BEL was acquired from Chengdu Alfa Biotechnology (China, with 98% purity by HPLC). Trimetazidine (TMZ) was procured from Nanjing Zenkom Pharmaceutical Co., Ltd. (China). ISO was obtained from Tokyo Chemical Industry (Japan). Antibodies for TβRⅠ (cat. no. GB11271), Histone H3 (cat. no. GB13102–1) and α-SMA for immunofluorescence analysis (cat. no. GB13044) were derived from Wuhan Servicebio (China). Antibodies for α-SMA used for western blotting detection (cat. no. ab32575), Smad2 (cat. no. ab40855), phospho-Smad2 (S467) (cat. no. ab53100), Smad3 (cat. no. ab40854), phospho-Smad3 (S423 + S425) (cat. no. ab52903), and Smad4 (cat. no. ab40759) were purchased from Abcam Technology (United Kingdom). Antibodies for p38 and phospho-p38 (Thr180/Tyr182) (cat. no. 8203) were purchased from CST (United States). The antibody for TβRⅡ (cat. no. A11788) was purchased from ABclonal Technology (United States). Antibodies for collagen Ⅰ (cat. no. AF7001), collagen Ⅲ (cat. no. AF0136), phospho-TβRⅠ (Thr204) (cat. no. AF8080), and phospho-TβRⅡ (Tyr284) (cat. no. AF8191) were purchased from Affinity Biosciences (United States). The antibody for NR4A1 (cat. no. abs137327) was obtained from Absin (China). The antibody for phospho-NR4A1 (Ser351) (cat. no. orb191558) was derived from Biorbyt Ltd. (United Kingdom). The antibody for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. 60004-1-Ig) was gained from Wuhan Proteintech (China). HRP-conjugated anti-rabbit and anti-mouse IgG (cat. nos. ZdR-5306 and ZdR-5307) antibodies were obtained from Beijing ZSGB Bioengineering Institute (China). Goat-anti-mouse-TRITC and goat-anti-rabbit-Alexa-488 were gained from Wuhan Proteintech (China). The 4′, 6-diamidino-2-phenylindole (DAPI) was purchased from Wuhan Servicebio (China). Recombinant human TGF-β1 was acquired from Gibco (United States). SB203580 (p38 inhibitor) and LY2157299 (TβRⅠ inhibitor) were obtained from MedChem Express (United States).

Male mice (18–22 g) were purchased from the Experimental Animal Center of Hebei Medical University (Shijiazhuang, China) and kept in a 12 h light/12 h dark cycle with free access to food and water. All experiments were according to the People’s Republic of China legislation for the use and care of laboratory animals. The animal care and study protocol were authorized by the Institutional Animal Care and Use Committee of Hebei University of Chinese Medicine (no. DWLL201802).

Mice were subjected to ISO injection to induce myocardial fibrosis after MI as described previously (Yang et al., 2017; Koga et al., 2019). Briefly, ISO (5 mg/kg/day) was injected subcutaneously into the mice for 7 days. The mice were then randomly assigned into five groups: control group (Control), model group (ISO), BEL intervention group (ISO + BEL 25 and 50 mg/kg/day, p. o., BEL 25 and 50 mg/kg), TMZ intervention group (ISO + TMZ 20 mg/kg/day, p. o., TMZ 20 mg/kg). The drug interventions were administrated for 21 days from the second day of the experiment. On day 22, the mice were anesthetized with 0.3% sodium phenobarbital and then sacrificed. The heart tissues were fixed with 4% paraformaldehyde for histopathological analyses, and the residual heart tissues were then frozen at −80°C for western blotting detection.

The heart tissues were embedded in paraffin wax after fixing in 4% paraformaldehyde for 24 h. Paraffin-embedded heart tissues were cut into 5 µm sections. Hematoxylin and eosin (HE; Sigma, United States) and Masson trichrome (Servicebio, China) were used to observe cardiac histopathological changes according to the manufacturer’s protocol. Briefly, cardiac sections were dewaxed to water and stained. Sections were then dehydrated, made transparent, and sealed. The morphological changes were observed and photographed using light microscopy (Leica, Germany).

