LTL powder was obtained from Hangzhou Tiancao Technology Co., Ltd. (Hangzhou, China); the purity was >98%. LPS freeze-dried powder was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); the purity was >99%. The two powders were dissolved in DMSO and the solution was filtered with a 0.22 µm membrane.

All animal experiments in the present study were approved by the animal ethics committee of Jiangsu Jiankang Vocational College. Hearts from Sprague Dawley (Nanjing Better Biotechnology Co., Ltd., Nanjing, China; http://www.biomart.cn/46372/index.htm) rats were used in single-cell cultures, as described previously (26). The rats (12 female, 3 male; age 8–10 weeks; average weight 300 g) had free access to water and food, and were maintained at 25°C, 12-h light/dark and 55–65% humidity. The neonatal rats were produced after the animals mated. Neonatal rats (n=5; age, 1–3 days) were used to isolate cardiomyocytes. The hearts were collected and minced into 1 mm³ sections. The pieces were dissociated with a buffer containing trypsin (0.05%) (Hyclone, Logan, UT, USA) and collagenase type II (0.4%) (Beijing Solarbio Science & Technology Co., Ltd) at 37°C for 30 min. The dissociation was stopped by the 5 min incubation of cold horse serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C. The isolated cells were cultured in Dulbecco's modified Eagle's medium/F12 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich; Merck KGaA). After three days, 0.1 mM bromodeoxyuridine (Sigma-Aldrich; Merck KGaA) was added into the medium to inhibit the growth of cardiac fibroblasts (27). Cells were cultured in a 90% humidified 37°C incubator with 5% CO₂. Finally, the morphology of the cultured rat cardiomyocytes was observed under inverted optical microscope (Nikon Corporation, Tokyo, Japan).

siRNAs were commercially purchased from Thermo Fisher Scientific, Inc. The sequences of selected regions to be targeted by siRNAs for β-catenin were: 5′-UGGUUGCCUUGCUCAACAATT-3′ (sense), 5′-UUGUUGAGCAAGGCAACCATT-3′ (antisense). Cells (1×10⁵ cells/ml) were transfected with 50 nM scramble siRNA (Negative control, NC) or β-catenin-siRNA by Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays were performed to assess the transfection efficiency. Cells transfected into β-catenin-siRNA were cultured for 24 h and then used for subsequent experiments.

Cardiomyocytes (2.5×103 cells/ml) were treated with 0.1% PBS (blank control group), 10% FBS (negative group, NC), LPS (100 ng/ml), LTL1 (50 mg/ml) and LPS (100 ng/ml), and LTL 2 (100 mg/ml) and LPS (100 ng/ml) for 8 h at 37°C. Subsequently, cells were used to detect the cell viability, the expression levels of α-actinin and LC3, and autophagy and Wnt signaling pathway-associated gene expression. Cardiomyocytes were treated with PBS, FBS, LPS, β-catenin siRNAs and LPS, and β-catenin siRNAs + LTL1 + LPS.

Total RNA was extracted from the treated cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. First-strand cDNA was synthesized using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc.). The mRNA expression levels were determined by qPCR using SYBR GREEN PCR Master Mix (Applied Biosystem; Thermo Fisher Scientific Inc.) and assayed by ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific Inc.). The thermocycling conditions were set as follows: 10 min pretreatment at 95°C for 10 min, denaturation at 95°C for 15 sec, annealing at 61°C for 30 sec, extension at 72°C for 30 sec (40 cycles) and finally a 7-min extension at 72°C. The data were analyzed using 2^−∆∆Cq method (28). The gene-specific primer sequences are listed in Table I; GAPDH was used as an internal loading control.

