Recombinant human TGF-β1, which was dissolved in deionized water, was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA). Antibodies against Akt (polyclonal; rabbit anti-rat; dilution, 1:1,000; cat. no. 9272), phosphorylated-Akt (monoclonal; rabbit anti-rat; dilution, 1:1,000; cat. no. 4060), and β-actin (monoclonal; rabbit anti-rat; dilution, 1:1,000; cat. no. 8457) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-α-actin (mouse monoclonal immunoglobulin M; dilution, 1:100; sc-58670) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). LY294002 and assay kits used to examine the release of lactate dehydrogenase (LDH), caspase-3 and caspase-9 were purchased from Beyotime Institute of Biotechnology (Haimen, China). Methanol, chloroform, dimethyl sulfoxide, fetal bovine serum (FBS), phosphate buffered saline containing 0.1% Tween-20 (PBS-T) and bovine serum albumin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

A total of 6 adult Wistar rats (age, 6 weeks; mean weight, 200 g) were obtained from the Experimental Animal Center of Harbin Medical University (Harbin, China). The experimental procedures applied in this study were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. The rats were conditioned at a controlled ambient temperature of 22±2°C with 50±10% relative humidity and a 12 h light-dark cycle (lights on at 8:00 am). All rats were provided with standard chow and water ad libitum.

Rats were sacrificed at 6 weeks of age by cervical dislocation. The outer diameter of pulmonary arteries (distal, 200–500 µm) was dissected from the lungs of adult Wistar rats under an optical microscope (SZ61; Olympus Corporation, Tokyo, Japan). The extracted segments were cut open, then the adventitia and endothelium of pulmonary arteries were stripped mechanically. PASMCs were dispersed according to previously established methods (25,26). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum in an atmosphere containing 5% CO₂ at 37°C, in a humidified incubator. Subsequently, anti-α-actin was used to determine the purity of the primary cultured PASMCs. The cells of passages 2–3 were used in all the experiments. Serum deprivation was used in order to induce apoptosis in PASMCs, thus the cells were incubated in DMEM without any serum for 24 h. Cells were divided into three groups: Control, SD, and SD + TGF-β1. Cells cultured in complete medium (DMEM + 10% FBS) were used as the control group. Cells cultured in DMEM without FBS were used as the model of serum deprivation, and cells in the SD + TGF-β1 group were serum-deprived and cultured with 10 ng TGF-β1 for 24 h.

In order to determine the mRNA expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) in PASMCs obtained from Wistar rats, qPCR was performed. The PASMCs were divided into three groups (Control, SD and SD + TGF-β1). LY294002 (10 µm) was used to block the PI3K/Akt signaling pathway. After 24 h following treatment, the RNA from 1×10⁶ cells from each group was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, reverse transcription was performed to obtain cDNA using a PrimeScript RT Reagent kit (RR037A) from Takara Biotechnology Co., Ltd. (Dalian, China). Reagents used from the kit included 2 µl 5X PrimeScript buffer, 0.5 µl PrimeScript RT Enzyme Mix I, 0.5 µl Oligo dT Primer (50 µM), 0.5 µl Random 6-mers (100 µM), 500 ng RNA and 10 µl RNase Free dH₂O. The reverse transcription thermocycling conditions were as follows: 37°C For 15 min and 85°C for 5 sec, then stored 4°C. An Applied Biosystems 7300 Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) was used to perform all our qPCR experiments and a BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to confirm the specificity of the primers. The total reaction volume was 20 µl containing: 1x SYBR Premix Ex Taq II (RP820A; Takara Biotechnology Co., Ltd.), 10 µM forward and reverse primers, 0.4 µl ROX reference dye (Takara Biotechnology Co., Ltd.), and 2 µl of cDNA. The qPCR conditions were as follows: 95°C for 30 sec 40 cycles of 95°C for 5 sec, and 60°C for 31 sec, followed by routine melting curve analysis. The sequences of the primers were as follows: Bcl-2 forward, 5′-CGGGAGAACAGGGTATGA-3′ and reverse, 5′-CAGGCTGGAAGGAGAAGAT-3′ (149 bp); Bax forward, 5′-ATCCACCAAGAAGCTGAG-3′ and reverse, 5′-GTAGAAGAGGGCAACCAC-3′ (184 bp); and β-actin forward:5′-AGGCCCCTCTGAACCCTAAG-3′ and reverse, 5′-CCAGAGGCATACAGGGACAAC-3′ (118 bp). Relative quantification of target gene expression was calculated using the 2^−ΔΔCq method (27). The ratio of Bcl-2/Bax was then calculated.