As per the previously mentioned method, CFs were isolated from 1 to 3 days old neonatal SD rats under sterile conditions by differential adherent method (Olson et al., 2005). Briefly, the hearts were aseptically removed from the neonatal rats and minced into small pieces with eye scissors in ice-cold PBS buffer (pH 7.4). The minced ventricles were digested for 5 min with 0.7 mg/ml collagenase Ⅱ (Solarbio, China) at 37°C and then the digestive liquid was then discarded. Next, the tissue fragments were further dissociated for several 5 min with 0.08% trypsin (Gibco, United States) without EDTA at 37°C. The digestion was repeated for 5–7 times until the fragments were completely digested. The cell suspension was then centrifugated, resuspended, and inoculated in culture bottles. After 1 h, the non-adherent cells were discarded, and the adherent cells were cultured in DMEM medium (Gibco, United States) supplemented with 10% fetal bovine serum (BI, Israel), 100 unit/ml penicillin (BI, Israel), and 100 μg/ml streptomycin (BI, Israel) in a humidified atmosphere containing 5% CO₂ at 37°C. CFs were identified by Vimentin immunofluorescence.

BEL was dissolved in DMSO, and the final concentration of DMSO (Solarbio, China) is 1/1,000. The CFs were divided into six groups: control, TGF-β1, TGF-β1 with 1‰ DMSO, TGF-β1 plus BEL 15, 30, and 60 µM groups. The CFs were pretreated with DMSO or BEL (15, 30, and 60 µM) for 0.5 h and then stimulated with TGF-β1 (10 ng/ml) for an additional 24 h.

SB203580 and LY2157299 were dissolved in DMSO, and CFs were incubated with DMSO, BEL (60 µM), SB203580 (0.5 µM), LY2157299 (10 µM), or SB203580 plus LY2157299 for 0.5 h. The samples were then administrated with TGF-β1 (10 ng/ml) for an additional 24 h.

CFs were inoculated into 96-well plates and grown to 70% confluence. Cells were cultured in a serum free medium for 18 h followed by administration with BEL (7.5, 15, 30, 60, and 120 μM) in the absence or the presence of TGF-β1 for 24 h. The cell viability was detected by CCK-8 kit (MedChem Express, United States). CCK-8 solution (20 μL) was added into the wells, and CFs were incubated for 3 h at 37°C. The OD values of each well were detected by Varioskan LUX Multimode Microplate Reader at the absorbance of 450 nm (Thermo Fisher Scientific, United States).

Immunofluorescence staining was performed to detect the expression of α-SMA, collagen Ⅰ, collagen Ⅲ, Smads2, 3, 4, NR4A1, phospho-Smads2, 3, and phospho-NR4A1. Briefly, cell climbing pieces were fixed in 4% paraformaldehyde and were permeabilized with 0.1% Triton X-100 for 20 min at room temperature (RT). The cells were subsequently blocked with 3% BSA for 90 min and then incubated with primary antibodies against α-SMA (1:500), collagen Ⅰ (1:100), collagen Ⅲ (1:100), Smad2 (1:50), Smad3 (1:50), Smad4 (1:50), NR4A1 (1:100), phospho-Smad2 (1:50), phospho-Smad3 (1:50), and phospho-NR4A1 (1:100) overnight in a humidity chamber at 4°C. Cells were subsequently incubated with species-specific secondary antibodies labeled with fluorescence for 1 h at 37°C. Cell nuclei were stained using DAPI. The sections were sealed with an anti-interferent fluorescent reagent. The cell slides were detected with Leica DM5000 B fluorescence microscope (Germany) or Leica SP8 confocal microscopy (Germany) using 40✕ or 63✕ magnification. Red fluorescence signal was measured at excitation and emission wavelengths of 550 and 570 nm. Green fluorescence signal was captured at excitation and emission wavelengths of 488 and 514 nm. DAPI was visualized at excitation and emission wavelengths of 360 and 461 nm. The results were quantified by Image Pro Plus 6.0 software (Media Cybernetics, United States).

