Animals and animal experimentation

The Laboratory Animal Welfare and Ethics Committee of Central South University attempted to authorize the study protocol, with IACUC number of CSU-2022-0001-0196, ensuring compliance with the ethical principles and welfare standards applicable to laboratory animals.

C57BL/6 mice and SD rats were acquired from the Department of Laboratory Zoology, Central South University and kept at the Central South University Department of Laboratory Zoology in a specific pathogen-free environment. Mice were acclimated to feeding for one week before lung fibrosis modelling and treatment. Mice were subjected to anesthesia utilizing sodium pentobarbital, and 50 µL of bleomycin (BLM; 3 mg/kg; Nippon Kayaku) was administered intratracheally, while controls received a corresponding volume of saline. Protodioscin (Solarbio) was diluted in a solvent consisting of saline, 5% Tween 80 (Macklin) (v/v), 40% polyethene glycol 300 (Macklin) and 5% dimethyl sulfoxide (Macklin).

To assess the therapeutic effect of protodioscin, 10 mice were randomly chosen from the total of 110 mice as the control group and they were injected with saline via the trachea before receiving intraperitoneal solvent injections on days 15–28. The remaining cohort of mice was subjected to the modeling procedure, employing a stratified sampling approach on day 14 based on individual body weights. Thereafter, an equivalent number of mice were systematically allocated to four distinct groups during the period spanning days 15 to 28. These groups comprised the low-dose treatment group, injected intraperitoneally with 1 mg/kg protodioscin; the medium-dose treatment group, injected intraperitoneally with 5 mg/kg protodioscin; the high-dose treatment group, injected intraperitoneally with 20 mg/kg protodioscin; the pulmonary fibrosis group, injected intraperitoneally with solvent. On day 29, all mice were anaesthetised with sodium pentobarbital, euthanised by femoral artery bloodletting, and lung tissues were removed for subsequent experiments.

To determine how a Nrf2 inhibitor (ML385) impacts the efficacy of protodioscin, 10 randomly-selected mice out of 100 were used as controls, and these were intratracheally injected with saline, followed by intraperitoneal solvent injections on days 15–28. The remaining animals were used as models, and for this purpose, they were stratified on day 14 according to their body weight before assigning an equal number of mice to the following three groups: in the specified experimental regimen, the high-dose cohort was administered intraperitoneal injections of 20 mg/kg protodioscin from days 15 to 28. Concurrently, the pulmonary fibrosis group underwent intraperitoneal injections of the designated solvent over the same duration. Moreover, the ML385 group received a distinctive treatment involving intraperitoneal injections of 30 mg/kg ML385 attained from Selleck Chemicals, followed by subsequent intraperitoneal injections of 20 mg/kg protodioscin three hours later. This intricate administration protocol was meticulously adhered to throughout the experimental timeline. On day 29, all mice were anaesthetised with sodium pentobarbital, euthanised by femoral artery bloodletting, and lung tissues were removed for subsequent experiments.

Extraction and culture of primary lung fibroblasts

After euthanasia of mice or rats, their thoracic cavities were exposed, and their lung tissues were rinsed with ice-cold PBS supplemented with 1% penicillin–streptomycin solution (Procell). The tissues were then cut into cubes and incubated with 1 mg/mL type I collagenase (Gibco) for 1h at 37 ℃ in a water bath. After centrifugation, the lung tissue was resuspended prior to filtration through a 70-µm filter. The resulting filtrate was centrifuged and after resuspension in red blood cell lysate (Solarbio), a 5-min incubation was performed on ice. The above-described procedure was reiterated by passing the amalgam through a 40-µm filter, centrifugation, and subsequent reconstitution in high-glucose Dulbecco's Modified Eagle Medium (Procell). This medium was supplemented with 1% penicillin–streptomycin solution (Procell) and 20% fetal bovine serum (FBS; Biological Industries). Primary lung fibroblasts, spanning the 3–7 generation range, were utilized for all experimental protocols. Ham's F-12 K medium attained from ProCell, inclusive of 1% penicillin–streptomycin solution and 10% FBS, was utilized for the cultivation of human fetal lung fibroblast 1 cells (HFL-1) attained from ProCell. Incubated under optimal conditions at 37 ℃ in a humidified incubator, these cell cultures were maintained in an environment with 5% carbon dioxide.

Hematoxylin and eosin (HE) and Masson staining, and Ashcroft scoring

The mouse lung tissues were subjected to thorough fixation utilizing 4% paraformaldehyde attained from Servicebio, and it was attempted to meticulously embed them in paraffin and section the tissues. These sections were thereafter subjected to staining procedures employing HE and Masson staining solutions, which both attained from Servicebio. The severity of lung fibres in each mouse was scored according to Ashcroft’s method [26].

Immunohistochemistry

Xylene was used to de-paraffinize paraffin sections and after ethanol-based dehydration and subsequent antigen repair, the membranes were lysed with 0.5% Triton X-100 (Servicebio). Endogenous peroxidase was also blocked by covering the tissues with an endogenous peroxidase blocker (Beyotime Biotechnology). It was attempted to carry out 30-min incubation with goat serum particularly at room temperature, after which overnight incubation of tissues was undertaken at 4 ℃ with 8-Hydroxy-2-deoxyguanosine (8-OHdG; Servicebio), col-I (Proteintech) and α-SMA (Proteintech) antibodies. A 1-h incubation was then performed at room temperature with goat anti-rabbit IgG monoclonal antibody (Proteintech) prior to staining with a DAB chromogenic solution and subsequently, with haematoxylin.

