ACPA (N-cyclopropyl-5Z, 8Z, 11Z, 14Z-eicosatetraenamide; C23H37NO; CAS: 229021-64-1; MW: 343.6, purity ≥98%) was obtained from Cayman Chemical Company. BLM (#RB003) was purchased from BioTang (Lexington, MA, USA). Recombinant TGF-β1 protein (#240-B) was manufactured by R&D Systems (Minneapolis, MN, USA). Primary antibodies against α-smooth muscle actin (α-SMA, #19245), Collagen (#72026), Smad2 (#5339), phospho-Smad2 (Ser465/Ser467, #18338), Smad3 (#9523), phospho-Smad3 (Ser423/425, #9520), Slug (#9585), Snail (#3879), and β-actin (#3700) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against CB₁R (#ab23703), TGF-β receptor Ⅱ, and Fibronectin (#ab2413) were purchased from Abcam Company (Cambridge, MA, USA). Primary antibody against CB₂R (#sc293188) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-rabbit IgG (Alexa Fluor 488 Conjugate) (#4412) was purchased from Cell Signaling Technology (Danvers, MA, USA). DAPI (#C1002), Forskolin (#S1612), Hematoxylin and Eosin (H&E) staining kit (#C0105), and BeyoClick™ EdU Cell Proliferation Kit were purchased from Beyotime (Shanghai, China). Masson staining Kit (#D026-1-3) was obtained from NanJing JianCheng (China). Reverse transcription kit and SYBR Green Real-Time PCR kit were purchased from Toyobo (Osaka, Japan). MTT was purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Six- to eight-week-old C57BL/6 mice were purchased from the Laboratory Animal Center Shanghai Laboratory Animal Center (SLAC, Shanghai, China). Mice were maintained in a climate-controlled room (25°C, 55% humidity, and 12 h light/darkness cycles), and all procedures were conducted with the use of protocols approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.

C57BL/6 male mice were randomly divided into three groups: the control (PBS + Vehicle) group, BLM (BLM + Vehicle) group, and ACPA (BLM + ACPA) group. ACPA was dissolved in a vehicle (1:1 mixture of PBS and ethanol). For the experiments (Figure 1C), the mice received an intratracheal injection (i.t.) of BLM (1.4 U/kg) or PBS at a volume of 20 μl per mouse after anesthetized with pentobarbital sodium (50 mg/kg) by intraperitoneal injection (i.p.). From the next day after BLM injection, the mice in the ACPA group were subjected to ACPA (3 mg/kg, i.p.), and the mice in the control and BLM groups received intraperitoneal injection of vehicle solution (1:1 mixture of PBS and ethanol) at a volume of 10 μl/g per body weight every other day (day 0–13). Animals (n = 7 in each group) were inspected daily in survival study, and death was recorded for each animal. Every animal found dead was necropsied. Surviving animals (n = 7 in control group; n = 5 in BLM group; n = 7 in ACPA group) were euthanized at day 14 to collect tissues. The left lungs from three mice of each group were fixed with polyoxymethylene for histological analysis. The remaining right lungs and lungs from other mice were collected for mRNA, protein, and hydroxyproline (HYP) measurements.

The left lungs from three mice of each group were fixed with 4 ml of 4% polyoxymethylene for 48 h, followed by dehydration in graded alcohol and embedding in paraffin. Then the paraffin-embedded sections were sliced into 5-μm sections and stained with H&E kit (Beyotime, Shanghai, China) or Masson staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions of the manufacturer. Stained sections were photographed with an Olympus BX51 microscope (Tokyo, Japan).