A western blotting assay was used to measure the expression of α-SMA, collagen Ⅰ, collagen Ⅲ, TβRⅡ, phospho-TβRⅡ, TβRⅠ, phospho-TβRⅠ, Smads2, 3, 4, phospho-Smads2, 3, p38, phospho-p38, NR4A1, and phospho-NR4A1; GAPDH was used as an internal reference, and Histone H3 was used as a nuclear internal reference. Tissue samples or cells were lyzed in RIPA buffer adding phenylmethyl sulfonyl fluoride (PMSF), protease inhibitor cocktail (Roche, Switzerland), and phosphatase inhibitors (Wuhan Servicebio, China). The nuclear and cytoplasmic proteins were extracted by Nuclear and Cytoplasmic Protein Extraction Kit (Shanghai Beyotime, China). The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Wuhan Servicebio, China). An equal amount of protein was separated using 10% SDS-PAGE and subsequently transferred from gel to polyvinylidene fluoride membranes. The membranes were interdicted with 5% defatted milk for 90 min at RT and individually incubated with primary antibodies α-SMA (1:5,000), collagen Ⅰ (1:1,000), collagen Ⅲ (1:1,000), TβRⅡ (1:1,000), phospho-TβRⅡ (1:3,000), TβRⅠ (1:1,000), phospho-TβRⅠ (1:3,000), Smad2 (1:2,000), Smad3 (1:10,000), Smad4 (1:5,000), phospho-Smad2 (1:1,000), phospho-Smad3 (1:2,000), p38 (1:1,000), phospho-p38 (1:1,000), NR4A1 (1:3,000), phospho-NR4A1 (1:500), GAPDH (1:10,000), and Histone H3 (1:10,000) at 4°C overnight. The membranes were incubated following the corresponding HRP-conjugated secondary antibody at RT for 60 min. The immunoreactive bands were examined with ECL (Invitrogen) reagents and the Fusion FX5 Spectra multifunction laser-scanning system (Vilber Lourmat, France). The protein expression was evaluated by ImageJ 3.0 software (National Institute of Health).

All data are expressed as the mean ± SEM. Statistical analyses were implemented with SPSS 21.0 statistical software. The significance of differences between groups were assessed using one-way ANOVA followed by Turkey's post hoc test and considered at a value of p < 0.05.

A mouse model of myocardial fibrosis was established by subcutaneous injection of ISO (5 mg/kg/day) for 7 days. HE staining demonstrated that cardiac fibers and tissue structure were obvious in the control group; disturbed cardiac fibers and necrotic cardiomyocytes were present in the ISO group. Compared with the ISO group, treatments with BEL and TMZ dampened ISO-induced cardiac damages (Figure 2A). Masson trichrome staining determined that collagen deposition in myocardial tissues was significantly increased in the model group induced by ISO. However, collagen deposition was reduced in mouse hearts treated by BEL and TMZ compared with the ISO treated group (Figure 2A).

Western blotting was used to quantify the contents of collagen Ⅰ, and collagen Ⅲ—the primary compounds of ECM in fibrotic heart. The results revealed that the expression of cardiac collagen Ⅰ, and collagen Ⅲ were obviously elevated in the ISO group compared with the control group while ISO-induced elevations in collagen I, and collagen III expression were decreased by BEL and TMZ administration (Figures 2B,C).

