Background
The endocannabinoid system is a promising therapeutic target in multiple fibrotic diseases. Endocannabinoids are internal lipid mediators that act on two known cannabinoid receptors (CBR), CB1R and CB2R [
1]. The activation of CB2R can protect against fibrosis in various organs, including the kidney [
2], heart [
3], skin [
4] and etc. Only a few studies have reported the role of CB2R in the development of pulmonary fibrosis (PF). CB2R-deficient mice are reported to experience earlier and augmented lung fibrosis [
5]. A recent study found that CB2R activation via JWH-133 can alleviate BLM-induced PF in mice [
6]. These findings indicate that targeting CB2R may serve as a therapeutic strategy for PF treatment.
Besides anti-fibrotic properties, CB2R has anti-inflammatory and anti-oxidative stress effects. For instance, CB2R activation can significantly decrease oxidative stress and downregulate the inflammatory cascade in various inflammatory diseases [
7]. CB2R activation can protect skeletal muscle against ischemia-reperfusion injury by ameliorating oxidative damage [
8]. Moreover, genetic deletion of CB2R can exacerbate acute inflammatory response and neutrophil recruitment [
9]. Meanwhile, numerous studies have shown that excessive oxidative stress and sustained inflammatory response in the lung can aggravate the fibrotic process [
10]. Therefore, the exact role of CB2R and the cellular pathways involved downstream of the CB2R in PF progression should be examined.
As a result, scientists have continuously developed various CB2R selective ligands, including selective agonists (tetrahydromagnolol [
11] and JWH-133 [
5,
6]) and inverse agonists (AM630 [
12], Sch225336 [
13], SR144528 [
14] and sulfonamides derivatives [
15]). The discovery of new scaffold CB2R ligands with better activity and controllable toxicity for potential therapeutic use remains attractive to researchers [
16]. Natural products are ideal compound sources for seeking new drug leads, due to their structural diversities and complexities [
17]. A natural product, Δ9-tetrahydrocannabinol (Δ9-THC) paved the way for understanding the functions of CBRs and the endocannobinoid system. Beside Δ9-THC directly separated from natural source, an array of its synthetic analogues was developed as CBR ligands. However, the source of Δ9-THC is limited, and the synthetic approach of its analogues is hampered by their structural complexity, largely limiting their further utilization and development.
Alternately, organic synthetic methodology studies have provided strategies to rapidly build structural diverse, complex chiral molecules with scaffolds that are frequently found in bioactive molecules [
18], including oxazoles, indoles, bridged carbocycles and spiro skeletons, etc. These “natural product-like” compounds incorporate the advantages of natural products such as diversity and complexity, along with the benefits of synthetic pharmaceutical agents such as accessibility. Although researches have indicated that the new compounds might have good bioactive potentials [
19], to further develop them as therapeutic agent’s remains unrealized. Therefore, there is still huge space in utilizing them for drug lead screening.
This study developed an in-house library covering 1600 chemicals obtained from various organocatalytic methodology studies reported by our group during 2012–2020 (representative structures in Fig.
1 A). The library was screened to identify new CB2R ligands. Virtual screening was first performed via molecular docking and dynamic simulation based on the reported CB2 X-ray structure since computational approaches have successfully been applied in CB2R ligand predictions [
20]. The synthetic YX-2102, a pyrano[2,3-
b]pyridine derivative was identified as a novel CB2R agonist. The potential therapeutic effects of YX-2102 on PF was then systematically investigated both in vivo and in vitro, as well as the underlying mechanisms. YX-2102 ameliorated lung fibrosis by suppressing EMT in a CB2R dependent manner via regulation of the Nrf2/Smad7 pathway, indicating that CB2R is a potential therapeutic target for PF treatment.
Methods
Reagents
Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (UT, USA). Penicillin and streptomycin were purchased from Beyotime (Jiangsu, China). Human recombinant TGF-β1 was provided from Peprotech (Rocky Hill, NJ). Bleomycin sulfate was supplied by Nippon Kayaku Co., LTD. (Japan). Information including source, catalog, dilution ratio, and storage conditions of primary/secondary antibodies are presented in Additional file
1: Table S1, and the structural information and source of the active compounds are listed in Additional file
1: Table S2.