The lungs of the euthanized C57BL/6 mice (6–8 weeks old) were perfused with 10 ml of normal saline, removed immediately into ice-cold Hanks’ balanced salt solutions, chopped finely with scissors, and digested with 2 mg/ml of collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) for 0.5 h. Then after washing with PBS three times, the extracted fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, MA, USA) supplemented with 10% fetal bovine serum (Sijiqing, Shanghai, China), 1% penicillin and streptomycin (Yishen, Shanghai, China) at 37°C in a 5% CO₂ incubator for 5 days. The medium was changed every 2 days. For the study of the effects of ACPA on the lung fibroblast in vitro, the cultured lung fibroblasts were incubated with serum‐free medium containing PBS or TGF-β1 (20 ng/ml) for 24 h, and treated with vehicle or ACPA 0.5 h before TGF-β1 stimulation.

The collagen contents in lung tissues were evaluated by HYP assay using an HYP assay kit (Nanjing Jiancheng, Nanjing, China). According to the instructions of the manufacturer, the lung tissues were homogenized in 1 ml of hydrolysate and heated in 1 ml of hydrolysate at 95°C for 20 min, and pH was adjusted from 6.0 to 6.8. The hydroxyproline was detected by incubation with chloramine T and p-dimethylaminobenzaldehyde, and the absorbance was measured at 550 nm by a Biotek ELx800 (Winooski, VT, USA).

RNA samples from lung tissues or cells were extracted using Trizol (Sigma-Aldrich, St Louis, MO, United States). cDNA was produced using qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the protocol of the manufacturer and used in subsequent real-time qPCR reactions. Quantitative real-time PCR was carried out to determine relative gene expression using the SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) on StepOne Plus (Thermo Fisher Scientific, Waltham, MA, USA) as specified by the manufacturer. The quantification of target genes was identified by comparing Ct values of each sample normalized with Ct value of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and reaction specificity was verified by melting curve analysis. The primer sequences for target genes in real-time PCR assay were obtained from HuaGene Company (Shanghai, China) as shown below: the primers GAPDH forward 5′-CAT​CAC​TGC​CAC​CCA​GAA​GAC​TG-3′ and reverse 5′-ATG​CCA​GTG​AGC​TTC​CCG​TTC​AG-3′, α‐SMA forward 5′-TGC​TGA​CAG​AGG​CAC​CAC​TGA​A-3′ and reverse 5′-CAG​TTG​TAC​GTC​CAG​AGG​CAT​AG-3′, collagen forward 5′-CCT​CAG​GGT​ATT​GCT​GGA​CAA​C-3′ and reverse 5′- CAG​AAG​GAC​CTT​GTT​TGC​CAG​G-3′, fibronectin forward 5′-CCC​TAT​CTC​TGA​TAC​CGT​TGT​CC-3′ and reverse 5′-TGC​CGC​AAC​TAC​TGT​GAT​TCG​G-3 ′, CB₁R forward 5′-ATC​GGA​GTC​ACC​AGT​GTG​CTG​T-3′ and reverse 5′-CCT​TGC​CAT​CTT​CTG​AGG​TGT​G-3′.

Protein samples from lung tissues or cultured cells were lysed and extracted with loading buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor cocktail (Roche Applied Science, USA). Protein concentration was measured using BCA Protein Assay kit (Beyotime, Shanghai, China) according to the protocol of the manufacturer. Protein samples were separated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked in 10% bovine serum albumin solution for 2 h at room temperature. Then the membranes were incubated with the appropriate primary antibodies overnight at 4°C, followed by the incubation with conjugated secondary antibodies at room temperature for 2 h. To visualize immune complexes, the membranes were stained using chemiluminescent substrate and imaged by a ChemiDoc MP System (Bio-Rad, MA, USA). The intensity of the protein band was quantified by ImageJ software and normalized to β-actin.

