarcinoma by regulating the ROS/ NRF2-autophagy axis pathway. We established a silica-induced mouse model to reproduce the key features of the disease and used in vitro experimental methods to study the interaction between macrophages and tumor cells. We found that silica induced the expression of inflammatory and immune molecules and further promoted tumor formation through the ROS/NRF2-autophagy axis pathway. When the NRF2 inhibitor (ML385) was used, the promoting effect of silica on tumor formation slowed down, suggesting that the NRF2 inhibitor may play a role in the treatment of silica-induced lung tumors.
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All donor blocks were obtained from archival pathological specimens collected between 2016 and 2018 at the Affiliated Huai'an NO.1 People's Hospital of Nanjing Medical University. The samples were deidentified prior to use and processed in compliance with approved protocols. The ethics committee of the Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University, KY-2024-250-01. The breeding, disposal, and application of experimental mice strictly follow the Regulations on the Administration of Laboratory Animals and international guidelines.
Construction of a silica-induced mouse model
Preparation of mice and silica suspension: Raise male C57BL/6 mice, 6-8 weeks old, in an animal room with a room temperature of 20-25 °C and a 12 h light / 12 h dark cycle. To prepare the silicon dust suspension, 300 mg of silicon dust powder was precisely weighed and suspended in 10 mL of sterile 1x phosphate-buffered saline. Subsequently, the mixture was vigorously vortexed and oscillated for 5 min, and then ultrasonically treated in a water bath ultrasonic machine for 30 min to ensure uniform dispersion of the particles and prevent aggregation. The final concentration of this reserve suspension is 30 mg/mL. Sterilize it by autoclaving at 121 °C for 30 min. Before each use, it is necessary to vortex and shake again for 2-3 min to resuspend any settled particles.
Mice inhaled silica suspension through the nose. The micropipette was used to slowly and dropwise introduce the solution into the nasal cavity of the mice. The mice were divided into 6 groups with 20 mice in each group. The inhalation volume of each group was 10 µL, 30 µL, 50 µL, 70 µL, 90 µL, and 200 µL, with a concentration of 30 mg/mL, once a day for 40 consecutive days. When perfusing, grasp the skin behind the mouse's ear with the thumb and index finger of the left hand, and fix the body and tail of the mouse with the other fingers. Ensure that the mouse is in a supine position with its head slightly tilted back to allow the suspension to be naturally inhaled into the respiratory tract. We found that there was no death event after the second day of inhalation of 10 µL, 30 µL, 50 µL, and 70 µL silica suspension in mice, while there was a death event after the second day of inhalation of 90 µL and 200 µL silica suspension in mice. Therefore, the maximum volume of silica inhalation in mice was 70 µL per day.
When handling crystalline silica, it must be carried out in a biosafety cabinet or fume hood, and N95 masks, dust-proof goggles, nitrile gloves, and protective clothing must be worn throughout the process. When preparing the suspension, the movements should be gentle, and a weighing chamber should be used to avoid the generation of aerosols. All silica contact waste should be collected in sealed containers with puncture resistance and disposed of in accordance with the regulations for hazardous chemical waste. It is strictly prohibited to mix it with general garbage or pour it into the sewer. Biological waste, such as cell culture medium, should be collected in special trash cans containing disinfectants and sterilized under high pressure. Animal carcasses and tissues should be placed in biological waste bags and sterilized under high pressure for harmless treatment.
Evaluate the success of the silica-induced mouse model. Assess animals at different time points after silica inhalation, namely on the 3rd, 14th, and 40th days. The mice were placed in professional animal euthanasia devices, and carbon dioxide was introduced at a filling rate of 30%-50% volume/min. They were continuously exposed until they stopped breathing. After confirming death, subsequent operations were carried out. The lung tissue was removed. Lung tissue was fixed in 4% paraformaldehyde solution at room temperature for 48 h. After fixation, the tissue was dehydrated with gradient alcohol, made transparent with xylene, and then embedded in paraffin. Continuous sections were made using a paraffin sectioning machine with a thickness of 4-5 µm for subsequent histological staining and immunohistochemical analysis. The lung tissue does not require decalcification treatment. Pathological examination of lung tissue was conducted to evaluate successful model generation. The success criteria of silicosis model construction were as follows: significant pulmonary inflammation, fibrosis, and the occurrence of silicosis nodules.
