Animals

Male C57BL/6 mice, aged eight weeks, were acquired and underwent adaptive feeding for a week at the Central South University Animal Center in Changsha, China. Following this period, anesthesia was administered to the mice, and their tracheas were subsequently injected with either silica (2.5 mg per mouse) or saline, marking the commencement of the experiment on day 0. The first batch of mice was administered SHI (12.5, 25, or 50 mg/kg) or saline by gavage over the next 14 consecutive days and sacrificed on days 14 or 48. The second batch of mice was administered SHI (12.5, 25, and 50 mg/kg) or saline by gavage from day 28 to day 42 and sacrificed on day 42. The mice in the first and second batches were randomly divided into five groups and treated as follows: (1) Control (CON) group, saline (intratracheal, i.t.) + saline (oral, p.o); (2) SiO2 group, silica + saline (p.o); (3) SiO2 + 12.5 mg/kg, silica + SHI (12.5 mg/kg, p.o); (4) SiO2 + 25 mg/kg, silica + SHI (25 mg/kg, p.o); (5) SiO2 + 50 mg/kg, silica + SHI (50 mg/kg, p.o). Six mice were in each group.

To observe the safety of SHI, mice were randomly divided into two groups and treated continuously for 14 days. The CON group received physiological saline (p.o.) and the SHI group received shionone (50 mg/kg, p.o.). Each group consisted of six mice.

SHI (National Institutes for Food and Drug Control, China, purity ≥ 99.5%) (Supplementary Fig. 1) was diluted in vehicle consisting of saline containing 5% (v/v) dimethyl sulfoxide (DMSO; provided by Shanghai Macklin Biochemical Co., Ltd., Shanghai, China). The methodology for all animal-based studies adhered strictly to the International Guidelines for Biomedical Research involving Animals.

Histological evaluation

A comprehensive analysis was conducted on lung tissue using a grading system from 0 to 4, which incorporated five distinct factors: the extent of mixed cellular response within alveoli, the proliferation of bronchoalveolar structures, presence of bleeding, accumulation of lipoproteins within alveoli, and formation of hyaline membranes. This evaluation generated a cumulative lung damage score, independently determined by three pathologists who were not informed about the experimental data [22].

Extraction and culture of primary peritoneal macrophages and primary mouse fibroblasts

To obtain primary peritoneal macrophages, mice were given a 3 mL intraperitoneal dose of 3% thioglycolate medium (Sigma-Aldrich, St. Louis, MO, USA). After allowing a period of four days for incubation, the mice then underwent a lavage using 15 mL of RPMI 1640 solution (Gibco, Waltham, MA, USA). The harvested cell blend was distributed evenly across culture dishes and then incubated at room temperature for 2 h.

For the isolation of primary lung fibroblasts, an approach using enzymatic cleavage was employed. The procedure began with the mice being anesthetized with sodium pentobarbital, followed by perfusion of their hearts with ice-cold PBS. The lungs were then excised, rinsed in cold PBS, and dissected into pieces measuring 1–2 cm^2. These sections were then subjected to enzymatic cleavage in a solution of DMEM containing 1 mg/mL collagenase I and incubated at 37 °C for an hour. Post-digestion, the mixture was strained through mesh filters of 70 and 40 μm, centrifuged, and the resulting sediment was resuspended in DMEM-high glucose (Gibco) enriched with 20% fetal bovine serum (FBS, Sigma-Aldrich) and 1% antibiotic–antimycotic solution. The fibroblasts were subsequently cultured at 37 °C in a moisture-saturated atmosphere containing 5% CO2.

In an environment where conditions were meticulously regulated, cellular cultures were sustained at a constant temperature of 37 ℃, under an atmospheric composition of 5% CO2. In detail, primary peritoneal macrophages were cultured in a medium enriched with 10% FBS (Gibco) and supplemented with a 1% antibiotic solution containing penicillin and streptomycin (Procell Life Science & Technology, Wuhan, China). In a parallel approach, primary mouse fibroblasts were nurtured in an environment that contained 20% FBS and an identical dosage of antibiotics. The study's methodology required subjecting certain cellular groups to various concentrations of transforming growth factor-beta 1 (TGF-β1, 10 ng/mL, PeproTech, Cranbury, NJ, USA) and SHI (in doses of 3, 10, or 30 μM) for a timeframe of 48 h.