Scratch wound migration assay and Transwell chamber migration assay were performed to detect the migration of primary lung fibroblasts. In scratch wound migration assay, lung fibroblasts were inoculated on six-well plates (1 × 10⁶ cells per well) and cultured with complete culture medium overnight until the cells adhere to the wall. Then the cells were washed three times with PBS and cultured in serum‐free medium. A 200-µl pipet tip was used for scraping the cell monolayer in two straight lines as a cross. The cells were washed three times with serum‐free medium to ensure that no cell debris remained in the scratch areas, and the cells were grown in serum-free medium with PBS + Vehicle, TGF-β1 (20 ng/ml) + Vehicle, or TGF-β1 (20 ng/ml) + ACPA (0, 10, 30 μM) for 24 h. Images were captured with a phase-contrast microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ software.

Lung fibroblasts (50,000 cells in 200 µl serum‐free medium PBS + Vehicle, TGF-β1 (20 ng/ml) + Vehicle, or TGF-β1 (20 ng/ml) + ACPA (0, 10, 30 μM) were transferred to the upper chambers, and 600 µl of DMEM supplemented with 10% fetal bovine serum was added to the lower chambers. After 24 h of incubation, the medium in the upper chambers was discarded, and the cells that traversed to the reverse face were carefully washed with PBS three times, fixed with the pre-prepared fixative (methanol: acetic acid = 2:1), and then stained with 0.1% crystal violet for 20 min. Pictures were taken with a microscope (Olympus, Tokyo, Japan).

Lung fibroblasts (1 × 10⁴ cells per well) plated on 96-well plates were treated with vehicle or ACPA (0, 10, 30, and 100 μM) for 24 h. Then 20 μl MTT solution (5 mg/ml) was added into the wells and incubated for 4 h. The culture supernatant was discarded, and 100 μl of DMSO was added into each well. OD absorbance at 490 nm was measured by a microplate reader (BioTek, Vermont, USA).

The EdU Cell Proliferation Kit was used to measure cell proliferation. Lung fibroblasts (5 × 10⁵ cells per well) were seeded on 12-well plates and cultured with complete culture medium overnight with the cells adhering to the wall. The cells were pretreated with different concentrations of ACPA (0, 30, 100 µM) for 30 min and then stimulated with TGF-β1 (20 ng/ml) for 24 h. According to the instructions of the manufacturer, fresh DMEM containing EdU (10 µM) was added to the wells. After a 2-h incubation, the EdU medium was discarded, and the fibroblasts were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 for 15 min. Next, the cells were incubated with Click Reaction solution for 30 min and stained with Hoechst for 10 min. Finally, images were captured using the microscope (Olympus, Tokyo, Japan).

Fibroblasts were plated in 12-well plates (3 × 10⁵ cells per well) overnight. The cells were treated with ACPA (30 μM) for 30 min before being challenged with 20 ng/ml of TGF-β1 for 24 h. The cells were fixed with 4% PFA for 15 min, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 5% BSA for 1 h. Then the cells were incubated with TGF-β receptor Ⅱ primary antibody overnight at 4°C. After washing with PBS three times, cells were incubated with Alexa 488-labeled secondary antibody for 2 h at room temperature, and then cells were stained with DAPI (5 μg/ml) to visualize the cell nuclei. Immunofluorescence images were recorded by a laser-scanning confocal fluorescence microscope (Leica Microsystem, Wetzlar, Germany).

Statistical analysis was performed using the Prism software (ver. 8.3; GraphPad, San Diego, CA, USA). Comparisons between different groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test, and Student’s t-test was used to compare differences between two groups. All experiment results were presented as the mean ± SEM. The referred experiment was performed independently for at least three times. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