Following myocardial injury, CFs transform to myofibroblast phenotype, which express contractile protein α-SMA, produce large amounts of collagen (in particular, collagen types I and III), and contribute to myocardial fibrosis (van Nieuwenhoven and Turner, 2013; Travers et al., 2016; Weber and Diez, 2016; Song et al., 2020). The level of α-SMA expression was examined by western blotting. The results demonstrated that α-SMA expression was significantly upregulated in mice with ISO-induced myocardial fibrosis compared with the control group. However, the upregulation of α-SMA expression induced by ISO was inhibited by BEL but not TMZ (Figure 2D). These results illustrated that BEL inhibited CFs activation, reduced collagen deposition, and ameliorated myocardial fibrosis.

BEL inhibits the proliferation and phenotypic transformation of CFs induced by TGF-β1.

It is well known that TGF-β1 contributes to myocardial fibrosis, and can induce the proliferation and activation of CFs. First, the CCK-8 kit was employed to detect the effect of BEL on TGF-β1-induced proliferation. CFs were pretreated with BEL (7.5, 15, 30, 60 and, 120 μM) for 0.5 h and then exposed to TGF-β1 (10 ng/ml) for 24 h. The results showed that the administration with BEL markedly inhibited CFs proliferation induced by TGF-β1 (Figure 3A). The role of BEL on CF viability without TGF-β1 stimulation was also investigated. CFs were administrated with BEL (7.5, 15, 30, 60, and 120 μM) for 24 h and examined by CCK-8 kit. The data showed that there were no significant differences by administrating BEL from 7.5–120 μM in comparison with the control group (Figure 3B). These results suggest that intervention with BEL at a concentration of 7.5–120 μM has no effect on CF viability while BEL administration could inhibit TGF-β1-induced CF proliferation.

The role of BEL on activated CFs induced by TGF-β1 was also investigated. The cultured CFs were exposed to TGF-β1 (10 ng/ml) in the absence or presence of BEL for 24 h. Immunofluorescence staining results revealed that the expression of α-SMA, collagen Ⅰ, and collagen Ⅲ were increased in CF stimulated with TGF-β1; these increases were decreased by BEL treatment (Figures 4A–D). Consistent with immunofluorescence staining, western blotting analysis further demonstrated that BEL decreased the expression of α-SMA, collagen Ⅰ, and collagen Ⅲ induced by TGF-β1 (Figures 4E–G). These results illustrated that BEL prevented TGF-β1-induced proliferation and activation of CFs.

The phosphorylation and activation of TGF-β receptors are key events in the signal transmission of TGF-β. The expression and phosphorylation of TβRⅡ and TβRⅠ were checked by western blotting. In CFs, TGF-β1 induced the phosphorylation and activation of TβRⅡ and TβRⅠ but did not affect the total expression of TβRⅡ and TβRⅠ comparing with the control group; this phosphorylation and activation of TβRs was limited by BEL administration (Figure 5). In vivo, the levels of phospho-TβRⅡ and TβRⅠ and TβRⅠ expression were significantly increased in the ISO group vs. the control group. However, ISO-induced increases in TβRⅠ expression and the phospho-TβRⅡ and TβRⅠ levels were decreased by BEL and TMZ (Figure 6). The results indicated that BEL inhibited the phosphorylation and activation of the receptors of TGF-β.

The TGF-β1/Smads signaling pathway plays a critical role in myocardial fibrosis (Hu et al., 2018). The effect of BEL on Smad proteins was investigated in the context of myocardial fibrosis. In vitro, immunofluorescence staining showed that the expression of Smads2/4 and the levels of phospho-Smads2, 3 were significantly elevated by TGF-β1 stimulation vs. the control group. These elevations were inhibited by BEL treatment (Figures 7A–F). Consistent with immunofluorescence staining, western blotting analysis indicated that BEL inhibited the expression of Smads2, 4 and the levels of Smads2, 3 phosphorylation (Figures 7G–I). In vivo, the results showed that the expression of Smad4 and the level of phospho-Smad3 were significantly elevated in the ISO group vs. the control group. However, BEL treatment decreased the Smad4 expression and the Smad3 phosphorylation level compared with the ISO group (Figure 8). These results demonstrated that BEL treatment inhibited the expression of Smads2 and 4 and the activation of Smads2 and 3 in CFs. These results indicated that BEL could inhibit TGF-β/Smads signaling pathway to alleviate myocardial fibrosis in CFs.

