ISO and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were products of Sigma Aldrich. TGF-β1 and SB431542 were purchased from MedChemExpress, Inc. Caffeic acid, oleanolic acid, ursolic acid, rosmarinic acid, rutin, hyperin, β-ecdysterone, chlorogenic acid, and ginsenoside Ro were purchased from HongYang, Biotech Co. (JiLin, China). Antibodies against TGF-β1, p-Smad2/3, type III collagen (COL3), alpha-smooth muscle actin (α-SMA), and vimentin as well as FITC-labeled rabbit IgG and Cy3-labeled mouse IgG were provided by Affinity Biosciences Ltd. (Jiangsu, China). The following items were purchased from the indicated vendors: the rat cardiac fibroblast (RCF) cell line, Beina-Chuanglian Biotechnology Co. (BNCC, Beijing, China) fetal bovine serum (FBS), YuSheng, Biotech Co. (Shanghai, China); high-glucose Dulbecco’s modified Eagle medium (DMEM), Gibco, YiXiang, Biotech Co. (Jilin, China); hydroxyproline content determination kit (alkaline hydrolysis method), Jiancheng, Biotechnology Co. (Nanjing, China); bicinchoninic acid (BCA) protein determination kit and enhanced chemiluminescence (ECL) color development kit, Beyotime Biotechnology Co. (Shanghai, China); and 3,3′-diaminobenzidine (DAB) immunohistochemistry (IHC) kit, Zhongshan Jinqiao Biotech Co. (Beijing, China).
2.2. Experimental animals
The special clean-grade male Wistar rats, weighing 180–200 g, were purchased from Liaoning Changsheng Biotechnology Co. Ltd. (Certificate No. SCXK (Liao) 2015–0001, Liaoning, China). The rats were housed in the Laboratory Animal Center of Changchun University of Chinese Medicine under standard laboratory conditions.
2.3. Animal model establishment and drug administration
After seven days of adaptive breeding, 40 Wistar rats were randomly divided into the following four groups: normal control group (Ctrl), model group (ISO), low-dose JDTL group (Jie L), and high-dose JDTL group (Jie H), with 10 rats in each group. Except for the Ctrl group, the rats in the other three groups were injected subcutaneously with ISO at 5 mg/kg body weight, while the Ctrl control group was injected subcutaneously an equal volume of saline. From the second day of model establishment, the experimental rats in the Jie L and Jie H groups were given JDTL decoction at 5 g/kg and 50 g/kg, respectively, by gavage daily for 7 consecutive days; meanwhile, the Ctrl and model groups were given (by gavage) an equal volume of normal saline daily.
To evaluate the effect of JDTL (alone) on normal rats, another batch of 30 Wistar rats was randomly divided into the following three groups: normal control group (Ctrl), low-dose JDTL (alone) group (Jie aL), and high-dose JDTL (alone) group (Jie aH), with 10 rats in each group. Rats in the Jie aL and Jie aH groups were given JDTL at 5 g/kg and 50 g/kg daily by intragastric administration, respectively; while the rats in the Ctrl group were intragastrically administered with an equal volume of normal saline daily for 7 consecutive days.
The rats were anesthetized and euthanized at 1–2 h after the last intragastric administration. Blood samples were collected, and the serum was inactivated at 56 ℃ for 30 min for future use.
2.4. High-performance liquid chromatography (HPLC) fingerprint analysis
The composition of the JDTL decoction (water extract) was analyzed by HPLC, according to a standard procedure. First, the water extract was separated via a ZORBAX SB-C18 column (4.6 mm × 250 mm, 5-µm pore size, Agilent, Santa Clara, CA, USA), with the following conditions: mobile phase, acetonitrile (A) and 0.4% phosphoric acid/water (B); column temperature, 30 °C; flow rate, 1 mL/min; UV detection wavelength, 203 nm; and injection volume, 5 µL. The authorized software Agilent 1200 was used for chromatographic data collection and analysis of specific components. Several chemicals, such as caffeic acid, oleanolic acid, ursolic acid, rosmarinic acid, rutin, hyperoside, β-ecdysterone, chlorogenic acid, and ginsenoside RO were used as standards to determine if they were components of the JDTL decoction.
2.5. Hematoxylin and eosin (HE) staining and Masson staining
The ventricular muscle tissues were collected from the experimental rats in each group and were used to make 5-µm-thick paraffin-embedded sections. After dewaxing with xylene, HE staining and Masson trichrome staining were performed on the ventricular muscle tissue sections. The images of the stained sections were recorded, and the degree of MF was analyzed using ImageJ software (U. S. National Institutes of Health, Bethesda, MD, USA).
