Normal rat kidney interstitial fibroblast (NRK-49F) cells were obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). The TGF-β1-induced in vitro fibrosis model was generated as described previously (13). Briefly, NRK-49F cells were seeded in collagen I-coated 96-well plates at a density of 5,000 cells per well and cultured at 37°C with 5% CO₂ for 72 h, after which the medium was replaced with serum-free medium. After a further incubation for 48 h, recombinant human TGF-β1 (5 ng/ml) was added with or without baicalin/baicalein (20, 40, 80 µM; National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) and the cells were incubated for an additional 48 h. The cells were then fixed in methanol overnight at −20°C and stained with a 0.1% Picrosirius red (PSR; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 4 h. The staining solution was removed and the cells were washed with 0.1% acetic acid, followed by the addition of 0.1 N sodium hydroxide. Antifibrotic activity was then assessed by measuring the optical density (OD) at 540 nm.

NRK-49F cells were seeded into a 96-well plate at a density of 5×10⁴ cells/well. Once the cells reached 80% confluence, the medium was replaced with serum-free medium. After an additional incubation for 48 h, the cells were treated with TGF-β1 (5 ng/ml) and baicalin/baicalein (20, 40, 80 µM) for 2 days. Cell viability was measured using the MTS and 5-bromo-2-deoxyuridine (BrdU) incorporation assays. For the MTS assay, 20 µl of MTS solution (5 mg/ml) was added to each well and the plate was incubated at 37°C for 4 h. The absorbance of the wells was then recorded at 490 nm. For the BrdU incorporation assay, the medium was removed and replaced with medium containing 10 µM BrdU. After incubation for 14 h, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 1 h. The DNA was denatured with 4 M HCl for 10 min at room temperature and the plate was blocked with 5% goat serum (Thermo Fisher Scientific, Inc.) containing 0.05% Triton X-100. BrdU incorporation was detected by incubation with a primary mouse monoclonal anti-BrdU antibody (1:100, B-2531; Merck KGaA) at 4°C overnight and an Alexa Fluor 555-labeled goat anti-mouse immunoglobulin G (IgG) secondary antibody (1:500, A021422; Thermo Fisher Scientific, Inc.) at 37°C for 30 min. Cell nuclei were counterstained with DAPI (Merck KGaA) at room temperature for 10 min. BrdU-positive cells were counted under a fluorescence microscope using ImageJ software (version 2.1; National Institutes of Health, Bethesda, MD, USA).

Apoptotic cells were detected using the Dead Cell apoptosis kit with Annexin V Alexa Fluor 488/propidium iodide (PI) (Thermo Fisher Scientific, Inc.). Briefly, NRK-49F cells were harvested by centrifugation at 800 × g (room temperature for 5 min) and the cell pellet was resuspended in Annexin V binding buffer at room temperature. After a 30 min incubation in the dark, the cells were washed with a buffer for fluorescence-activated cell sorting and incubated with PI solution prior to flow cytometric detection. Early (Annexin V-positive, PI-negative) and late (Annexin V-positive, PI-positive) apoptotic cells were analyzed using the WinMDI software version 2.9 (The Scripps Research Institute, La Jolla, CA, USA).

Immunofluorescence staining was performed to detect the expression of mothers against decapentaplegic homolog 3 (SMAD3) protein and α-smooth muscle actin (α-SMA). Briefly, NRK-49F cells were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized for 5 min using 0.1% Triton X-100. After washing with PBS, the cells were blocked with 5% bovine serum albumin (Merck KGaA) in PBS containing Tween-20 at room temperature for 1 h, then incubated with primary mouse anti-α-SMA (1:400, A5228; Merck KGaA) and rabbit anti-phosphorylated-SMAD3 (1:200, ab52903; Abcam, Cambridge, MA, USA) antibodies overnight at 4°C. Subsequently, the cells were incubated with Alexa Fluor 555-conjugated anti-mouse IgG (1:500, A021422; Thermo Fisher Scientific, Inc.) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, ab150077; Abcam) antibodies at room temperature for 1 h, then stained with DAPI at room temperature for 10 min prior to fluorescence microscopy.

NRK-49F cells were treated with or without baicalin/baicalein (20–80 µM) for various periods of time (24–96 h). Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. First-Strand complementary DNA was then synthesized from the total RNA by reverse transcription using the GoScript™ Reverse Transcription system (Promega Corp., Madison, WI, USA) according to the manufacturer's protocol. qPCR was conducted using TaqMan^® Gene Expression assays (TGF-β1, Rn00572010_m1; COL1A1, Rn01463848_m1; COL1A2, Rn00584426_m1; Thermo Fisher Scientific, Inc.) and a 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, in order to quantify rat collagen type I α 1 (COLIA1) and 2 (COLIA2) mRNA expression. The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min; followed by 40 cycles of denaturation at 95°C for 10 sec, 60°C for 15 sec and 72°C for 15 sec. GAPDH was used as the reference gene. Relative quantification of gene expression was performed using the 2⁻∆∆^Cq method.

After a 48 h treatment with baicalin/baicalein (20–80 µM), total protein was extracted from NRK-49F cell lysate and the protein concentration was determined using the bicinchoninic acid assay. Protein samples (20 µg) were loaded and separated on a 10% gel using SDS-PAGE, and then electrotransferred to nitrocellulose membranes. After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with rabbit anti-rat polyclonal COL1 primary (1:1,000, ab34710; Abcam) and mouse anti-rat monoclonal GAPDH (1:1,000, MAB374; Merck KGaA) primary antibodies overnight at 4°C, followed by incubation with a secondary antibody (mouse anti-rabbit IgG-HRP, sc-2357-CM, and chicken anti-mouse IgG-HRP (sc-2954; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 1:5,000 dilutions for 1 h at room temperature. Protein bands were then detected using SignalFire ECL Reagent (Cell Signaling Technology, Inc., Danvers, MA, USA).