Measurement of hydroxyproline

The hydroxyproline assay kit attained from the Nanjing Institute of Construction Biotechnology, was employed in accordance with the provided guidelines. Mouse lung tissue underwent homogenization, followed by the sequential addition of various reagents. Subsequent centrifugation particularly at 3500 rpm for 10 min resulted in the supernatant, and it was attempted to transfer it to a 96-well plate for absorbance measurements at 550 nm. These recorded values were subsequently employed in the calculation of hydroxyproline content.

Lung tissue ROS assay

A histochemical pen was used to draw circles around frozen sections before the introduction of a fluorescent quencher. After adding ROS staining solution into the circle, a 30-min incubation was performed at 37 ℃. After staining with DAPI for 10 min at room temperature, the sections were sealed with fluorescence quenching sealer.

Malondialdehyde (MDA) and glutathione (GSH) levels and superoxide dismutase (SOD) activity measurements

Assay kits for glutathione, superoxide dismutase, and malondialdehyde were obtained from the Nanjing Institute of Construction Biotechnology and used following the provided directions. Mouse lung tissues were homogenised, various reagents were added sequentially, and the supernatants were collected after centrifugation to measure the absorbance at the corresponding wavelengths; SOD activity as well as GSH and MDA content were finally obtained by calculation.

Determination of cell viability and cell supernatant lactate dehydrogenase (LDH)

Cell Counting Kit-8 (CKK-8) assays were performed using a kit (Elabscience) in accordance with the provided directions to detect cell viability, while determining lactate dehydrogenase level particularly in the cell supernatants was undertaken through a lactate dehydrogenase assay kit (Nanjing Institute of Construction Biotechnology). It was attempted to plate the cells onto 96-well plates and subjected to a 24-h incubation period with varying concentrations of protodioscin. Thereafter, the detection reagent was introduced, and the incubation of cells was subsequently undertaken at 37 ℃. The absorbance values at 450 nm for each well were then recorded.

Western blotting (WB)

The extraction of total protein from mouse lung tissues or cells involved the utilization of RIPA lysis buffer containing protease and phosphatase inhibitors, including phenylmethylsulfonyl fluoride. Subsequent determination of the total protein concentration was undertaken through a BCA kit that was attained from Cwbio. Thereafter, the proteins were electrophoresed on 10% SDS-PAGE gels followed by transfer to PVDF membranes. The membranes were blocked with 5% skim milk, followed by incubation with antibodies against β-actin, α-SMA, Collagen I, Fibronectin 1 (FN1), CTGF, HO-1, NQO1, Nrf2, Lamin B1, p62, Phospho-P62 (Ser349), Keap1, NBR1, and PRKCI (all from Proteintech) overnight at 4 ℃. The 2-h incubation of membranes was then undertaken particularly at room temperature with goat anti-mouse or goat anti-rabbit IgG monoclonal antibodies, with the level of protein expression eventually visualized and assessed using an ECL developer.

Total RNA extraction and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)

Upon completion of total RNA extraction from both lung tissues and cells using TRIzol reagent attained from Thermo Fisher Scientific, reverse transcription into cDNA was undertaken through a reverse transcription kit attained from Thermo Fisher Scientific. Subsequently, it was attempted to carry out qRT-PCR on a Bio-Rad CFX96 Touch Real-time PCR Detection System, which attained from Bio-Rad, on the basis of the amplification conditions outlined below: 2-min initial denaturation at 95 ℃, followed by 40 cycles, each comprising a 3-s denaturation at 95 ℃ and a 30-s extension at 60 ℃. The reaction was completed with a melting curve of 60–95 ℃. Table 1 lists the different primers (Sangon Biotech) used in the experiment.

Table 1 Primers used in the experiment.Cellular reactive oxygen species assay

The 30-min incubation of cells was undertaken particularly at 37 ℃ with the ROS probe DCFH-DA attained from Beyotime Biotechnology. Subsequently, the cells underwent two additional washes with DMEM. The ensuing procedures involved either observation under a fluorescence microscope or collection for flow cytometry.

SiRNA transfection

After the cell seeding plate, when the cell density reached 60–80%, control SiRNA, p62 SiRNA, Nrf2 siRNA (Santa Cruz) or NBR1 siRNA (Ribobio) was mixed with Lipofectamine 2000 as the transfection reagent (Invitrogen) and incubated for 12 h to detect the transfection effect or for subsequent intervention.

Co-immunoprecipitation assays

An agarose Co-immunoprecipitation Assays (co-IP) kit (ABBKINE) was used as instructed by the manufacturer. Collected cells were lysed before determining the protein concentration. Immunoprecipitation was performed after removing non-specific binding and detected by WB.

Statistical analysis

It was attempted to carry out statistical analysis through GraphPad Prism 8.3.1 software. The expression of data was undertaken in form of mean ± standard deviation, and pairwise comparisons were carried out using t-test. For the purpose of making comparisons involving multiple groups, one-way ANOVA, followed by Tukey's test was employed. A P threshold below 0.05 was suggestive of statistical significance.