Brahma-related gene 1 acts as a profibrotic mediator and targeting it by micheliolide ameliorates peritoneal fibrosis

Eight-to-ten-week-old male C57BL/6J mice were obtained from Southern Medical University Animal Center (Guangzhou, China). The animal protocol was reviewed and approved by the Animal Experimental Ethics Committee at Southern Medical University (Approval No. LAEC-2020-166). A mouse model of peritoneal fibrosis was established by daily intraperitoneal injection of 3 ml of standard PD fluid composed of 4.25% glucose(Baxter HealthCare, Deerfield, IL) for 4 weeks [27, 28]. To investigate the role of BRG1, in vivo overexpression of BRG1 in mice was performed by ultrasound-microbubble-plasmid containing mouse BRG1 expression plasmid (pBRG1) [16] or empty plasmid(Vector) treatment on days 0 and 14 over 28 days [28]. To investigate the role of DMAMCL in BRG1 overexpression-induced peritoneal fibrosis, five groups of mice were treated as follows: (1) the control group received daily intraperitoneal injection of 3 ml saline and ultrasound-mediated vector plasmid gene transfer treatment and daily intragastric administration of saline; (2) mice in the PD group received daily intraperitoneal injection of 3 ml PD fluid and ultrasound-mediated vector plasmid gene transfer treatment and daily intragastric administration of saline; (3) mice received PD fluid and ultrasound-mediated BRG1 expression plasmid gene transfer treatment and daily intragastric administration of saline; (4) mice received PD fluid and ultrasound-mediated vector plasmid gene transfer treatment and daily intragastric administration of DMAMCL (25 mg/kg); and (5) mice received PD fluid and ultrasound-mediated BRG1 expression plasmid gene transfer treatment and daily intragastric administration of DMAMCL (25 mg/kg).The detailed experimental design was shown in Fig. 5a.After the 28-day PD fluid treatment, the mice were sacrificed. Parietal and visceral peritoneal tissues were collected for further analysis.

The thickness of the submesothelial layer was determined on 4-um paraffin-embedded tissue sections stained with Masson’s trichrome staining according to a routine procedure. The thickness of the submesothelial tissue was calculated by measuring the average distance from the superficial mesothelial cell layer to the muscle from 15 independent measurements for each animal. Immunohistochemical staining was conducted as previously described [27]. Paraffin sections (4-um thick) were subjected to deparaffinization in xylene and rehydrated in a series of decreasing alcohol concentrations. Subsequently, the sections were exposed to sodium citrate antigen retrieval solution at a sub-boiling temperature for 8 min in a microwave oven to restore the antigen. To inhibit the endogenous peroxidase of the samples, 3% H2O2 was applied for 15 min in a dark environment. Following this, the sections were incubated in PBS containing 3% BSA for 60 min at room temperature. After overnight incubation with primary antibodies at 4 °C, the samples were treated with secondary antibody for 30 min at room temperature. Finally, the tissue sections were exposed to diaminobenzidine (DAB) for 3 min at room temperature. Antibodies used were described in Additional file 1: Table S1.

Two different cell types were analyzed: primary rat peritoneal mesothelial cells (RPMCs) and immortalized human peritoneal cells (HMrSV5 cells). Primary RPMCs were derived from trypsin/ ethylenediaminetetraacetic acid digestion method from peritoneal tissue obtained from male SD rats (120 g–150 g). After centrifuged and washed, cells were cultured in DMEM/F-12 medium (Gibco)) containing 10% fetal bovine serum(Gibco), 1% antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin,Gibco) and 1% Insulin-Transferrin-Selenium (Gibco) at 37 °C in 5% CO2. HMrSV5 cells (kindly provided by Professor Xueqing Yu, Sun Yat-Sen University,Guangzhou, China) were cultured in DMEM/F12 with 10% fetal bovine serum and 1% antibiotics at 37 °C in 5% CO2. For TGF-β1 treatment, cells were grown to ~ 70% confluence and starved with serum free DMEM/F-12 medium for 12 h and then treated with 5 ng/ml recombinant human TGF-β1 (R&D Systems, Minneapolis, MN)) for 48 h. For PD fluid treatment, serum-starved cells were cultured in conventional PD fluid (Baxter HealthCare, Deerfield, IL) for 48 h with different concentrations. Cells incubated in medium without stimuli or with medium diluted 1:2 with sterile saline served as a control for TGF-β1–stimulated and PD fluid-stimulated cells, respectively. In some experiments, cells were treated with TGF-β1 and cotreated with BRG1 inhibitor PFI-3 (2 μM, Selleck Chemicals), MCL (1.25 μM, 2.5 μM, 5 μM) in a serum-free medium for 48 h.