CB₁R has been found to be linked to multiple pathologic processes in fibrosis, including pulmonary fibrosis (Garcia-Gonzalez et al., 2009; Marquart et al., 2010; Lazzerini et al., 2012; Bronova et al., 2015; Lecru et al., 2015; Chiurchiù et al., 2016; Cinar et al., 2017; Correia-Sá et al., 2020a; Correia-Sá et al., 2020b), but the actual function of CB₁R activation in pulmonary fibrosis remains controversial. In the present study, we first examined the expression of CB₁R in the lung tissues of a murine pulmonary fibrosis model by i.t. injection of BLM (1.4 U/kg). We found that the mRNA levels of CB₁R significantly increased in the lung tissues of mice with pulmonary fibrosis compared with normal mice (Figure 1A), which indicated that overexpressed CB₁R might play certain role in the pathologic processes of pulmonary fibrosis. Then we observed the influences of CB₁R activation on mice with BLM-induced pulmonary fibrosis using the highly selective CB₁R agonist ACPA (Figure 1B) to stimulate CB₁R. The murine model of pulmonary fibrosis was established by i.t. injection of BLM, and the function of ACPA for pulmonary fibrosis was evaluated by i.p. injection (Figure 1C). Compared with the mice in normal control group, the pulmonary fibrosis mice subjected to BLM i.t. injection exhibited significantly diminished body weight, an important indicator of pathological severity following BLM challenge, while ACPA treatment largely rescued the weight loss of pulmonary fibrosis mice (Figure 1D). Similarly, the survivorship curve also manifested that ACPA treatment improved the survival rate of pulmonary fibrosis mice (Figure 1E). In addition, HE staining demonstrated extensive inflammatory cell infiltration and severe pulmonary lesions in lung tissues of mice with BLM-induced pulmonary fibrosis, which were apparently reduced by ACPA administration (Figure 1F). Collectively, these data showed that CB₁R overexpression in lung tissues and activation stimulated by ACPA would help to mitigate pathological changes and improve survival of pulmonary fibrosis mice.

Pulmonary fibrosis is a group of lethal disease characterized by deposition of a pathological ECM, we further evaluated the efficacy of CB₁R-selective agonist ACPA on collagen deposition and the expression of fibrotic makers in BLM-induced experimental pulmonary fibrosis in vivo (Figure 2). Histologic assay of Masson staining demonstrated a dramatic collagen accumulation, the indicative of fibrosis, in BLM-stimulated mice, while ACPA treatment strongly attenuated deposition of pulmonary collagen (Figure 2A). Consistently, HYP, the main component of collagen, was also significantly reduced by treatment compared with that in the BLM group (Figure 2B). In line with the results of Masson staining and HYP assay, the productions (protein levels) of ECM, e.g., collagen, fibronectin, and α-SMA, which are biomarkers for fibrotic levels, remarkably decreased in ACPA group (Figures 2C–F). Similarly, the transcription (mRNA levels) of collagen, fibronectin, and α-SMA were also downregulated by ACPA (Figures 2G–I). Collectively, these results showed that CB₁R-selective agonist ACPA protected pulmonary fibrosis mice against BLM-induced pulmonary fibrosis with the inhibition of ECM production.

Fibroblasts are the primary cells that build and maintain the ECM. Increased activation of lung fibroblast is central for the initiation and maintenance of unremitting deposition of ECM and fibrotic lesions in pulmonary fibrosis (Xia et al., 2014; DePianto et al., 2015; Kasam et al., 2020). In this study, we investigated the expression of CB₁R in lung fibroblasts isolated from BLM-challenged mice and found that CB₁R mRNA levels were significantly increased in the isolated lung fibroblasts from mice with BLM-induced pulmonary fibrosis compared with control mice (Figure 3A), which is in accordance with the increased CB₁R mRNA levels in lung tissues of pulmonary fibrosis mice (Figure 1A). TGF-β1 is one of the most potent and well-studied profibrotic inducers of pulmonary fibrosis that triggers and greatly enhances the fibrogenic activity of lung fibroblasts (Fernandez and Eickelberg, 2012; Martinez et al., 2017; Aschner et al., 2020; Lee et al., 2020). We isolated primary mouse lung fibroblasts to examine the expression of CB₁R and ECM proteins induced by TGF-β1. Primary mouse lung fibroblasts were incubated with PBS or TGF-β1 for 24 h after pretreated with vehicle or ACPA with different concentrations. As shown in Figures 3B–D, both mRNA and protein levels of CB₁R in primary mouse lung fibroblasts demonstrated a time-dependent augmentation with the stimulation of TGF-β1, suggesting that TGF-β1 promotes CB₁R expression in lung fibroblasts. By contrast, CB₂R expression was much lower in lung fibroblasts and its level was not observably changed by TGF-β1 or ACPA treatment (Supplementary Figures S1A–E). Consistent with BLM-induced pulmonary fibrosis, the expression of ECM protein collagen, fibronectin, and α-SMA in primary fibroblasts also increased enormously in response to TGF-β1 stimulation. Although ACPA (10, 30 μM) alone did not affect the expression of ECM proteins (Figure 3I) or exhibit obvious cytotoxicity to fibroblasts (Figure 3J), when administrated with TGF-β1, it significantly reduced the augmentation of ECM proteins induced by TGF-β1, which are also considered as fibrosis markers, in a dose-dependent manner (Figures 3E–H). Taken together, these results revealed that lung fibroblasts expressed CB₁R when stimulated by TGF-β1 and CB₁R activation, in turn, inhibiting the excessive expression of ECM proteins in TGF-β-challenged lung fibroblasts.