Experimental animals and treatments
Male Sprague-Dawley rats (6–8 weeks old, weight: 180–200 g) were obtained from the Laboratory Animal Center of the Army Medical University (Chongqing, China). The Institutional Animal Care and Use Committee of the Army Medical University approved all animal experiments. The PF model was established as previously described [
21]. Briefly, rats were administered with a single intratracheal instillation of 5 mg/kg BLM (in saline). The sham-operated rats underwent the same procedure, except for intratracheal instillation of normal saline instead of 5 mg/kg BLM. The rats were randomly divided into 4 groups (6 animals each): (1) Sham group (sham-operated rats received vehicle only), (2) YX-2102 group (sham-operated rats treated with YX-2102 alone), (3) BLM group (BLM-induced rats received the vehicle), and (4) BLM + YX-2102 group (BLM-induced rats treated with YX-2102). The vehicle was composed of a 0.9% saline: dimethyl sulfoxide (DMSO): Tween-80 (18:1:1). The YX-2102 was dissolved in this vehicle. The rats were intraperitoneally injected with 25 mg/kg YX-2102 daily or an equal volume of vehicle. The dose administered were selected based on the results of the preliminary experiments (Additional file
1: Fig. S1). All rats were sacrificed via cervical dislocation on day 7 (to observe the acute inflammatory responses) or day 21 (to observe the pulmonary fibrosis). Blood samples were then collected via cardiac puncture, and the serum was stored at − 80 °C for further experimentation. The bronchoalveolar lavage fluid (BALF) was collected, then the whole lung lobes were removed for histopathological and immunohistochemical (IHC) analyses (left lobes) or molecular and biochemical analyses (right lobes). All experiments followed relevant guidelines for the care and use of animals.
Cell culture and treatments
Human alveolar epithelial adenocarcinoma cell line A549 (Cell Bank of the Chinese Academy of Sciences) and rat alveolar type II cell line RLE-6TN (American Type Culture Collection) were cultured in DMEM containing 10% FBS at 37 °C in a 5% CO2 atmosphere. All chemical compounds were dissolved in DMSO to a 10 mg/mL stock concentration, then stored at −20 °C. The cells were serum-starved for 18 h, then preincubated with the chemical compounds (YX-2102, JWH-133 or XL-002) at indicated concentrations for 2 h, and subsequently incubated with TGF-β1 (5 ng/mL) for 24 or 48 h. Total RNA was extracted and subjected to quantitative Real-Time PCR analysis. Proteins were extracted from cell lysates and were subjected to western blotting (WB) analysis. All measurements were performed at least 3 replicates.
Histology, immunohistochemistry (IHC) and immunofluorescence (IF) assays
To visualize the complete tissue architecture and collagen deposition, hematoxylin and eosin (H&E) and Masson’s trichrome staining were processed according to a reported literature [
22‐
24]. The alveolitis and fibrosis score was evaluated based on at least 10 randomly selected fields under microscope, the severity of alveolitis and fibrosis was semi-quantified as described by Szapiel et al. [
25] and Ashcroft et al. [
26]. Immunohistochemical staining and immunofluorescence experiments of lung tissue sections or cultured cells were performed following established procedures [
22‐
24].
Quantitative RT-PCR (qRT-PCR)
The qRT-PCR experiments were performed following the procedures reported previously [
24]. The specific primer sequences for qRT-PCR are shown in the Additional file
1: Table S3.
Enzyme-linked immunosorbent assays (ELISA)
ELISA was used to assess the levels of serum TGF-β1 via the ELISA kits (EK0514, Boster Bioengineering Institute, China), following the manufacturer’s instructions.
Western blotting analysis
Tissues or cells were lysed using RIPA buffer (Beyotime, Jiangsu, China) on ice for 20 min, then quantified using a BCA assay kit (Sangon Biotech, China). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins, then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp, USA) via electrophoresis (Bio-Rad, USA). The membranes were blocked with 5% non-fat milk in PBS with 0.05% Tween-20, then incubated with respective primary antibodies at 4 °C overnight. The protein bands were then stained with horseradish peroxidase (HRP)-conjugated secondary antibodies, and the immunoreactive proteins were visualized using the enhanced chemiluminescence reagent (ECL; Thermo Fisher, USA). Images were obtained using ChemiDoc™ Touch Imaging System (Bio-Rad, USA) and analyzed using Image Lab packages (Bio-Rad).
RNA interference
The siRNA targeting human CB2R (sc-41,586, Santa Cruz, USA) and control siRNA were transferred into A549 cells at 10 µM concentration using lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. After 6 h, the medium was replaced with a complete medium and cells were cultured for another 48 h, then collected for further experiments.
Hydroxyproline assay
Lung hydroxyproline (Hyp) content is a tissue marker for collagen. Herein, Hydroxyproline Analysis Kit (Jiancheng, China) was used to measure lung Hyp content, following the manufacturer’s instructions. The content was expressed as micrograms Hyp per milligram wet weight (µg/mg).