Levels of myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH)

The lung tissue was ground and homogenized to tested with Myeloperoxidase assay kit, Malondialdehyde (MDA) assay kit, Superoxide Dismutase (SOD) assay kit, glutathione (GSH) assay kit (Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions.

Measurement of the lung hydroxyproline

To measure the hydroxyproline content within lung tissues, we employed a method that made use of a Hydroxyproline Assay Kit, which was provided by the Nanjing Institute of Built-up Biotechnology in Nanjing, China. This approach required the processing of lung tissue samples (1 mg to 9 mL, weight/volume) with a digestion solution at a ratio of 5 to 1 (volume/volume), which were then incubated in a water bath set to maintain a steady temperature of 37 °C for a duration of 3 h. Following this step, an application solution was introduced to the specimens, which were then subjected to incubation at 60 °C within a water bath. The next phase involved centrifugation at 3500 rpm for a quarter-hour at a cold temperature setting of 4 °C. To determine the hydroxyproline concentration, we measured the optical density at a 550 nm wavelength.

ROS and mitoROS assay

In the study focusing on reactive oxygen species (ROS) and mitochondrial ROS (MitoROS), analysis was conducted on primary macrophages and fibroblasts. The detection of ROS was carried out using 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA), acquired from Thermo Fisher Scientific. For assessing mitochondrial ROS, we applied the MitoSOX assay from the Nanjing Jiancheng Bioengineering Institute, located in Nanjing, China. Cells, after incubating for half an hour, were washed three times with PBS. This process allowed for the measurement of ROS and MitoROS levels through fluorescence microscopy with equipment provided by Nikon, headquartered in Tokyo, Japan.

Flow cytometry

Regarding the assessment of ROS through flow cytometry, primary cells received a pretreatment with H2DAFDA for 30 min at 37 ℃. Following a thrice-repeated PBS wash, the cells were suspended anew for ROS level evaluation via flow cytometry, utilizing the LSR Fortessa system from BD, located in Santa Clara, CA, USA. Analysis of the collected data was facilitated through the employment of FlowJo version 10 software, developed by FlowJo in Eugene, OR, USA.

Polarization of macrophage

Primary peritoneal macrophages were stained with allophycocyanin (APC) Anti-Mouse CD80 (Elabscience, Wuhan, China) and fluorescein isothiocyanate (FITC) Anti-Mouse CD206 (Elabscience) to determine the proportion of polarized macrophages. Flow cytometric analysis of the samples was carried out utilizing the LSR Fortessa device as previously mentioned. The acquired data underwent evaluation through the FlowJo software, version 10.

Immunofluorescence

In the initial procedure, macrophages and fibroblasts were immobilized using a 4% paraformaldehyde solution for a quarter of an hour. Following this, the permeabilization of the cells was achieved by treating them with 0.5% Triton X-100 for 20 min, and thereafter, they were submerged in goat serum for half an hour. Overnight, at a temperature of 4 °C, the fibroblasts were incubated with specific antibodies targeting Nrf2 (obtained from Abcam, Cambridge, UK), P65 (procured from Proteintech, Rosemont, IL, USA), and alpha-smooth muscle actin (α-SMA; sourced from Proteintech). Post-incubation, a washing step with PBS was performed, after which the cells were exposed to either CoraLite488-conjugated Goat Anti-Rabbit IgG(H + L) or CoraLite594-conjugated Goat Anti-Rabbit IgG(H + L) (both supplied by Proteintech), and kept at 37 °C for an hour. Finally, the cells were examined under a fluorescence microscope equipped with a Zeiss ApoTome attachment (produced by Carl Zeiss, Oberkochen, Germany).

Nuclear and cytoplasmic protein extraction

To begin the process of extracting nuclear and cytoplasmic proteins, the cells were first rinsed once with PBS before being dislodged with a cell scraper. This was followed by a centrifugation step to facilitate the separation of nuclear and cytoplasmic proteins, making use of a Nuclear Protein Extraction Kit from Solarbio (Beijing, China).