Cells were transiently transfected with BRG1-specific small interfering RNA (siRNA) (designed and synthesized by GenePharma, Shanghai, China) or siNC using lipofectamine 2000 reagent (Invitrogen, MA, U.S.A.). The siNC was used as  a negative control. Cells were transfected with pEZ-M14-FLAG plasmid containing full length BRG1, plasmid containing BRG1 mutant(asparagine 1540 was mutated into alanine,N1540A), BRG1 deletion mutant expression plasmids [N-terminal domain (1–765), C-terminal domain(1301–1647) or ATPase domain (766–1300)], plasmid containing H3 lysine 14 full length(H3K14WT) and two mutant plasmids (H3K14Q and H3K14R with lysine 14 mutated to glutamine and arginine, respectively) (all from GeneCopoeia) using Lipofectamine 3000(Invitrogen). The sequence of siBRG1 were shown in Additional file 1: Table S2.

Total protein sample from tissues or cells were extracted with RIPA lysis buffer (KeyGen, Nanjing, China) according to the routine procedure. Cytoplasmic and nuclear proteins from RPMCs were separated by a commercial protein separation kit (P0028, Beyotime, China) according to the manufacturer’s protocol. Antibodies used were listed in Additional file 1: Table S1.

Cells cultured on coverslips were fixed with 4% formaldehyde followed by permeabilization with 0.1% Triton X-100. And then these slides were blocked with 5% goat serum for 1 h at room temperature. Subsequently, they were incubated with primary antibodies. After washing, slides were incubated with fluorescent-labeled secondary antibodies for 1 h at room temperature, and then nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (BestBio, Shanghai, China). Images were taken with confocal microscope (Nikon, Tokyo, Japan). Antibodies used were listed in Additional file 1: Table S1.

Total RNAs were isolated with RNAiso reagent (Agbio, China) and converted into cDNA using a Reverse Transcription System kit according to the instructions of the manufacturer (Agbio). And then the amplified product was used in Quantitative real-time-PCR ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The sequences of the primers were presented in Additional file 1: Table S3.

Cells were lysed on ice with NP-40 buffer for 20 min and then were centrifuged at 12,000 ×g for 15 min at 4  °C. The supernatants were collected and incubated with primary antibody and rocked overnight at 4  °C. Then, protein A/G (sc2003, Santa Cruz Biotechnology) was added and cells were incubated at 4  °C on a rocker platform for 4 h. The immunocomplexes were washed for three times and mixed with the loading buffer for degeneration at 100  °C for 10 min. The bound proteins were subjected to immunoblotting analysis. Antibodies used were described in Additional file 1: Table S1.

Whole-cell lysates were collected and equally divided into two parts. The one was incubated with unlabeled MCL (200 μM; negative control), and the other was incubated with biotin-MCL (200 μM), as described previously [29]. After incubation overnight at 4  °C, prewashed 30 µL streptavidin beads (Thermo Fisher Scientific, Waltham, MA, USA)were added to each samples and incubated for another 4 h at 4  °C. Then the beads were washed five times with PBS and the bead-bound proteins were eluted, followed by western blotting analysis. Antibodies used were described in Additional file 1: Table S1.

Molecular docking was adopted to understand the potential interactions between MCL and BRG1. Discovery Studio 2017 R2 (BIOVIA Software, Inc., San Diego, CA, U.S.A.) software was employed in the present study. The structure of MCL was optimized by Minimize protocol. The X-ray crystal structure of BRG1 was downloaded from the RCSB Protein Data Bank. CDOCKER protocol was applied to run molecular docking. Other parameters were set as default.

Data were expressed as mean ± SEM. Statistical analyses of the data were performed using SPSS 22. 0 (SPSS Inc, Chicago, IL). Independent-Student’s t test was employed for comparisons between two groups. One-way ANOVA followed by Least significant difference (LSD) test or Dunnett’s T3 procedure for multiple comparison was used for groups of three or more. Statistical significance was defined as P < 0. 05.

