Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Wisent Inc., St Bruno, QC, Canada) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/ml penicillin and 100 mg/ml streptomycin (both, purchased from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and cultured in a 37°C humidified incubator supplemented with 5% CO₂. For the transfection assay, cells were plated at 1.0×10⁵ cells/well and were transfected with small interfering (si)RNA of β-catenin (Invitrogen; Thermo Fisher Scientific, Inc.) or Stealth RNAi^® negative control duplexes (NC) (cat. no. 12935300, Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The following β-catenin siRNA oligo sequences were used: 5′-UGUAGCAGGAGAUUAUGCAGCGUGG-3′ (forward) and 5′-CCACGCUGCAUAAUCUCCUGCUACA-3′ (reverse). H9c2 cells were stimulated with 200 µM H₂O₂ for 2 h prior to an MTT assay and with 100 µM H₂O₂ for 30 min prior to the western blotting assay, to establish the myocardial oxidative damage model. 293T cells were co-transfected with 1 ng/well V5-tagged Wnt3a (Wnt3a), 30 mM LiCl, 0.5 µM BIO or 40 ng/well β-cateninΔN (β-catenin truncation mutant with N-terminal region deletion), as well as TOPFlash for 48 h before detecting RLU (relative light units) values in each experimental group. Negative control group was transfected with TOPFlash only and positive control group was transfected with wnt3a or activator of Wnt/β-catenin signaling pathway (LiCl, BIO or β-cateninΔN), as well as TOPFlash but not treated with baicalin. Wnt3a was provided by X. He and C. Niehrs (German Cancer Research Center) (24–27). TOPFlash reporters were provided by RT. Moon (University of Washington, School of Medicine) (28). LiCl, BIO and NaCl were purchased from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany. Baicalin was purchased from Chengdu Must Bio-Technology Co., Ltd.; Chengdu, Sichuan, China.

A TOPFlash/Renilla reporter gene assay was performed as previously described (24,29). Cells were seeded in 96-well plates at 4×10³ cells/well and transfected with 40 ng TOPFlash plasmids for Wnt and β-catenin and 10 ng Renilla luciferase control reporter vector. A total of 48 h following transfection, cells were lysed with cell lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% nadeoxycholale, 0.5 M EDTA, 1% Nonidet 40, 5 mM DTT, 10 µg aprotinin, 10 µg leupeptin, 10 mM PMSF) and screened for luciferase activity using the Dual-Luciferase Reporter Assay system according to the manufacturer's protocol (Promega Corporation, Madison, WI, USA).

For total cellular protein extraction, cells were lysed with a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% nadeoxycholale, 0.5 M EDTA, 1% Nonidet 40, 5 mM DTT, 10 µg aprotinin, 10 µg leupeptin, 10 mM PMSF) on ice for 15 min and centrifuged at 4,000 × g for 15 min at 4°C, prior to the collection of the supernatant. Nuclear extracts were prepared as follows: Cells were resuspended in 400 µl hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂; 10% Nonidet; 5 M DTT; 10 µg Aprotinin; 10 µg Leupeptin; 10 mM PMSF) and incubated on ice for 10 min, then centrifuged at 1,000 × g for 5 min at 4°C. Following separation, the pellet was re-suspended in the lysis buffer on ice for 20 min. Following centrifugation at 4,000 × g for 10 min at 4°C, the supernatant (nuclear extract) was collected.

Concentrations of proteins were quantified using the bicinchoninic acid method. Subsequently, 30 µg proteins per lane were subjected in 10% sodium dodecyl polyacrylamide gel (Sangon Biotech Co., Ltd., Shanghai, China) using 1× SDS-PAGE running buffer (cat. no. P0014; Beyotime Institute of Biotechnology, Haimen, China), and then transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) using 1× western transfer buffer (cat. no. P0021A; Beyotime Institute of Biotechnology). Following blocking with 5% skimmed dry milk in TBS (20 mM Tris-HCl pH 7.4, 150 mM NaCl) with 0.1% Tween 20 (TBST) for 1 h at room temperature, membranes were incubated with the following polyclonal primary antibodies: Rabbit anti-Axin-2 (1:5,000; cat. no. ab32197; Abcam, Cambridge, UK), mouse anti-c-Myc (1:5,000; cat. no. ab32, Abcam), rabbit anti-β-catenin (1:1,000; cat. no. 8480; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-apoptosis regulator Bcl-2 (Bcl-2) (1:1,000; cat. no. 2870; Cell Signaling Technology, Inc.), rabbit anti-apoptosis regulator BAX (Bax) (1:1,000; cat. no. 2772; Cell Signaling Technology, Inc.), rabbit anti-cleaved caspase-3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.) and mouse anti-GAPDH (1:1,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc., Chicago, IL, USA) at 4°C overnight. The membranes were subsequently washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:25,000; cat. no. 31460; Thermo Fisher Scientific, Inc.) or rabbit anti-mouse IgG secondary antibody (1:25,000; cat. no. 27025; Thermo Fisher Scientific Inc.) at room temperature for 1 h. Following washing by TBST, the membranes were placed in enhanced chemifluorescence solution (EMD Millipore) for 1 min. Immunoreactive bands were visualized by an Image Quant LAS 500 Imager (GE Healthcare Life Sciences, Shanghai, China), and the densitometry of bands were calculated by Quantity One software (version 4.62; Bio-Rad laboratories Inc., Hercules, CA, USA).