Cells (5×10⁵ cells/ml) were lysed on ice in radioimmunoprecipitation assay buffer (cat. no. 8990; Pierce; Thermo Fisher Scientific, Inc.) buffer containing protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). Pierce bicinchoninic Protein Assay kit (Thermo Fisher Scientific, Inc.) was applied to analyze the concentration of proteins. Total protein (30 µg) was separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and incubated with primary antibody overnight at 4°C. Next day, the membranes were as required incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX. USA) or chicken anti-goat lgG-HRP (1:1,000; cat. no. sc-516086; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. The protein expression levels were measured using an enhanced chemiluminescence system (Amersham Pharmacia Biotech; GE Healthcare, Chicago, IL, USA). The results were analyzed by Image Lab Software version 4.1 (Bio-Rad Laboratories, Inc.). Primary antibodies used were as follows: Anti-GAPDH (1:1,000; cat. no. sc-20358; Santa Cruz Biotechnology); anti-LC3 [two forms: LC3I and LC3II (29,30)]; 1:3,000; cat. no. ab64781; Abcam, Cambridge, UK); anti-Atg12 (1:1,000; cat. no. ab155589; Abcam), anti-autophagy-related 4b cysteine peptidase (Atg4b; 1:1,000; cat. no. 5299; Cell Signaling Technology, Inc., Danvers, MA, USA); anti-vacuolar protein sorting-associated protein 34 (Vps34; 1:1,000; cat. no. V9764; Sigma-Aldrich; Merck KGaA); anti-B cell lymphoma 2 interacting protein 1 (Bnip1; 1:300; cat. no. sc-1713); anti-Wnt2 (1:300; cat. no. sc-50361; Santa Cruz Biotechnology, Inc.); anti-glycogen synthase kinase 3β (GSK3β; 1:5,000; cat. no. ab32391; Abcam, Cambridge, UK) anti-phosphorylated-glycogen synthase kinase (p-GSK3β; 1:1,000; cat. no. 5558; Cell Signaling Technology, Inc.) and anti-β-catenin antibody (1:1,000; Abcam, cat. no. ab2982).

MTT assay was carried out to determine the cell viability. The treated cardiomyocytes (2×10³ cells/well) were first seeded into 96-well plates and then cultured in cell incubators for 6 h at 37°C. After being cultured, 20 µl solution MTT (5 mg/ml, cat. no. M-2128, Sigma-Aldrich; Merck KGaA) was added. After 4 h of incubation at 37°C, 10 µl DMSO was added to dissolve the formazan product by incubating for 10 min at room temperature. Subsequently, the plates were measured by a microplate reader at 490 nm absorbance. Each condition was determined in quintuplicate from three independent experiments.

The treated cardiomyocytes (3×10⁴ cells/ml) were washed with PBS gently and then fixed with 4% paraformaldehyde at 4°C for 20 min. After blocking with 10% goat serum (Gibco™; cat. no. 16210064; Thermo Fisher Scientific, Inc.), cells were incubated with primary antibodies anti-α-actinin (1:1,000; cat. no. ab9465; Abcam) and anti-LC3 (1 µg/ml; cat. no. ab48394; Abcam) overnight at 4°C. After being washed, cells were incubated with a fluorescence-conjugated secondary antibody (4 µg/ml; Alexa-Fluor^® 594 goat anti-rabbit antibody IgG; A-11037; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI; 1:1,000; Thermo Fisher Scientific, Inc.) for 15 min at 37°C, and the images were then obtained using a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The cell surface area (green of fluorescence staining) was calculated by formula (axb)/2 (a: The longest distance through the nucleus; b: The shortest distance through the nucleus) and analyzed by Image Pro-Plus 6.1 software (National Institutes of Health, Bethesda, MD, USA).

All experiments were performed at least three times. Numerical data are shown as the mean ± standard error. Statistical analysis was performed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) and statistical significance was assessed by analysis of variance with Dunnet's post-test comparison. P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential biological roles of LTL in cardiomyocytes, myocardial cells were isolated from newborn rats. The morphology of the cultured rat cardiomyocytes was observed with a microscope and most cells were typical myocardial cells (Fig. 1A). To determine the effect of LTL on cell viability, cardiomyocytes were treated with 0.1% PBS (blank control group), 10% FBS (negative group, NC), LPS alone (100 ng/ml), LTL1 (50 mg/ml) + LPS (100 ng/ml) and LTL 2 (100 mg/ml) + LPS (100 ng/ml) for 8 h. MTT assay was used to detect cell viability. The results indicated that LPS significantly decreased the viability of rat cardiomyocytes (P<0.001), however, such an effect was reversed by LTL (P=0.0487; Fig. 1B).