To assess the expression of BRG1 in a mouse model of PD, we initially conducted the immunohistochemical staining and found that compared to the saline-injected mice, the expression of BRG1 was notably augmented, particularly in the nuclei, in parietal peritoneal tissues from PD mice (Fig. 1a). In addition, western bolt analysis revealed that in comparison to saline group, the expression of BRG1 was increased in visceral peritoneum from PD mice, along with an accumulation of ECM proteins such as a-SMA and Fibronectin (Fig. 1b and Additional file 1: Fig. S1a).

We then examined the BRG1 expression in vitro stimulation with TGF-β1 or 4.25%PD fluid respectively. TGF-β1 substantially increased synthesis of ECM-related proteins, such as Fibronectin and a-SMA, in HMrSV5 cells (Fig. 1c and Additional file 1: Fig. S1c, S1d). Of note, BRG1 expression was increased after 48 h by TGF-β1 stimulation (Fig. 1c and Additional file 1: Fig. S1b). Consistently, PD fluid also enhanced the expression of BRG1, Collagen I, TGF-β1, while decreasing E-cadherin level in HMrSV5 cells (Fig. 1d and Additional file 1: Fig. S1e–S1h).

We further isolated primary rat peritoneal mesothelial cells (RPMCs) and incubated them with TGF-β1 or PD fluid respectively. Primary cultured RPMCs of the third passage exhibited a polygonal cobblestone-like appearance (Fig. 1e) and expressed both mesenchymal marker (Vimentin) and epithelial marker (E-cadherin), but not myofibroblast marker (a-SMA) (Fig. 1f). Compared to the control group, TGF-β1 stimulation increased the expression of mesenchymal marker Vimentin and profibrotic factors such as Fibronectin and Collagen I, while the level of epithelial marker E-cadherin decreased, indicating the occurrence of MMT in RPMCs (Fig. 1g and Additional file 1: Fig. S1i). Of note, BRG1 was also elevated when exposed to TGF-β1 stimulation (Fig. 1g and Additional file 1: Fig. S1i). Consistently, compared to the saline group, incubation with 4.25%PD fluid augmented the expression of BRG1, Collagen I, a-SMA, TGF-β1, while reducing the expression of E-cadherin (Fig. 1h and Additional file 1: Fig. S1j). These data indicate that the elevation of BRG1 might be involved with peritoneal fibrosis.

To investigate the role of BRG1 on the fibrotic responses in peritoneal mesothelial cells, HMrSV5 cells were transfected with either a BRG1 expression vector (pBRG1) or an empty vector. RT-qPCR (Additional file 1: Fig. S2a) and western blot analysis (Fig. 2a, b) were employed to evaluate the transfection efficacy. BRG1 overexpression dramatically increased synthesis of ECM-related protein, such as Fibronectin, Collagen I and a-SMA (Fig. 2a–e). We then transfected primary RPMCs with a BRG1 expression vector (pBRG1) or an empty vector, and the transfection efficacy was assessed using RT-qPCR (Additional file 1: Fig. S2b) and western blot analysis (Fig. 2f, Additional file 1: Fig. S2c). Results of western bolt revealed that BRG1 overexpression upsurged the TGF-β1 expression while decreasing E-cadherin expression (Fig. 2f and Additional file 1: Fig. S2c). Immunofluorescence staining additionally confirmed the augmentation of Fibronectin and a-SMA in RPMCs upon BRG1 overexpression (Fig. 2g).

Moreover, we knocked down BRG1 expression using an siRNA strategy and BRG1 inhibitor PFI3. RPMCs were transfected with control (siNC) or BRG1-specific siRNA (siBRG1), followed by exposure to TGF-β1. Efficient BRG1 knockdown was confirmed by RT-qPCR (Additional file 1: Fig. S2d) and western blot analysis (Fig. 2h, i). Exogenous TGF-β1 increased abundance of BRG1 (Fig. 2h, i). By contrast, BRG1 interference markedly abolished the synthesis of Vimentin, Collagen I, Fibronectin induced by TGF-β1 and restored the expression of E-cadherin (Fig. 2h, i). Immunofluorescence staining also confirmed the attenuation of Fibronectin and a-SMA when BRG1 was inhibited in TGF-β1-stimulated cells (Fig. 2j). PFI-3 is a selective chemical probe to target SMARCA bromodomains and impede the action of BRG1 by disrupting its connection with acetylated histone [30]. Results of western blot analysis revealed that PFI-3 largely inhibited the overproduction of Vimentin and Collagen I by TGF-β1 in RPMCs, without influencing the expression of a-SMA (Fig. 2k, Additional file 1: Fig. S2e). These data suggest that BRG1 might be an essential determinant in fibrotic responses of peritoneal mesothelial cells.