H9c2 cells were seeded in 96-well plates at a density of 4×10³ cells/well. MTT (0.5 mg/ml; Beyotime Institute of Biotechnology) was added and cells were incubated for 4 h at 37°C prior to the addition of dimethyl sulfoxide. The absorbance was measured at a wavelength of 490 nm according to the manufacturer's protocol (SpectraMax^® M2/M2e Multiskan; Molecular Devices LLC, Sunnyvale, CA, USA). All experiments were performed in quintuplicate.

All data are expressed as the mean ± standard deviation. All statistical analyses were performed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). Statistical significance was concluded by one-way analysis of variance, followed by the Fisher post hoc test for multiple comparisons and unpaired t-test for between two groups comparisons. All experiments were replicated at least three times. P<0.05 was considered to indicate a statistically significant difference.

The TOPFlash/Renilla reporter gene assay was used to evaluate the effect of baicalin on the activation of the Wnt/β-catenin pathway in 293 cells. As presented in Fig. 1, Wnt/β-catenin signaling was significantly activated upon stimulation with Wnt3a, LiCl, BIO and β-cateninΔN. However, baicalin induced a specific and significant inhibitory effect on the activation of TOPFlash with in a dose-dependent manner, demonstrating that baicalin efficiently inhibits the Wnt/β-catenin signaling pathway.

To evaluate the cardioprotective effects of baicalin against H₂O₂-induced cardiotoxicity, H₂O₂-stimulated H9c2 cells were treated with different concentrations of baicalin and cell viability was assessed using an MTT assay. Stimulation with H₂O₂ led to a significant decrease in H9c2 cell viability, which was significantly reversed by the treatment with baicalin in a dose-dependent manner (Fig. 2A). Treatment with baicalin alone exhibited no effect on the viability of H9c2 cells even at increased concentrations (Fig. 2B). Therefore, baicalin can protect against oxidative damage of cardiomyocytes in vitro, without causing apparent cytotoxicity.

The effect of baicalin on H₂O₂-induced apoptosis of H9c2 cells was examined using western blot analysis. As presented in Fig. 3, H₂O₂ upregulated the expression of cleaved caspase-3 and increased the pro-apoptotic Bax/Bcl-2 ratio in H9c2 cells, indicating pro-apoptotic property of H₂O₂. However, baicalin significantly inhibited H₂O₂-induced cardiomyocyte apoptosis, in a dose-dependent manner (Fig. 3).

To investigate the mechanism underlying the anti-apoptotic and cardioprotective activities of baicalin, its effect on the expression levels of β-catenin and its downstream targets axin-2 and c-Myc in H9c2 cells was determined. As presented in Fig. 4A, baicalin treatment significantly suppressed H₂O₂-induced expression of β-catenin, axin-2 and c-Myc. Additionally, siRNA-mediated downregulation of β-catenin expression prevented apoptosis in H₂O₂-treated H9c2 cells (Fig. 4B). LiCl, a GSK-3β inhibitor, commonly used to stabilize β-catenin, was used to further verify above-observed results (30–32). As presented in Fig. 5, LiCl increased β-catenin expression, which in turn induced apoptosis of H9c2 cells. However, LiCl-induced injury in H9c2 cells was significantly neutralized by the treatment with baicalin (Fig. 5). Taken together, the results of the present study indicate that baicalin exerts an anti-apoptotic activity, likely through the suppression of the Wnt/β-catenin pathway.