2.6. IHC staining
The collected myocardial tissue of the experimental rats was fixed with 4% paraformaldehyde for IHC staining. The expression of TGF-β1, p-Smad2/3, and COL3 in the different groups was analyzed using a standard IHC staining procedure (primary antibody dilution, 1:300). The secondary antibody was then added, according to the instructions of the IHC kit (PV-6000; Zhongshan Jinqiao Biotech Co., Beijing, China). The staining results were visualized by the DAB chromogenic method.
2.7. Cell culture and experimental groups
RCFs were cultured with high-glucose DMEM containing 10% FBS. Cells in the logarithmic growth phase were divided into five groups: normal control group, TGF-β1 stimulation group, low-dose JDTL group, high-dose JDTL group, and SB431542 group. The cells in the normal control group were cultured in medium containing 2% normal rat serum. In the TGF-β1 stimulation group, TGF-β1 was added to the medium containing 2% normal rat serum at a final concentration of 10-8 µg/mL. In the low-dose and high-dose JDTL groups, the cells were cultured in medium containing 2% normal rat serum, low-dose or high-dose JDTL, and TGF-β1 (10-8 µg/mL). In the SB431542 group, both TGF-β1 (10-8 µg/mL) and SB431542 (10-5 µg/mL) were added into the medium containing 2% normal rat serum.
2.8. Cell viability assay
RCFs were plated into 96-well plates at a density of 2 × 103 cells/well. The corresponding medium of each group, as mentioned above, was added to the wells of the different groups, and the cells were incubated with 5% CO2 at 37 ℃ for 24 h. The proliferation activity of the cells was determined using an MTT assay, which is a colorimetric assay that involves the reduction of the tetrazolium dye MTT into its insoluble formazan). The optical density (OD) of each well was measured with a microplate reader at a wavelength of 490 nm to determine the viability of the RCFs for evaluating their proliferation potentials. Meanwhile, the blank control wells (with culture medium only, but without cells) were used for zeroing. The cell proliferation activity was calculated as follows: cell proliferation activity (%) = 100 × (OD of experimental group [– blank control group] / (OD of control [– blank control group]).
2.9. Wound-healing assay
RCFs were placed into a six-well plate at a cell density of 4 × 105 cells/well and cultured to near confluence. In the wells of the different groups, the old culture medium was replaced with the corresponding medium of each group, as mentioned above. At 24 h after treatment, the cells were scratched and washed with phosphate-buffered saline three times to remove the suspended cells, followed by an additional 24-h culture in the corresponding medium of each group. The image of each well was recorded with an optical microscope. Comparison of the width of the scratch between the different groups was performed to evaluate the wound-healing ability of the treated cells by using ImageJ software.
2.10. Hydroxyproline content determination
To assess the degree of cell fibrogenesis, hydroxyproline released in the cell culture supernatant of the RCFs was measured by a spectrophotometric method (alkaline water method).
2.11. Immunofluorescence staining
Both cultured cells and collected tissue samples on the glass slide were fixed with 4% formaldehyde and then incubated with mouse anti-α-SMA or vimentin antibodies (1:50) at 4 ℃ overnight. The next day, the slides were incubated with secondary antibody coupled with FITC or Cy3 (1:1000) at room temperature in the dark for 1 h, followed by DAPI nuclei staining for 10 min. Finally, the stained slides were examined using a fluorescence microscope. The images of the stained slides were recorded and analyzed using ImageJ software.
2.12. Western blot analysis
The cultured cells were lysed with radioimmunoprecipitation assay buffer and centrifuged at 12,000 rpm for 15 min before the supernatant was collected. The protein concentration was measured with a BCA protein assay kit. After denaturation at 95 ℃ for 5 min, the protein samples (100 µg each) were separated by electrophoresis on 10% or 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk and then incubated with primary antibodies (1:1500 dilution) against total Smad2/3 and p-Smad2/3, respectively, at 4 ℃ overnight. The next day, after incubation with secondary antibody (1:1500 dilution) at room temperature for 1 h, the bands on the membrane were visualized by ECL detection. The western blot results were analyzed using ImageJ software.
2.13. Statistical analysis
In this study, all data were expressed as the mean ± standard deviation (X ± s). SPSS19.0 software (IBM Corp., Armonk, N.Y., USA) was used for statistical analysis. Group comparison was performed by one-way analysis of variance. The least significant difference method was used for pairwise comparison between groups. A value of p < 0.05 was considered as a significant difference.