Transfection with small interfering RNA (siRNA)

In the case of transfecting primary fibroblasts with small interfering RNA (siRNA), the procedure involved the use of siRNA designed to target Nrf2 along with a control siRNA, both sourced from Santa Cruz Biotechnology (Dallas, TX, USA). Lipofectamine RNAiMAX, a product of Invitrogen (Carlsbad, CA, USA), served as the vehicle for this transfection. The efficacy of the gene silencing was subsequently confirmed through western blot analysis.

Western blotting

To isolate total proteins, lung tissues or cells underwent homogenization in RIPA buffer (Solarbio). The concentration of proteins was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). After this initial step, proteins with a 10% concentration were then subjected to separation via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). In order to prevent non-specific binding, these membranes were initially soaked in a blocking solution consisting of 5% skim milk in PBS mixed with 0.1% Tween 20 (PBST). Following this, they were incubated at 4 °C overnight with primary antibodies directed against β-actin (1:5000 dilution, Proteintech), NF-κB P65 and its phosphorylated counterpart (1:1000 for each, obtained from Pho-NF-κB by Cell Signaling Technologies, Danvers, MA, USA), IκBα and its phosphorylated state (1:2500 and 1:10000 respectively, Abcam), HO-1 (1:1000, Proteintech), NQO-1 (1:1000, Proteintech), Collagen I (1:1000, Abcam), α-SMA (1:20000, Proteintech), and Lamin B1 (1:5000; Proteintech), NLR family pyrin domain containing 3 (NLRP3, 1:1000, Abcam), Apoptosis associated speck like protein containing CARD (ASC, 1:1000, Abclonal, Wuhan, China), Caspase-1 p10 (1:1000, Abcam), CD80 (1;1000, Cell Signaling Technology), CD206 (1:1000, Cell Signaling Technology), and Nrf2 (1:1000, Cell Signaling Technology). Following an in-depth wash with PBST, the samples were treated with horseradish peroxidase-tagged secondary antibodies for a two-hour period at ambient temperature. These antibodies included Rabbit Anti-Mouse IgG H&L (1:5000, Zen-Bio, Chengdu, China) and Goat Anti-Rabbit IgG H&L (1:5000, Zen-Bio). Subsequently, protein bands were detected utilizing Millipore's Luminata™ Crescendo chemi-luminescent horseradish peroxidase reagent, Burlington, VT, USA. The visual recording of these protein markers was achieved with a GeneGnome XRQ imager (Syngene, Cambridge, UK).

Real-time quantitative PCR (qPCR) analysis

RNA was extracted from samples of lung tissue and cells utilizing TRIzol Reagent furnished by TransGen Biotech, located in Beijing, China. Post-extraction, the conversion to cDNA was carried out with a Reverse Transcription Kit procured from Novoprotein, based in Suzhou, China. Quantitative PCR analyses were conducted using SYBR GREEN, also supplied by Novoprotein, and carried out on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The primers used in this process were acquired from Shanghai Sangong Biotechnology Co., Ltd., as specified in Table 1.

Table 1 Primer sequences for qPCRRespiratory function

Mice were anesthetized prior to tracheal intubation. Breathing frequency, pulmonary ventilation, lung compliance, and airway resistance of the mice were determined using the Max II system (Buxco Electronics, Inc., Wilmington, NC, USA).

Statistical analyses

When analyzing the data, findings are presented as the average with the addition or subtraction of the standard deviation. This statistical evaluation was enabled through GraphPad Prism software (version 9.0), originating from San Diego, CA, USA. Initially, the Shapiro–Wilk test was applied to determine if the data distributions were normal. For datasets exhibiting a normal distribution, an analysis of variance (ANOVA) was undertaken to discern differences across multiple groups, with subsequent application of Tukey’s test for detailed comparisons between pairs. Conversely, the Kruskal–Wallis test was employed for datasets deviating from a normal distribution pattern. The examination of survival rates was performed utilizing the log-rank test, with a p-value less than 0.05 deemed indicative of statistical significance.