Besides TGF-β/Smads canonical signaling, p38 plays an important role in fibrosis (Hu et al., 2016; Liu et al., 2017). The levels of p38 expression and phosphorylation were examined by western blotting in the cultured CFs. The results showed that p38 was significantly phosphorylated and activated by TGF-β1 stimulation. However, BEL treatment inhibited the activation of p38, but the expression of total p38 did not change (Figure 9A). Interestingly, in ISO-induced injury heart tissue, the level of p38 phosphorylation was downregulated vs. the control group; the downregulation was upregulated by BEL treatment but not TMZ administration (Figure 9B). These results illustrated that BEL prevented the phosphorylation of p38 in CFs.

BEL may downregulate the levels of phospho-p38 and phospho-Smad3; but it is unclear whether the inhibitory roles of BEL on p38 signaling suppress Smad3 activation. CFs were pretreated with DMSO, BEL (60 µM), SB203580 (0.5 µM), and LY2157299 (10 µM) or SB203580 plus LY2157299 for 0.5 h. They were then stimulated with TGF-β1 for 24 h. Western blotting analyzed Smad3 expression and phospho-Smad3 level. The results showed that TGF-β1-induced an increase in Smad3 phosphorylation that was significantly downregulated by BEL, LY2157299, and SB203580 plus LY2157299 but not SB203580 (Figure 10A). Further, the expression of α-SMA, collagen Ⅰ, and collagen Ⅲ were detected. The results showed that the elevations in α-SMA, collagen Ⅰ, and collagen Ⅲ induced by TGF-β1 were decreased by BEL, SB203580, LY2157299, and SB203580 plus LY2157299 (Figures 10B–D). The inhibition of p38 does not affect phosphorylation of Samd3. This study indicated that BEL could inhibit phenotypic transformation of CFs by inhibiting the canonical TGF-β pathway and non-canonical signaling pathway respectively.

NR4A1 is a feedback molecule for regulating TGF-β signaling. In fibrotic disease, NR4A1 transferred from the nucleus to the cytoplasm losing its negative feedback regulation effect on TGF-β signaling. At the same time, p38 activation promotes the transformation of NR4A1 from the nucleus to the cytoplasm. To illuminate the relationship of NR4A1 and p38 signaling, CFs were pretreated with BEL or SB203580 for 0.5 h then stimulated with TGF-β1 for 24 h. Western blotting analysis demonstrated that BEL and SB203580 inhibited TGF-β1-induced the upregulation of phospho-NR4A1 (Figure 11B). Immunofluorescence staining indicated that TGF-β1 promoted NR4A1 and phospho-NR4A1 cytoplasmic localization while pretreatments with BEL and SB203580 limited phospho-NR4A1 transformation (Figure 11A). Further western blotting showed NR4A1 expression and phosphorylation levels in the cytoplasm and nucleus. The results illustrated that phospho-NR4A1 level was enhanced in the cytoplasm by TGF-β1. However, BEL and SB203580 decreased the enhancements of phospho-NR4A1 in the cytoplasm (Figure 11C), and BEL also enhanced nuclear retention of NR4A1 (Figure 11D). These data illustrated that BEL impeded NR4A1 cytoplasmic localization and restored the activation of NR4A1 to inhibit TGF-β1 signaling. Therefore, the inhibitory role of BEL on CFs activation was exerted by suppressing NR4A1 from the nucleus to cytoplasmic localization.

In summary, our data suggest that BEL can ameliorate myocardial fibrosis by inhibiting the proliferation and phenotypic transformation of CFs; and these inhibitory effects of BEL are related to the regulation of TGF-β/Smads and p38 pathway and prevention of NR4A1 cytoplasmic localization that negatively regulated TGF-β signaling in CFs.