Micro-computed tomography (CT)
The rats were anaesthetized using isoflurane and the lungs were imaged using a Quantum FX Micro-CT scanner (PerkinElmer Inc., MA) as previously reported [
27]. The Mimics 17.0 (Materialise Software, Belgium) was used to convert the acquired data into 3D models to show airways, lung lobes and fibrosis.
Bronchoalveolar lavage fluid (BALF)
The BALF was obtained by cannulating the trachea and infusing with 10 mL ice-cold 0.9% saline thrice. About 60–80% of fractions were recovered. The BALF was centrifuged (2000 rpm, 10 min, 4˚C), and the obtained supernatant was stored at − 80 °C for further experimentation. The cell sediments were re-suspended and stained with a modified Wright–Giemsa solution (Jiancheng, Nanjing, China). Differential cell counts were determined using 400 lung inflammatory cells.
Molecular docking
The crystal structure of CB2R was obtained from RCSB Protein Data Bank (ID: 5ZTY) [
20]. Before docking, the protein structure was prepared as reported [
28,
29], using SYBYL-X 2.0 software and PDB2PQR Server [
30]. The binding pocket was generated based on the ligand 9JU with default settings. The Surflex-Dock in SYBYL-X 2.0 software (SFXC mode) was used for the docking of the compound library CB2R. The following parameters were set on: pre-dock minimization/post-dock minimization/consider ring flexibility/molecule fragmentation/soft grid treatment. Obtained docked complexes with the highest scores (top 5) were subjected for molecular dynamic simulation (MD). The binding interactions between the ligand and CB2R were characterized using LigPlot+ [
31].
MD simulation, trajectory analysis and calculation of binding free energies
The structure preparation, MD simulation, the trajectory analysis and binding free energies calculation were performed using different modules of AMBER14. All detailed procedures and parameter settings followed the previous articles reported by our group [
28,
29].
CB2R binding assay
The affinities of compounds with human CB2 receptors were determined using a radioligand binding assay as previously described [
25] [using [3 H](−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) as CB receptor radioligand].
Statistical analysis
The data are shown as the mean ± SEM, and significance was established when P < 0.05. One-way analysis of variance (ANOVA) followed by the LSD or Dunnett’s test was used for pairwise comparisons.
Discussion
Previous studies have identified numerous CB2R ligands with different scaffolds, including the natural polyphenol compound, tetrahydromagnolol [
11], polycyclic chromene JWH-133 [
5], indole derivative AM630 [
12], and substituted imidazole, SR144528 [
15]. These agonists or antagonists have demonstrated good binding affinity with CB2R and pharmacological activities against various pathologies, including fibrosis and non-small cell lung cancer [
39]. These studies demonstrated the therapeutic potential of developing new CB2R ligands for the treatment of several diseases.
Here, we identified a novel CB2R agonist YX-2102 through in silico screening of an in-house library consisted of over 1600 + molecules with high structural diversity and complexity. The structure of YX-2102, which contains a pyrano[2,3-
b]pyridine scaffold with continuous stereocenters, is rarely found in commercial compound libraries. Similarity comparison between YX-2102 and 1000 high affinitive CB2R ligands was less than 50%, suggesting it is quite different from the reported ligands in scaffolds (Additional file
1: Fig. S6). Despite its high molecular complexity and unusual structural features, YX-2102 can be easily synthesized via well-established routes, with high yield and good enantioselectivity. Hence, where structural optimization is needed, YX-2102 is easily modifiable to obtain derivatives with better activity. These results also demonstrated the superiority and potential of the organic synthetic methodology-based library in drug lead discovery.
We also showed that YX-2102 administration significantly ameliorated BLM-induced PF in rats, and alleviated early inflammatory response induced by bleomycin by inhibiting M1 macrophage polarization. Moreover, our results indicate that YX-2102 retarded TGF-β1-induced EMT in alveolar epithelial cells in a CB2R-dependent manner, at least partially by enhancing Nrf2-mediated Smad7 elevation, highlighting CB2R as a potential therapeutic target against fibrotic diseases.
