Experimental materials

High-sugar DMEM medium and fetal bovine serum were purchased from Gibco, USA; Trypsin was purchased from Wuhan Kerui Co., Ltd; Trizol was purchased from Takara, Japan; Reverse transcription and real-time fluorescence quantitative PCR kit was purchased from vazyme, Nanjing, China; RIPA lysis solution was purchased from Beyotime, Shanghai, China; PVDF membrane was purchased from Millipore, USA ECL luminescent solution was purchased from Google Bio; ThermoFisher incubator was purchased from ThermoFisher, USA; Biomicroscope was purchased from Olympus, Japan; Real-time fluorescence PCR instrument was purchased from Bio-Rad, USA; BLM was purchased from Pfizer; Recombinant human TGF-β1 was purchased from PeproTech, USA. Trigonelline Hydrochloride was purchased from Selleck, Shanghai, China (Fig. 1A).

Fig. 1

Silicon dioxide and BLM-induced mouse model administration times. A Chemical structure of trigonelline hydrochloride; (B) silica-induced pulmonary fibrosis model and administration time; (C) BLM-induced mouse pulmonary fibrosis model and administration time

Experimental mice

The male, and of wild-type C57BL/6 background 8–10 weeks old, (Beijing Viton Lihua Laboratory Animal Technology Co.) were selected in this study. The animal breeding and experiments were conducted in the Specific Pathogen-Free Laboratory Animal Center in the Research Building of Tongji Hospital, Huazhong University of Science and Technology. The animal experiments were conducted in accordance with the principles of the National Institutes of Health and were approved by the Animal Research Committee of Tongji Hospital, Huazhong University of Science and Technology (TJH-202312005).

Animal model

The SiO2-induced fibrosis model involved randomly dividing mice into four groups: (1) PBS + PBS group, (2) PBS + Trig (10 mg/kg) group, (3) SiO2 + PBS group, and (4) SiO2 + Trig (10 mg/kg) group. SiO2 (200 mg/kg in 50 mg/mL PBS) or PBS was administered for endotracheal perfusion, as previously described [32]. The starting day of the model was designated as day 1. Starting on day 28, mice in the PBS + Trig (10 mg/kg) group and SiO2 + Trig group were administered Trig (10mg/kg) intraperitoneally for 14 consecutive days. Meanwhile, mice in the SiO2 + PBS group and the PBS + PBS group received an equal amount of PBS. On day 42, the mice were euthanized to analyze the pulmonary fibrosis phenotype (Fig. 1B).

The BLM-induced fibrosis model involved randomly dividing all mice into four groups: (1) PBS + PBS group, (2) PBS + Trig (10 mg/kg) group, (3) BLM + PBS group, and (4) BLM + Trig (10 mg/kg) group. Pulmonary fibrosis was induced using a high-pressure nebulizing needle (BJ-PWM; BioJane Trading Ltd., Shanghai, China) with 1.5 mg/kg BLM in 50μL PBS by intratracheal injection, as previously reported [33]. Mice receiving equal amounts of PBS served as controls. The starting day of the model was set as day 1. Starting from day 14, mice in the PBS + Trig (10 mg/kg) and BLM + Trig (10 mg/kg) groups were intraperitoneally administered Trig (10 mg/kg) once a day for 7 days. Meanwhile, mice in the BLM + PBS group and the PBS + PBS group received equal amounts of PBS. On day 21 after BLM exposure, mice were euthanized to assess pulmonary fibrosis (Fig. 1C).

Human lung tissue

Specimens were collected from patients who underwent surgical resection of lung tissues at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The patients had a clinical diagnosis of a lung mass that required investigation, no fibrosis was observed on imaging, and postoperative pathological diagnosis confirmed a benign lesion. Normal lung tissues greater than 5 cm distal to the site of the lesion were retained. Informed consent was obtained from the patients after the study received approval from the Ethics Committee of Tongji Hospital (TJ-IRB20231297).

Human lung tissue specimens were aseptically transferred to EP tubes, dissected from the trachea into pieces. The pieces were then spread onto 10 cm petri dishes and cultured using high-sugar DMEM medium supplemented with 10% Gibco serum. The dominant cells in the culture were human lung tissue fibroblasts. To identify the primary human lung fibroblasts, fibroblast specific protein 1 (FSP1) immunostaining was conducted, and positive results indicated that they were fibroblasts (Supplementary Fig. 1).