The migratory capacity of fibroblasts by responding to cytokines and chemokines, e.g., TGF-β1, is important for the progression of pulmonary fibrosis (Kasam et al., 2020). To ascertain whether CB₁R activation participates in modulating TGF-β1-triggered cell migration of primary lung fibroblasts, we conducted scratch wound migration assay and Transwell migration assay on primary lung fibroblasts. Primary fibroblasts were pretreated with CB₁R-selective agonist ACPA (0, 10, 30 μM) before stimulating with TGF-β1 for 24 h. In EdU cell proliferation assay, TGF-β1 significantly stimulate the proliferation of fibroblasts, as obviously increased EdU-positive cells were seen in TGF-β1 group than those in the control group, and it was reduced by ACPA (10, 30 μM) treatment (Figures 4A, D). Consistently, TGF-β1 significantly spurred fibroblasts to migrate to the scratch guidelines in scratch wound healing assay, while ACPA alleviated the fibroblast migration with the high concentration (30 μM) of ACPA showing a nearly radical inhibition (Figures 4B, E). Moreover, numbers of transmigrating fibroblasts were much higher among TGF-β1-challenged fibroblasts than controls, while ACPA (30 μM) obviously decreased the migration of the lung fibroblasts toward fetal bovine serum (Figures 4C, F). Thus, we concluded that CB₁R-selective agonist ACPA restrained cell proliferation, migration, and motility of lung fibroblasts triggered by TGF-β1.

In the present study, we revealed that CB₁R-selective agonist ACPA substantially suppressed ECM production both in the lung tissues of mice with pulmonary fibrosis and in TGF-β-challenged lung fibroblasts, indicating that CB₁R activation downregulated TGF-β signaling mediated the expression of ECM proteins in pulmonary fibrosis. To manifest this, we investigated the activation of canonical TGF-β-Smad2/3 signaling and the levels of its downstream effectors in TGF-β1-challenged primary lung fibroblasts with ACPA treatment. Western blot showing that TGF-β1 stimulation resulted in the activation of canonical TGF-β-Smad2/3 signaling, assessed by the elevated p-Smad2 and p-Smad3 (Figures 5A–C), followed by the expression of downstream transcription factors Snail and Slug (Figures 5A, D, E), key regulators that mediated TGF-β-Smad signaling triggered the overexpression and excessive production of ECM proteins in pulmonary fibrosis. However, CB₁R-selective agonist ACPA exerted a notable inhibition on elevated p-Smad2, p-Smad3, snail, and slug in lung fibroblasts spurred by TGF-β1 (Figure 5). These data indicate that CB₁R activation acted as a negative regulation of canonical TGF-β-Smad2/3 signaling, downregulated TGF-β triggered Snail and Slug expression, and subsequent overexpression of ECM proteins in lung fibroblasts.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.