Bovine serum albumin (BSA), D-glucose and rabbit anti-GAPDH antibody were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rabbit anti-profilin-1 and anti-Rho antibodies were purchased from Abcam (Shanghai, China), mouse anti-RAGE antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and rabbit anti-p65 antibody was obtained from Bioworld Technology, Inc. (St. Louis Park, MN, USA). Blood glucose and total cholesterol (TC) detection kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The procollagen type III N-terminal peptide (PIIINP) ELISA kit was purchased from Uscn Life Sciences, Inc. (Wuhan, China). TRIzol reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The reverse transcription kit was obtained from Thermo Fisher Scientific, Inc. The QuantiFast SYBR Green PCR kit was from Qiagen GmbH (Hilden, German). The profilin-1 short hairpin RNA (shRNA) adenovirus vectors and blank control adenovirus vectors were designed and synthesized by Hanbio Biotechnology Co., Ltd. (Shanghai, China).

AGEs were prepared according to the protocol described by Wu et al (22). Briefly, BSA (10 mg/ml) was incubated in 0.2 M PBS with D-glucose (90 g/l) containing 100 U/ml penicillin and 100 µg/ml streptomycin in the dark at 37°C for 12 weeks. After 12 weeks incubation, the preparations were dialyzed three times for 18 h at 4°C against PBS (pH 7.4) each time to remove free glucose, and were subsequently separated into aliquots and stored at −20°C prior to use.

Male (n=30; weight, 150±10 g; age, 1 month) Sprague-Dawley rats were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The rats were raised at an ambient temperature of 24°C with 12-h light/dark cycle and 50±5% humidity, and free access to a standard chow diet and water. Rats were randomly divided into the following four groups (n=6 each): Control, rats were treated with vehicle saline; AGEs, rats were treated with AGEs; AGEs + S, rats were treated with AGEs and profilin-1 shRNA adenovirus vectors; and AGEs + V, rats were treated with AGEs and blank control adenovirus vectors. For transfection efficiency detection, the remaining 6 rats were randomly divided into two groups (n=3 each) for 4 weeks treatment: Ad-vector group, rats were treated with blank control adenovirus vector; Ad-profilin-1 shRNA group, rats were treated with adenovirus vector containing profilin-1 shRNA. AGEs (50 mg/kg/day) was administered by tail vein injection for 8 weeks, with the same volume of saline injected as a control. Ad-profilin-1 shRNA or blank control vector was injected twice by tail vein at a dose of 3×10⁹ plaque forming units with an interval of every 4 weeks, beginning with the initial AGEs injection. The experimental procedures and protocols performed in the present study were approved by the Medicine Animal Welfare Committee of Xiangya Hospital, Central South University (Changsha, China), conforming with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication 85–23, revised 1996) (23).

The cardiac contractile function in vivo was determined after 8 weeks of daily administration of AGEs (50 mg/kg/day) using a BL-420E Data Acquisition and Analysis system (Chengdu TME Technology Co., Ltd., Chengdu, China). Following anesthesia with pentobarbital sodium (60 mg/kg; intraperitoneal), all rats were examined and, using the left ventricular long axis view, the following parameters were measured: Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS).

After 8 weeks of daily administration of AGEs (50 mg/kg/day), 800 µl blood samples were collected and centrifuged for 10 min at 4°C and 3,000 × g. Supernatants were stored at −80°C until the analysis of biochemistry parameters. The whole heart was harvested immediately following blood sample selection, the atrium and right ventricle were removed, and left ventricle samples were divided into the following three groups: One group was fixed with 4% paraformaldehyde at room temperature for 1 week and embedded in paraffin. Paraffin sections were used for Masson's trichrome staining and immunohistochemistry; the second group was homogenized by TRIzol reagent for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis; the final group was homogenized in radioimmunoprecipitation assay lysis buffer (P0013B; Beyotime Institute of Biotechnology, Beijing, China) and 0.1 mmol/l phenylmethylsulfonyl fluoride (ST506; Beyotime Institute of Biotechnology) for western blot analysis.

Metabolic parameters, including blood glucose and TC, were detected using relevant kits. The serum levels of PIIINP were detected using an ELISA kit (cat. no. SEA573Ra), according to the manufacturer's instructions.

Total protein from left ventricle tissue samples was extracted, as described above for the preparation of serum and tissue samples, and measured using a BCA protein assay kit. Following heating at 95°C for 5 min, 20 µg/sample lysates were separated by 10 or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk in TBST buffer (0.1% Tween-20 in TBS) for 90 min at room temperature, and subsequently incubated with primary antibodies against profilin-1 (cat. no. ab124904, 1:1,500 dilution), p65 (cat. no. BS1253, 1:1,000 dilution), RAGE (cat. no. sc-365154, 1:600 dilution), Rho (cat. no. ab40673, 1:1,000 dilution) and GAPDH (cat. no. G9545, 1:5,000 dilution) at 4°C overnight. Following primary antibody incubation, membranes were subsequently washed and incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. CW0102, 1:5,000 dilution; CW Biotech, Co., Ltd., Beijing, China) or anti-rabbit IgG (cat. no. CW0103, 1:5,000 dilution; CW Biotech) secondary antibodies for 1 h at room temperature. Potent ECL kit (cat. no. 70-P1425; MultiSciences Biotech Co., Ltd., Hangzhou, China) was used to visualize proteins. The signal was detected and ratios of the target protein against the GAPDH control were calculated using the Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total RNA was extracted from left ventricle tissue samples using TRIzol reagent, according to the manufacturer's instructions. The concentration and purity of isolated RNA was subsequently detected. cDNA was generated from 1 µg total RNA using a high-capacity cDNA reverse transcription kit (4368814; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Reverse transcription was performed as follows: 25°C for 10 min, 37°C for 2 h, 85°C for 5 min and stored at 4°C. GAPDH primers were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and all other primers were synthesized and purified by Sangon Biotech Co., Ltd., as listed in Table I. cDNA was quantified using a QuantiFast SYBR Green PCR kit and qPCR was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Briefly, the amplification was performed with an initial step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec and extension at 60°C for 30 sec for each target gene. All amplification reactions for each sample were performed in triplicate and the results were expressed as the ratio of target genes to GAPDH mRNA using the 2^−ΔΔCq method (24).