Western blot analysis

RIPA lysis buffer (Beyotime, Shanghai, China) was used to homogenize cells and lung tissues. Protein blot analysis was performed following standard protocols [34]. Primary antibodies used were collagen 1 (Proteintech, Wuhan, 1:1,000), fibronectin (ABclonal, Wuhan, 1:1,000), α-smooth muscle actin (Cell Signaling Technology, USA, 1:1,000), and GAPDH (Proteintech, Wuhan, 1:1,000), Smad2/3 (Cell Signaling Technology, USA, 1:1,000), p-Smad2 (ABclonal, Wuhan, 1:1,000), p-Smad3 (ABclonal, Wuhan, 1:1,000), Akt(Cell Signaling Technology, USA, 1:1,000),p-Akt(Cell Signaling Technology, USA, 1:1,000), mTOR(ABclonal, Wuhan, 1:1,000),p- mTOR (ABclonal, Wuhan, 1:1,000),p85(Cell Signaling Technology, USA, 1:1,000) andp-p85(Cell Signaling Technology, USA, 1:1,000). A chemiluminescent substrate system was used for detection, and ImageJ software was used to analyze the gray values.

Quantitative RT-PCR assays

RNA was extracted from cells and lung tissues using TRIzol reagent (Takara, Dalian, China). The yield and quality of the RNA samples were detected using a NanoDrop2000 spectrophotometer (ThermoScientific). Subsequently, cDNA synthesis was performed using HiScript-III-RT SuperMix (Vazyme, \Wuhan). Quantitative RT-PCR analysis was performed on a CFX96 RT-PCR Detection System (Bio-Rad) using SYBR qPCR Master Mix (Vazyme). The cycling conditions applied were an initial denaturation for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C each. Normalization of data was performed using the Ct method, presenting the average of normalized transcript levels. Actb RNA levels were used as a reference for data normalization. The following primers were used for PCR amplification: mouse-Fn1 (5’-GTG TTT TCC ACG GAT GCT G-3’, 5’-TGC CCA CTG CTG ACT TAG G-3’), mouse-col1a1 (5’-GCG AGT GCT GTG CTT TCT G-3’, 5’-AGG ACA TCT GGG AAG CAA A-3’), and mouse-Actb (5’-TAT GCT CTC CCT CAC GCC A-3’, 5’-CGC ACG ATT TCC CTC TCA G-3’).

Cellular immunofluorescence

The fibroblasts were fixed with 4% formaldehyde for 5 min and washed three times with PBS. Next, the cells were exposed to a 10% normal donkey serum solution in PBS for 1 h to block non-specific binding. Subsequently, the cells were incubated overnight at 4 °C with primary antibodies against Collagen 1 (1:100), α-SMA (1:100), and Fibronectin (1:100). After primary antibody incubation, the cells were subjected to the corresponding secondary antibody dressing. Finally, the cell nuclei were stained with DAPI and observed under an Olympus fluorescence microscope.

Immunohistochemical analysis

The left lung was perfused with 4% paraformaldehyde via endotracheal infusion for 24 h. It was then paraffin-embedded and cut into 5μm sections. The sections were stained using established techniques, including H&E staining, Sirius red, and Masson staining [35]. For immunofluorescence staining, the sectioned tissues were co-incubated using antibodies to Collagen 1 (1:100), α-SMA (1:100), and Fibronectin (1:100), followed by incubation of the fluorescent secondary antibody, and photographs were taken using an Olympus fluorescence microscope.

Cell migration

Fibroblasts were cultured in tissue culture dishes with appropriate medium and 10% fetal bovine serum until they reached 70–80% density. The cells were then pretreated with medium containing 1% serum. A 200μL pipette tip was used to create a cross-shaped linear scratch in the cell culture dish. The changes were recorded under a microscope at 0, 12, and 24 h and analyzed using ImageJ software.

EdU assay

The impact of Trig on fibroblast proliferation was investigated utilizing the Cell Bright EdU-Apollo488 in vitro kit (RiboBio), following previously established protocols [36]. In brief, fibroblasts underwent treatment with pbs, L-Trig (40 μM), or H-Trig (80 μM) for a duration of 24 h. Then, cells were exposed to 100 μL of EdU solution (50 μM) for 2 h, followed by fixation with formaldehyde (4%) for 30 min. Subsequently, fluorescently labeled Apollo488 solution was added to the reaction mixture and incubated for another 30 min. Finally, nuclei were counterstained with DAPI and visualized under a microscope (Leica Microscopy, Germany).

Statistical analysis

Statistical differences were compared using Prism software (Prism 9; GraphPad Software, CA, USA). When comparing multiple groups, we used one- or two-way analysis of variance (ANOVA) combined with Tukey's multiple comparisons test for data that conformed to normal distribution. All experiments were performed with at least three independent replications. The data are expressed as mean ± standard deviation. p < 0.05 was considered statistically significant.