In order to prove the role of LTL on cardiomyocyte hypertrophy and formation of autophagosomes, cultured cardiomyocytes were treated with PBS, FBS, LPS, LTL1 and LPS, and LTL2 and LPS for 8 h. The expression levels of ANP and BNP were increased by LPS compared with the control (P<0.01; Fig. 1C). Cell size was measured through surface area calculation [(axb)/2] under fluorescence microscopy following anti-α-actinin staining. Data demonstrated that LPS increased α-actinin (a cardiomyocyte specific marker and a hypertrophic marker gene) (31,32) expression levels. However, the effect produced by LPS was reversed by LTL (Myocyte area: From 7.9 to 5.7 µm², P<0.05; From 7.9 to 4.0 µm², P<0.01; Figs. 1C and 2A). It was indicated that LTL suppressed LPS-induced cardiomyocyte hypertrophy. Similarly, by observing the specific green fluorescence labeling at the autophagosome membranes (LC3II) and the cytoplasmic labeling (LC3I), and quantifying the amount of autophagosomes by densitometry (29,33), it was found that LPS increased LC3 expression levels, which was reversed by LTL, suggesting that LTL suppressed LPS-induced formation of autophagosomes (P<0.05; Fig. 2B).

In addition, the effects produced by LTL on autophagy-associated genes were further analyzed. Cardiomyocytes were treated with PBS, FBS, LPS, LTL1 + LPS, or LTL2 + LPS for 8 h. Expression levels of LC3 and autophagy-associated genes (Atg12, Atg4b, Vps34 and Bnip1) were measured by RT-qPCR and western blot assays. Results demonstrated that LC3 expression levels were notably upregulated in the LPS group in comparison with that of the NC group, and that LC3II expression levels were downregulated and LC3I expression was upregulated in both LTL + LPS groups, compared with the LPS alone group (P<0.05; Fig. 3A and B). However, LPS promoted autophagy-associated gene (Atg12, protein level of Atg4b, Vps34 and Bnip1) expression levels, which was then inhibited by LTL (P<0.05; Fig. 3C and D). LTL exhibited an abnormal effect on the mRNA expression of Atg4b in that low concentrations of LTL demonstrated a promoting effect and high concentrations of LTL demonstrated an inhibitory effect. This finding requires further research.

Wnt/β-catenin signaling pathway serves a conservative signaling pathway, and exists in various organisms (34,35). In addition, Wnt/β-catenin signaling pathway serves a dual role in the process of heart development (36,37). In the present study, the expression levels of Wnt signaling pathway-associated genes were investigated (Wnt2, β-catenin and p-GSK3β) using RT-qPCR and western blot assays. LPS significantly increased the expression levels of Wnt2, β-catenin and p-GSK3β, however, such an increase was inhibited by LTL (P<0.05; Fig. 3E and F).

As shown in previous results, LTL decreased LPS-mediated Wnt signaling pathway-associated genes. The knockdown efficiency of β-catenin in cardiomyocytes using RT-qPCR and western blot assays was established. The results indicated that the mRNA expression levels of β-catenin were decreased in si-β-catenin group compared with that of control group (P<0.05; Fig. 4A). Western blot results demonstrated that in comparison with the NC group, the protein expression levels of β-catenin decreased notably in the si-β-catenin group (P<0.05; Fig. 4B and C).

To further investigate the exact roles of β-catenin in LPS-induced cardiomyocyte hypertrophy and the formation of autophagosomes in them, cardiomyocytes were treated with PBS, FBS, LPS, β-catenin siRNAs + LPS, and β-catenin siRNAs + LTL1 + LPS for 8 h. Immunofluorescence staining was then performed to measure the expression levels of α-actinin and LC3. The results demonstrated that LPS induced α-actinin expression levels, which was then reversed by silencing β-catenin by siRNA (From 7.5–5.5 µm²) and by siβ-catenin with LTL1 co-treatment (From 7.5–5.0 µm²). This may suggest that silencing of β-catenin and siβ-catenin with LTL1 co-treatment suppressed LPS-induced cardiomyocyte hypertrophy (P<0.05; Fig. 5A). Similarly, LPS was observed to increase LC3 expression levels, which was then reversed by the silencing of β-catenin and by siβ-catenin with LTL1 co-treatment, suggesting this may have inhibited LPS-induced formation of autophagosomes (P<0.05; Fig. 5B). Though there is no difference in the effects of siRNA with or without LTL1 co-treatment, the inhibitory effects of LTL1 co-treatment with siRNA was slightly stronger compared with siRNA treatment alone. If the concentration of LTL is increased, the inhibitory effects may be more pronounced and this needs to be investigated further.