Paraformaldehyde-fixed paraffin sections were cut from the left ventricle at a thickness of 4 µm and were stained using a Masson's trichrome stain kit (BSBA-4079A; Zhongshan Golden Bridge Biotechnology Co., Beijing, China), according to the manufacturer's instructions, to evaluate ventricular remodeling. Immunohistochemistry was performed as described previously (14). Briefly, left ventricle sections, at a thickness of 4 µm, were incubated overnight at 4°C with the primary antibody against profilin-1 (1:200 dilution), and subsequently incubated with a biotin conjugated goat anti-rabbit immunoglobulin-G secondary antibody (cat. no. ZB-2010; Zhongshan Golden Bridge Biotechnology Co., Beijing, China) at a 1:1,000 dilution at 37°C for 20 min. Following washing in PBS, sections were developed using 3,3′-diaminobenzidine tetrahydrochloride obtained from Zhongsha Golden Bridge Biotechnology Co. (cat. no. ZLI-9018) and counterstained with 50% hematoxylin for 15 min at room temperature. Subsequently, sections were mounted with neutral balsam. Masson's trichrome stain and immunohistochemistry pictures were visualized under a light microscope (magnification, ×200; Nikon Corporation, Tokyo, Japan).

Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) either by unpaired student's t-test for two groups or one-way analysis of variance followed by Newman-Keuls post-hoc test for multiple groups. P<0.05 was considered to indicate a statistically significant difference.

As presented in Table II, no significant differences were observed in the body weight, blood glucose and TC of different groups of rats following 8 weeks of treatment.

To determine whether chronic injection of AGEs has an effect on cardiac injury, the present study focused on the primary characteristics of DCM, including cardiac function, fibrosis and hypertrophy. Compared with the control group, echocardiography demonstrated that left ventricle systolic function, including LVEF and LVFS, was significantly decreased in the AGEs group (Fig. 1). In addition, the AGEs group developed cardiac fibrosis, as evidenced by markedly increased collagen deposition in the heart tissue sections, particularly around vessels, indicated by blue color in Masson's trichrome staining (Fig. 2A). Furthermore, PIIINP expression in the serum, and MMP-2 and −9 mRNA expression in left ventricle tissues, was significantly increased in the AGEs group compared with the control (Fig. 2B-D). As demonstrated in Fig. 2E and F, AGEs significantly increased the expression of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) compared with the control, which indicates cardiac hypertrophy. As expected, chronic delivery of AGEs significantly increased the expression of profilin-1 compared with the control group, as determined by immunohistochemistry, western blot analysis and RT-qPCR (Fig. 3). No marked differences were observed between AGEs and AGEs + V groups (Figs. 1–3).

To investigate the role of profilin-1 in AGE-induced cardiac dysfunction, hypertrophy and fibrosis, the present study knocked down profilin-1 expression by intravenous delivery of adenovirus expressing profilin-1 shRNA. On day 6 after the second injection of adenovirus, 50 and 40% decreases in profilin-1 expression at protein and mRNA levels were observed, respectively (Fig. 4A and B). Knockdown of profilin-1 expression improved cardiac systolic function (Fig. 1) and reduced cardiac fibrosis, as indicated by reduced collagen deposition (Fig. 2A) and reduced PIIINP, MMP-2 and MMP-9 expression (Fig. 2B-D), compared with the AGEs-only group. In addition, silencing profilin-1 expression significantly inhibited AGE-induced cardiac hypertrophy, as indicated by reduced ANP and β-MHC expression (Fig. 2E and F) compared with the AGEs-only group.

Increasing evidence has indicated that AGEs activate various signaling pathways, including NF-κB and Rho/Rho-associated protein kinase (ROCK), and induces the expression of genes associated with diabetic complications by binding to RAGE (2,25,26). Due to the important role of the NF-κB signaling pathway in regulating oxidative stress, fibrosis, hypertrophy and apoptosis, and as the Rho/ROCK pathway in the actin cytoskeleton are reported to be involved DCM, p65 and Rho were selected for further investigation in the present study. Following chronic treatment with AGEs for 8 weeks, a significant increase in the expression of RAGE, p65 and Rho at the protein level was observed in left ventricle heart tissues, compared with the control group (Fig. 5). Notably, blockade of profilin-1 significantly reduced the expression of RAGE, p65 and Rho compared with the AGEs-only group (Fig. 5).