Analysis of immune cell infiltration in the silica-induced mouse model
The lung tissues of mice at day 14 after silica inhalation were stained with fluorescent markers containing CD3e (1:200 dilution ratio), CD8a (1:200 dilution ratio), CD86 (1:200 dilution ratio), F4/80 (1:200 dilution ratio), and Nk1.1 (1:200 dilution ratio) to visualize immune cells. Fix cell or tissue sections and block with 5% bovine serum albumin (BSA) for 1 h to reduce non-specific binding. Use normal serum of the same species origin as the blocking antibody, dilute serum with PBS at a 1:10 ratio, and add 100 µL of diluted serum into the sample. Then, block at room temperature for 30 min to allow serum to block endogenous antibodies. After sealing, gently rinse 2x with PBS. The primary antibodies containing CD3e (1:200 dilution ratio), CD8a (1:200 dilution ratio), CD86 (1:200 dilution ratio), F4/80 (1:200 dilution ratio), and Nk1.1 (1:200 dilution ratio) were incubated overnight at 4 °C or at room temperature for 1-2 h. After washing with PBS, the fluorescent secondary antibody was added and incubated in the dark for 1 h.
Masson staining and immunohistochemical analysis
Paraffin sections were dewaxed with xylene and hydrated with gradient alcohol, and then antigen retrieval was carried out in sodium citrate buffer with pH 6.0 by high-pressure thermal remediation. For high-pressure thermal remediation, place the tissue slices soaked in repair buffer in a microwave oven at medium heat for 8-12 min. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution for 20 min, and then the non-specific binding site was blocked with 5% BSA at room temperature for 30 min. Add the primary antibodies NRF2 (1:200), CDK1 (1:400), and VDAC1 (1:500), and incubate overnight at 4 °C. After washing 3x with PBS, add HRP-labeled goat anti-rabbit secondary antibody (1:500) and incubate at room temperature for 1 h. Paraffin sections were subjected to DAB staining, hematoxylin counterstaining, dehydration transparency, and neutral gel sealing to obtain the corresponding molecular immunohistochemistry results. When DAB is used for color development, the moderate color development should be brownish yellow to brownish brown, and the background should be clear. Both excessive (dark brown) and insufficient (light yellow) color development require optimization of the color development time.
The degree of pulmonary fibrosis was semi-quantitatively analyzed using the Ashcroft scoring method15: Masson-stained sections were observed under a 100x magnification microscope, and the lung tissue was randomly divided into 8 regions. Each region was scored according to the degree of fibrosis (0-8 points), with the final score being the average of all regions: 0 points (normal lung tissue), 1-2 points (mild fibrosis), 3-4 points (moderate fibrosis), 5-8 points (severe fibrosis).
The immunohistochemical results were quantitatively analyzed using Image software. Five high-magnification fields were randomly selected, and the average optical density value (MOD) of each field was measured as an indicator of protein expression level.
Examining the impact of silica and ML385 on autophagy and ROS in RAW264.7 cells
Both RAW264.7 and A549 cells (provided by Anhui University of Science and Technology) were cultured using DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin bispecific antibody solution. The 1 x 107 cells were uniformly cultured in a constant temperature incubator at 37 °C and 5% CO2. 5 µL of 30 mg/mL silica suspension was added to 1 x 105 cells/mL of RAW264.7 cells. After incubation for 24 h, the cells were rinsed with PBS 3x, and the autophagy and oxidative stress were detected by ROS, LC3, and lysosome probes. At the same time, ML38516, an inhibitor of NRF2, was used to inhibit the expression of NRF2 (final concentration of ML385: 5 mM/L). Autophagy and oxidative stress were detected according to the above experimental steps. The oxidative stress and autophagy flow of the two groups were statistically analyzed.