HEK293 cells (obtained from National Infrastructure of Cell Line Resource, Beijing, China) were seeded in a 96-well plate at a density of 5,000 cells per well and transfected with the SMAD-binding element (SBE)-SEAP plasmid using Fugene HD transfection reagent (Promega Corp.), as described previously (19), and the pSEAP basic plasmid as a negative control (both from Clontech Laboratories, Inc., Mountainview, CA, USA) prior to treatment with TGF-β1 and baicalin/baicalein. The SEAP signal was quantified using the SensoLyte^® p-nitrophenyl phosphate (pNPP) SEAP Reporter Gene assay kit (AnaSpec, Fremont, CA, USA) according to the manufacturer's protocol. Briefly, after treatment with baicalin/baicalein (20–80 µM) for 48 h, cell supernatant was collected and heated at 65°C for 30 min to inactivate non-specific alkaline phosphatase activity. The assay buffer was then added and the mixture was incubated for 5 min at room temperature. Subsequently, pNPP substrate buffer was added and the mixture was incubated for 1 h at room temperature. OD values were then measured at 405 nm.

All data are presented as the mean ± SEM. The statistical significances of differences between groups were analyzed via one-way analysis of the variance followed by a post hoc Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using GraphPad Prism software (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA).

The effects of baicalin and baicalein on NRK-49F cell viability were investigated using the MTS and BrdU incorporation assays. In the MTS assay, TGF-β1 significantly increased NRK-49F cell viability compared with the control group (P<0.001), while baicalin and baicalein dose-dependently inhibited this TGF-β1-induced increase in cell viability at concentrations ranging from 20–80 µM (Fig. 1A). The results of the BrdU incorporation assay confirmed that TGF-β1 promoted DNA synthesis in NRK-49F cells, whereas baicalin and baicalein significantly reduced the percentage of BrdU-positive cells (Fig. 1B and C).

An Annexin V/PI double staining assay was performed to investigate the effects of baicalin and baicalein on NRK-49F cell apoptosis (Fig. 1D). Compared with the control group, the percentage of early apoptotic cells increased after 48 h of treatment with TGF-β1. When the cells were treated with baicalin or baicalein (40 or 80 µM), there was no notable increase in the number of Annexin V-positive cells. These data suggest that baicalin and baicalein inhibit NRK-49F cell proliferation without inducing notable cell apoptosis.

The accumulation of extracellular matrix material is considered a key event in the pathogenesis of renal fibrosis. To investigate the potential role of baicalin and baicalein in the process of TGF-β1-induced NRK-49F cell activation, cells were activated with TGF-β1 and stained with PSR to analyze total COLI deposition. The COLI protein accumulation induced by TGF-β1 was significantly downregulated after baicalin (40–80 µM; P<0.05; Fig. 2A) or baicalein (20–80 µM; P<0.001; Fig. 2B) treatment compared with the control group. By contrast, baicalin and baicalein caused no effect on COLI deposition in normal unactivated NRK-49F cells (Fig. 2A and B), suggesting that they specifically antagonized TGF-β1 activation. In addition, the mRNA and protein expression of COLI was analyzed by RT-qPCR analysis and western blotting, respectively. The expression levels of COLIA1 and COLIA2 mRNA were significantly increased after TGF-β1 treatment (P<0.001); however, their expression was significantly inhibited by baicalin (40–80 µM; P<0.01; Fig. 2C and D) and baicalein (20–80 µM; P<0.001; Fig. 2E and F) in a dose-dependent manner. The western blotting analysis confirmed that the upregulated expression of COLI protein (~140 kDa) following TGF-β1 activation was inhibited by baicalin (Fig. 2G) and baicalein (Fig. 2H).

The TGF-β1 signaling pathway is considered to be a major signaling pathway in renal fibrosis. In the present study, the endogenous expression of TGF-β1 mRNA by NRK-49F cells was quantified by RT-qPCR analysis (Fig. 3A-D). The results revealed that the endogenous expression of TGF-β1 by NRK-49F cells increased after TGF-β1 treatment, peaking at 48 h (Fig. 3D). Baicalein dose-dependently (40–80 µM; Fig. 3B) and time-dependently (24–96 h; Fig. 3D) inhibited the endogenous expression of TGF-β1, whereas baicalin caused weaker, but still notable, downregulation (Fig. 3A and C). The activation of SMAD3 protein was also investigated by immunostaining (Fig. 3E) and the SEAP reporter gene assay (Fig. 3F and G). In the immunostaining assay, higher phosphorylated SMAD3 and α-SMA signaling was observed after TGF-β1 activation. The activated SMAD3 protein was accumulated in the cell nucleus and was reduced dose-dependently by treatment with baicalin or baicalein. In the SEAP reporter gene assay, the SBE-SEAP plasmid was transfected into HEK293 cells prior to treatment with TGF-β1 and baicalin/baicalein. TGF-β1 significantly induced SEAP reporter activity, whereas baicalin (40–80 µm; P<0.001; Fig. 3F) and baicalein (20–80 µm; P<0.01; Fig. 3G) significantly antagonized the SMAD3 signaling pathway in a dose-dependent manner.