Activation of TGF-β1 signaling is implicated in the development and progression of peritoneal fibrosis [5, 7, 31]. Data from Fig. 3a, c revealed that overexpression of BRG1 amplified TGF-β1-increased-phosphorylation of Smad2 and Smad3. Moreover, Smad2 and Smad3 became phosphorylated and activated with TGF-β1 in RPMCs, yet this process is impeded by knockdown on BRG1, as illustrated in Fig. 3b, d. Immunofluorescent staining revealed that following TGF-β1 stimulation, Smad2 and Smad3 were accumulated in the nuclei of RPMCs (Fig. 3e). However, BRG1 inhibition largely abolished the TGF-β1-triggered Smad2 and Smad3 nuclear accumulation (Fig. 3e). We also explored the distribution of Smad2 and Smad3 between cytoplasm and nucleus. Western blot results revealed the nuclear localisation of Smad2 and Smad3 in RPMCs exposed to TGF-β1 was impeded when BRG1 was inhibited (Fig. 3f, g). However, although TGF-β1 induced the phosphorylation of ERK1/2, P38 MAPK and JNK, knockdown of BRG1 had no impact on these molecules in RPMCs (Additional file 1: Fig. S3).

Given that the potential pro-fibrotic role of BRG1 in TGF-β1-stressed peritoneal mesothelial cells, we further explore the possibility of targeting BRG1 in the intervention of peritoneal fibrosis. Our previous study has uncovered that MCL is an effective anti-fibrotic compound in a mouse model of peritoneal fibrosis and in TGF-β1-stimulated HMrSV5 cells [27]. In this study, we explored the effect of MCL in BRG1- induced fibrotic responses. Interestingly, western blot results revealed that BRG1 overexpression significantly promoted the Vimentin, Fibronectin, Collagen I synthesis, which was remarkably reversed upon MCL treatment (Fig. 4b–g). Besides, TGF-β1 induced the phosphorylation of Smad2 and Smad3, whereas MCL hampered this process (Fig. 4h–j). Immunofluorescent staining revealed that Smad2 and Smad3 were accumulated in the nuclei after TGF-β1 stimulation, which were abolished by MCL treatment (Fig. 4c). Moreover, BRG1 overexpression increased the expression of TGF-β1 and phosphorylation of Smad3. Nevertheless, MCL abolished the BRG1-triggered TGF-β1 expression and Smad3 phosphorylation (Fig. 7i, Additional file 1: Fig. S4e, S4f). These data above imply MCL might be an anti-fibrotic compound by targeting BRG1.

In vivo, to further investigate the functional role of DMAMCL, the pro drug of MCL, in BRG1-induced peritoneal fibrosis, mice undergoing daily intraperitoneal injection of PD fluid were subjected to BRG1 overexpression plasmid through ultrasonic microbubbles at day 0 and day 14. Subsequently, these mice were treated with daily DMAMCL or saline. The efficiency of ultrasound-microbubble-mediated BRG1 overexpression in mice was shown by western blot analysis (Fig. 5d). Masson’s trichrome staining showed that compared to the normal group, the PD mice had thicker matrix deposition and mice that underwent PD and overexpression of BRG1 had thickest peritoneal fibrous layer among the five groups (Fig. 5b, c). DMAMCL alleviated the PD-induced matrix deposition and remarkably rescued the matrix deposition upon BRG1 overexpression (Fig. 5b, c). Synthesis of ECM-related proteins like a-SMA, Collagen I, Fibronectin were higher in PD mice and the most abundant in BRG1-overexpression-PD mice. DMAMCL repressed these ECM-related proteins level and upon BRG1 overexpression, reversed their expression (Fig. 5d–h). The similar results of Fibronectin and Collagen I synthesis were observed by immunohistochemical staining (Fig. 5i). Moreover, PD fluid exposure increases TGF-β1 expression and its downstream signaling, which was manifested by increasing the phosphorylation of Smad2 and Smad3 in PD mice. This effect was further aggravated upon BRG1 overexpression. Nevertheless, DMAMCL administration repressed TGF-β1-Smad2/3 signaling and reversed TGF-β1-Smad2/3 pathway when BRG1 was overexpressed.