Immunohistochemical analysis of VDAC1, CDK1, p62, and NRF2 expressions in lung adenocarcinoma tissue
The expressions of VDAC1, CDK1, p62, and NRF2 were checked in 30 patients. Tissues with lung adenocarcinoma and adjacent healthy tissues were obtained from the Affiliated Huai'an NO.1 People's Hospital of Nanjing Medical University and were analyzed by immunohistochemistry (IHC). The results of IHC and pulmonary fibrosis were statistically analyzed. Collect patient data according to patient information in the hospital system. The specific experimental steps are as follows: Paraffin sections were dewaxed with xylene and hydrated with gradient alcohol, and then antigen remediation was carried out in sodium citrate buffer with pH 6.0 by high-pressure thermal remediation. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution for 20 min, and then the non-specific binding site was blocked with 5% BSA at room temperature for 30 min. Add the primary antibodies NRF2 (1:200), CDK1 (1:400), p62 (1:500), and VDAC1 (1:500), and incubate overnight at 4 °C. After washing 3x with PBS, add HRP-labeled goat anti-rabbit secondary antibody (1:500) and incubate at room temperature for 1 h. DAB color development, hematoxylin counterstaining, dehydrated transparent, neutral gum sealing. When DAB is used for color development, the moderate color development should be brownish yellow to brownish brown, and the background should be clear. Both excessive (dark brown) and insufficient (light yellow) color development require optimization of the color development time.
Cell migration and invasion analysis
The effect of the ML385 and supernatant from the co-incubation of silica with RAW264.7 cells on cell migration and invasion after being incubated with the lung adenocarcinoma cell line A549 was studied. The supernatant of RAW264.7 cells co-cultured with silica for 24 h was added to A549 cells. The cells were divided into the silica group, the silica+ML385 group, and the PBS group. The silica group: 5 µL of 30 mg/mL silica suspension was added to 1 x 105 cells/mL of RAW264.7 cells. After incubation for 24 h, remove the cell supernatant for later use. The silica+ML385 group: 5 µL of 30 mg/mL silica suspension and a final concentration of 5 mM/L ML385 were added to 1 x 105 cells/mL of RAW264.7 cells. After incubation for 24 h, remove the cell supernatant for later use. PBS group: 5 µL of PBS was added to 1 x 105 cells/mL of RAW264.7 cells. After incubation for 24 h, remove the cell supernatant for later use. During the scratch assay, A549 cells were first seeded in 12-well plates at a density of 5 x 105 per well. After the cells grew to 95%-100% fusion, scratches were made on the monolayer cells using the tip of a 200 µL sterile pipette. The exfoliated cells were gently washed off with PBS. Subsequently, the culture conditions were changed to include 1% FBS basic medium and the supernatants of each treatment group to maintain cell viability during the 7-day experimental period. Images of the same position were collected under an inverted microscope at 0 h (Day 0) and on the 7th Day (Day 7) after the scratch, respectively. Finally, the scratch area was quantitatively analyzed, and the scratch closure rate was calculated using ImageJ software to evaluate the effect of the supernatant from different treatment groups on the migration ability of A549 cells.
In the cells pass through pores of specific size invasion assay, an 8 µm polycarbonate membrane chamber was used, with the upper chamber membrane pre-coated with gel simulating the extracellular environment diluted at a ratio of 1:8 to construct a basement membrane barrier. A549 cells were resuspended in a suspension made by mixing serum-free medium with the supernatants of each treatment group in a 1:1 ratio and then inoculated in the upper chamber (2 x 104 cells per chamber). In the lower chamber, a solution made by mixing a medium containing 20% FBS with the supernatants of the corresponding treatment groups (Silica+ML385 group, Silica group, and PBS group) in the same proportion was added as a chemical attractant. The supernatants of the corresponding treatment groups were collected by suction through a pipette gun in RAW264.7 Cell phagocytosis of silica. After the cells were continuously incubated at 37 °C and 5% CO2 for 7 days, the non-invasive cells in the upper chamber were removed with cotton swabs, and the cells that had penetrated the membrane and reached the lower chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Finally, the number of invasive cells was randomly selected from 5 fields of view by an optical microscope.