Mounting evidence has implicated the cannabinoid system in lung homeostasis and disease [
43]. CB1R and CB2R are thought to have different and sometimes opposing roles in the development of tissue fibrosis [
2,
44]. The CB1R is mainly present in the central nervous system while the CB2R with a dynamic range of expression levels in different cell types of human tissues, including immune and hematopoietic cells, epithelial cells, myocytes, fibroblasts, and skin keratinocytes [
45]. Hence CB2R is considered a promising therapeutic target in the treatment of various diseases associated with inflammation and tissue injury. However, few studies have elucidated the role of cannabinoid receptors in PF. Pharmacologic inhibition of CB1R enhances radiation- [
46] and BLM-induced pulmonary fibrosis [
44]. In contrast, recent studies show that CB2R activation using a specific agonist, JWH133, suppressed nicotine-induced mouse interstitial lung fibrosis [
47] and BLM-induced pulmonary fibrosis [
6]. Consistently, using YX-2102, a novel selective CB2R agonist, we show that CB2R activation protects against BLM-induced lung fibrosis. Past studies identified cannabinoid receptors on structural cells and most inflammatory cells in the lung [
43]. Our data show that CB2R is highly expressed in alveolar epithelial cells and in bleomycin-induced PF rat lung tissue, especially in the fibrotic area. Taken together, these data implicate CB2R in PF progression and highlight it as a promising target for the identification of novel therapies against PF.
Numerous studies show that CB2R activation exhibits anti-inflammatory effects and it has been recognized as a potential target for several inflammatory diseases [
7,
48]. Sustained inflammation plays a key role in the pathogenesis of pulmonary fibrosis. Inflammatory cells in the lungs, including lymphocytes, neutrophils, and macrophages, are important sources of various inflammatory mediators and influence the onset and progression of PF. Intratracheal instillation of bleomycin causes acute lung injury, and the ensuing inflammatory response is implicated in fibrosis. Here, we found that intrapulmonary exposure to bleomycin markedly enhances the number of inflammatory cells and associated inflammatory mediators, while YX-2102 administration alleviates the inflammatory responses induced by bleomycin. Notably, the expression of the anti-inflammatory cytokines was significantly enhanced by YX-2102. These results reaffirmed the anti-inflammation properties of CB2R during the early stages of pulmonary fibrosis.
Nevertheless, the mechanism by which CB2R modulates inflammatory responses during pulmonary fibrosis had not been determined. In the airway and lung microenvironment, macrophages are intricately involved in inflammation and fibrosis. Infiltrating macrophages at sites of lung tissue injury were activated and polarized into M1 or M2 subpopulations, with M1 macrophages being pro-inflammatory/anti-fibrotic and M2 macrophages being pro-fibrotic or regulatory [
40]. It has been reported that the anti-inflammatory effect of CB2R may be mediated by regulating macrophage polarization. Numerous studies indicate that CB2R activation attenuates inflammation via reducing M1 macrophage polarization and enhancing M2 polarization. In contrast, Du et al. reported that CB2R alleviates inflammation by suppressing M1 macrophages, rather than upregulating M2 macrophages [
36]. Our data have shown that YX-2102 markedly decreased the number of M1 macrophages and increased M2 macrophages. However, because M2 macrophages promote tissue fibrosis and CB2R inhibits fibrosis, M2 polarization upon CB2R activation seems counterintuitive. There are several potential explanations for this. (i) Excessive inflammatory responses in the early stages of BLM-induced lung injury/fibrosis that aggravate tissue damage. Thus, reduction of early infiltrating pro-inflammatory M1 macrophages upon CB2R activation may mitigate the severity of subsequent fibrosis. (ii) The current classification of M1/M2-polarized macrophages may be overly simplistic. Actually, both M1 and M2 macrophages are intricately involved in the progression of PF and their contributions to this disease remain elusive. (iii) A timely switch and dynamic balance of M1 and M2 macrophages are needed to maintain tissue homeostasis. It may be presumed that CB2R activation by YX-2102 suppresses the accumulation of M1 macrophages, which in turn, led to enhancement of the relative proportion of M2 macrophages in the lung. These speculations will be the focus of future investigations. Our findings indicate that YX-2102 may improve lung fibrosis via CB2R-mediated inhibition of M1 polarization, thereby significantly reducing early lung inflammation.