We further delve deeper into the mechanism of MCL in related to BRG1-induced fibrotic responses. As previous studies showed, the binding of BRG1 to acetylated histones H3K14ac is a crucial factor in its gene regulation [32, 33]. The role of BRG1-H3K14ac complex in fibrotic responses was explored in vitro. HMrSV5 cells were co-transfected with BRG1 expression vector and H3K14 different mutation vectors, in which lysine 14 was mutated into glutamine (H3K14Q) to mimic acetylation and mutated into arginine (H3K14R) to inhibit acetylation. H3K14Q mutant augmented the expression of Fibronectin and Collagen I, which was not observed in H3K14R mutant. Moreover, BRG1 overexpression further promoted the expression of Fibronectin and Collagen I when co-transfected with H3K14Q, whereas H3K14R co-transfection rescued this effect (Fig. 6a–e). Furthermore, the increase of TGF-β1 by BRG1 was further augmented in H3K14Q mutant, while it was abolished in H3K14R mutant (Fig. 6a, f). Additionally, BRG1-induced Smad2 phosphorylation was further elevated in H3K14Q mutant, whereas it was blocked in H3K14R mutant (Fig. 6a, d). Subsequently, we examined the influence of MCL on the binding of BRG1 and H3K14ac. Interestingly, co-immunoprecipitation analysis showed the BRG1-H3K14ac interaction was abolished by incubation with MCL (Fig. 6g), suggesting that MCL’s ability to prevent peritoneal fibrosis may be through disrupting the BRG1 interaction with H3K14ac.

To explore the mechanism of MCL disrupting BRG1 interaction with H3K14ac, cell lysates were incubated with MCL-biotin probe or negative probe, and mixtures were separately pulled down with streptavidin-coated agarose beads, followed by western blot analysis. Pull down analysis validated the interaction between MCL and BRG1 in both HMrSV5 cells lysates and RPMCs lysates (Fig. 7a, b). We next performed cellular thermal shift assay to confirm MCL binding to BRG1 (Fig. 7c). BRG1 is composed of multiple domains, most of which have been identified by primary sequence analysis and include an conserved catalytic ATPase domain, as well as a conserved C-terminal bromodomain, AT-hook motif and the N-terminal region housing QLQ, HSA and BRK domains[34]. To next investigate the direct bind site between MCL and BRG1, HMrSV5 cells were transfected with three BRG1 domain deletion mutants (N-terminal domain (1–765), C-terminal domain (1301–1647) or ATPase domain (766–1300)) (Additional file 1: Fig. S4a) and cells lysates were incubated with MCL-biotin probe. Pull-down assay showed that MCL interacted with C-terminal domain of BRG1 (Fig. 7d). And the predicted conformation of MCL-bound human BRG1 protein was further evaluated by computational modeling analysis (Fig. 7e). Of note, MCL has the potential to interact with the conserved asparagine 1540 residue (N1540) in the C-terminal bromodomain of BRG1 via two traditional hydrogen bonds, by mutating the N1540 of BRG1 to alanine (N1540A), we ascertained that asparagine 1540 of BRG1 was indispensable for its interaction with MCL. This was confirmed by the data that the BRG1 N1540A mutant completely eliminated the interaction between MCL and BRG1 (Fig. 7f). Furthermore, co-immunoprecipitation analysis showed that the interaction between BRG1 and H3K14ac was decreased after the mutation of BRG1 N1540 (Fig. 7g). These suggest that MCL disrupt interaction with H3K14ac may through its interaction with N1540 of BRG1.

Western bolt analysis revealed that, unlike BRG1 overexpression group, which promoted synthesis of Fibronectin, Collagen I, a-SMA, and activated TGF-β1-Smad3 signaling, the BRG1 N1540A mutant was unable to produce these effects (Fig. 7h-7i, Additional file 1: Fig. S4b–g). Moreover, while MCL treatment assuaged the effects of BRG1-induced-ECM deposition and TGF-β1-Smad3 signaling, the mutation of BRG1 N1540A prevented any further effects of MCL (Fig. 7h, i, Additional file 1: Fig. S4b–g). Altogether, these data above indicated that MCL inhibited BRG1-induced ECM deposition and TGF-β1-Smads signaling partially by binding directly to BRG1 at the N1540 residue.