Analysis of the impact of silica and ML385 on A549 cells' apoptosis using flow cytometry
Cell preparation and grouping: A549 cells treated with the supernatants of each treatment group (PBS group, silica group, silica + ML385 group) were collected. The cells were gently washed 2x with pre-cooled PBS, then resuspended in 1x binding buffer, and the cell density was adjusted to 1 x 106 cells per tube/100 µL. Then, 5 µL of Annexin V-FITC and 5 µL of PI staining solution were added to the cell suspension, gently vortexed to mix, and incubated at room temperature in the dark for 15 min. After incubation, 400 µL of 1x binding buffer was added to each tube and immediately tested on the machine. Detection was carried out using a flow cytometer. Excited by a 488 nm laser, the fluorescence signal of Annexin V-FITC was collected through the FITC channel, and the fluorescence signal of PI was collected through the PE channel. Before the experiment, single-staining samples were used for fluorescence compensation adjustment to eliminate spectral overlap. When conducting data analysis, first delineate the target cell population in the FSC-A/SSC-A scatter plot and eliminate fragments. Subsequently, cell adhesions were excluded using the FSC-H/FSC-A scatter plot; Finally, a gate was set up to distinguish between living cells, early apoptotic cells, late apoptotic/necrotic cells, and mechanically damaged cells. The data was obtained and analyzed using FlowJo V10 software.
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Silica mouse model
In Figure 1, the mice were administered silica intranasally at a dosage of 30 mg/mL and 70 µL for 40 consecutive days, which ensured that no death events occurred in the mice and the silica mouse model could be successfully established. Meanwhile, mice gavage with PBS were used as negative controls. The aim was to eliminate the influence of the operation and the solvent itself and ensure that the phenotype was specifically induced by silica. The succes...
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Lung adenocarcinoma is one of the main causes of cancer-related deaths worldwide, and its incidence and mortality rates are increasing year by year17. Silica is classified as a class 1 human carcinogen by the International Agency for Research on Cancer, and the risk of lung adenocarcinoma increases with cumulative occupational exposure18,19,20,21. Previous studies have p...
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The authors have nothing to disclose.
Thank you to the team members for their support and contribution to this study. This study was supported by the Key Laboratory of Industrial Dust Deep Reduction and Occupational Health and Safety of Anhui Higher Education Institutes (NO. AYZJSGXLK202202006), Shanghai Pudong New Area People's Hospital Talent Introduction Start-up Fund (NO. PRYYH202501).
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| BeyoGold Transwell | Beyotime Biotechnology | FTW067-48lns | Transwell |
| CD3e | BD Bioscience | 561827 | FITC Hamster Anti-Mouse CD3e(145-2C11) |
| CD86 | BD Bioscience | 105013 | CD86 |
| CD8a | BD Bioscience | 100713 | CD8a |
| CDK1 | Abcam | ab133327 | Anti-CDK1 |
| Cell apoptosis detection kit | Beyotime Biotechnology | C1062L | apoptosis detection |
| DAPI | Beyotime Biotechnology | P0131-25ml | DAPI |
| Embedding machine | P.S.J MEDICAL | BM450A | Embedding machine |
| F4/80 | BD Bioscience | 123109 | F4/80 |
| Fully automatic tissue dehydrator | Leica Biosystems | ASP3005 | Fully automatic tissue dehydrator |
| Glass microscope slides | Citotest | 250124A1 | Glass microscope slides |
| H&E dye | Beyotime Biotechnology | C0105M | H&E dye |
| IHC Kit | Absin Biotechnology | abs996-5ml | IHC Kit |
| LC3 probe | Beyotime Biotechnology | C3018M | LC3 probe |
| Low Profile Microtome Blades | Thermo Fisher | 3052835 | Low Profile Microtome Blades |
| lysosome probe | Beyotime Biotechnology | C1046 | lysosome probe |
| Marker pen | Deli | SK109 | Marker pen |
| Masson dye | Beyotime Biotechnology | C0189M | Masson dye |
| Matrix-Gel | Beyotime Biotechnology | C0371-5ml | Matrix adhesive |
| Microtome | Leica Biosystems | HistoCore BIOCUT | Microtome |
| ML385 | Abcam | ab287109 | NRF2 inhibitor |
| NK1.1 | BD Bioscience | 561117 | NK1.1 |
| NRF2 | Abcam | ab1809Y | Anti-NRF2 |
| p62 | Abcam | ab20735 | Anti-p62 |
| Paraffin wax | Solarbio | YA0012 | Paraffin wax |
| Reactive oxygen species Assay kit | Beyotime Biotechnology | S0033M | ROS probe |
| Silica | Sigma Aldrich | S5631 | Crystalline silica |
| VDAC1 | Abcam | ab34726 | Anti-VDAC1 |
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