EMT plays a critical role in pathogenesis of fibrosis in many organs, including lung [
49,
50]. It is a highly active process where epithelial cells lose their epithelial E-cadherin and gains mesenchymal markers such as fibronectin and α-SMA [
51]. Fibronectin is required for collagen matrix assembly and α-SMA is an important biomarker of activated myofibroblasts, both of which were believed to be the key contributors to organ fibrosis [
52]. Following YX-2102 treatment, the decreased expression of fibronectin and α-SMA concomitant with an elevation in E-cadherin levels implied that YX-2102 might ameliorates the degree of lung fibrosis through inhibiting the process of EMT. Furthermore, of multiple stimuli involved in pulmonary fibrosis, TGF-β1 is considered the master regulator of pathological fibrosis and is a widely studied profibrotic factor involved in driving EMT [
53]. The TGF-β1/Smad pathway has been implicated in the progression of PF. Upon binding with its receptor, TGF-β1 triggers the phosphorylation of Smad2/3 for their activation. The Smad2/3 dimer and Smad4 form complexes and then translocate into the nucleus. In nucleus, the Smad complex suppresses the expression of E-cadherin through transcription factors Snail1 and Slug. The transcriptional effects of TGF-β/Smad signaling also indirectly drive EMT by inducing the expression of Twist, Zeb1 and Zeb2. These events result in downregulation of epithelial markers and the upregulation of mesenchymal genes [
54]. However, the involvement of CB2R and its role in TGF-β1-induced EMT have not been previously studied. Here, we show that CB2R activation by YX-2102 significantly repressed changes in cellular EMT markers by reducing the activation Smad2/3 and inhibiting the translocation of Smad3:Smad4 complex into the nucleus in response to TGF-β1 stimulation. Additionally, YX-2102 markedly reduced the mRNA levels of the EMT transcription factors, Snail and Slug. These results are consistent with observations that in BLM-induced PF rats, EMT-related markers and TGF-β1/Smad signaling was suppressed by YX-2102. Consistently, JWH-015, another CB2R agonist, is reported to inhibit macrophage-induced EMT in A549 cells by downregulating epidermal growth factor receptor (EGFR) and its targets [
39]. Thus, we presumed that YX-2102, a CB2R agonist may inhibit EMT by inducing biological mediators that disturb TGF-β/Smad signaling, which may account for the fibrosis alleviation.
To investigate the mechanism by which YX-2102 inhibits TGF-β1-induced EMT, we assessed the level of Smad7, a negative regulator of TGF-β-signaling. Smad7 inhibits TGF-β-induced transcriptional responses by inhibiting TGF-β-mediated Smad2/3 phosphorylation or interfering with Smad-DNA interaction. Decreased Smad7 expression in fibrotic lung tissues corresponds with increased TGF-β1/Smad pro-fibrotic signaling, which is a critical event in PF [
55]. Our results are consistent with past findings that Smad7 is significantly suppressed in a bleomycin-induced PF mouse model. We also found that YX-2102 restored Smad7 expression in TGF-β treated alveolar epithelial cell in a CB2-dependent manner, in vitro. Correspondingly, treatment with YX-2102 significantly elevated the level of Smad7 in lung tissues of rats after bleomycin instillation. A past study found that CB2R activation markedly increased the level of Smad7 during skin wound repair in mice [
56]. To our knowledge, this is the first study linking CB2 activation to induction of Smad7 in alveolar epithelial cells.
Nrf2 is a redox-sensitive transcription factor that protects against oxidative stress injury and inflammation. Studies have shown that Nrf2 protects from PF. Nrf2 has been reported to protect against PF by regulating cellular redox levels [
57]. Additionally, Nrf2-mediated redox imbalance promotes profibrotic myofibroblast phenotypes, resulting in persistent fibrosis in lungs of aged mice [
58]. Given the effect of CB2R on oxidative stress, we investigated if YX-2102 influences Nrf2 expression in alveolar epithelial cells, and found that activating CB2R with YX-2102 markedly enhanced Nrf2 expression and promotes Nrf2 translocation into the nucleus. These effects were inhibited using a selective CB2R antagonist. Numerous studies have reported crosstalk between CB2R and Nrf2 signaling pathway [
8,
42]. CB2R activation ameliorates myocardial fibrosis by accelerating Nrf2 translocation into the nucleus and suppressing the TGF-beta1/Smad3 pathway in a Nrf2-dependent manner [
42]. Interestingly, recent studies found that Nrf2 inhibits TGF-β1-induced EMT and lung fibrosis by regulating snail expression [
59]. Furthermore, Nrf2 is reported to positively regulate Smad7 expression and the Nrf2-Smad7 axis plays a critical role in the prevention of renal and cardiac fibrosis [
60,
61]. Thus, although the precise mechanisms remain to be explored, past studies and our findings imply that the inhibitory effect of CB2R activation with YX-2102 on TGF-β-induced EMT and pulmonary fibrosis might be partly ascribed to Nrf2-mediated smad7 elevation that serve to perturb the TGF-beta pathway during this process. However, this is a potential mechanism by which CB2R activation by YX-2102 inhibits EMT. Further in vitro and in vivo studies are needed to elucidate the mechanism underlying and to determine its feasibility in future applications.
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