The cells were cultured in 6 well plates, and 1×10⁶ cells were added to each well. Cells were divided into three groups: Control, SD, and SD + TGF-β1. Their growth was arrested for 24 h prior to adding 10 ng TGF-β1 under serum deprivation conditions (SD + TGF-β1 group). LY294002 (10 µm) was used to block the PI3K/Akt signaling pathway. Cells cultured in complete medium were the control group. The cells were then washed three times with ice-cold phosphate-buffered saline, 24 h after the treatment was applied. Subsequently, the cells were treated with 200 µl lysis buffer, containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA and 2 mM PMSF, and then incubated for 30 min on ice. The lysates were sonicated for 1 min and centrifuged at 17,000 × g for 15 min at 4°C. The protein concentrations in the supernatant were determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). The western blot protocol followed to determine the protein expression of the samples was similar to that reported in a previous study (28). Briefly, 50 µg protein was electrophoresed on SDS polyacrylamide gels, and then transferred onto polyvinylidene diflfluoride membranes (Merck Millipore, Darmstadt, Germany). Then, 5% bovine serum albumen was used to block the membrane for 1 h at room temperature. The membrane was then incubated with phosphorylated-Akt and Akt primary antibodies at 4°C overnight. After washing for 35 min with PBS-T, the membrane was incubated with an alkaline phosphatase-conjugated secondary antibody (monoclonal; goat anti-rabbit; dilution, 1:5,000; #7074; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. After washing for 35 min with PBS-T, immunoreactivity was detected using an enhanced chemiluminescence western blotting detection kit (Amersham Biosciences, Piscataway, NJ, USA) and exposed to X-ray film.

The MTT assay was performed according to the method published by Ma et al (26), in order to determine the cell viability. Briefly, 1×10⁴ cells were added to each well in 96-well culture plates and prepared using the same method as used in the western blot analysis. After incubation at 37°C for 24 h following treatment, the cells were incubated for 4 h in 0.5% MTT (Beyotime Institute of Biotechnology). The supernatant was then removed, and 150 µl dimethyl sulfoxide was added to each well. The plates were then agitated on a plate shaker for 10 min at room temperature. A spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) was used to read the absorbance at 490 nm. The measured absorbance value was used to represent the number of living cells.

The expression levels of LDH were determined using a Cytotoxicity Detection kit (Beyotime Institute of Biotechnology). The experiments were carried out as previous studies (26). Briefly, 100 µl culture medium and an equal volume of LDH substrate solution was added to the culture medium for 30 min at room temperature. The reaction was stopped by adding 0.1 M NaOH to the mixture, and a spectrophotometer (Epoch 2; BioTek Instruments, Inc.) was used to detect the absorbance at 440 nm.

The cleavage of two chromogenic caspase substrates was determined, including the caspase-3 substrate Ac-DEVD-pNA (also known as N-acetyl-Asp-Glu-Val-Asp p-nitroanilide) and the caspace-9 substrate Ac-LEHD-pNA (also known as N-acetyl-Leu-Glu-His-Asp p-nitroanilide). The experimental procedures followed the manufacturer's protocols and a previously published method (29). Briefly, 50 µg total protein was added to 50 µl reaction buffer (Beyotime Institute of Biotechnology), which contained 10 µl Ac-DEVD-pNA (2 mM) or 10 µl Ac-LEHD-pNA (2 mM), and the samples were incubated at 37°C for 2 h. The absorbance of yellow pNA cleaved from their corresponding precursors was measured using a spectrophotometer (Epoch 2; BioTek Instruments, Inc.) at 405 nm. The absorbance was used to represent the activity of caspase-3/9.

Statistical analysis was performed using SPSS version 15.0 for Windows (SPSS, Inc., Chicago, IL, USA). Experiments were performed in triplicate. All values were represented as the mean ± standard error of mean. One-way analysis of variance and t-test analysis (two-tailed) were used to determine the statistical significance of differences between the means of different groups. P<0.05 indicated a statistically significant difference.

MTT assay was applied to determine the effects of TGF-β1 (10 ng) on cell viability of PASMCs. Serum-deprivation was used to induce cell apoptosis. As shown in Fig. 1A, the decrease in cell viability due to serum deprivation was reduced following TGF-β1 treatment in a dose-dependent manner. TGF-β1 significantly improved cell viability when used at a concentration of ≥10 ng. The effects of TGF-β1 on cell death were also examined, and serum deprivation was found to result in increased release of LDH, which was reversed by the addition of 10 ng TGF-β1 (Fig. 1B; P<0.05). The results suggest that TGF-β1 improves cell viability and inhibits cell death in a dose-dependent manner in PASMCs.

Caspase-3 and caspase-9 are synthesized by the precursor proteins procaspase-3 and procaspase-9, respectively, in response to apoptotic stimuli; subsequently, they are then activated, triggering cell apoptosis (30). Therefore, the activity of caspase-3 and caspase-9 was examined to determine whether TGF-β1 inhibited the apoptosis of PASMCs. As shown in Fig. 2, the activity of caspase-3 and caspase-9 was significantly greater in untreated serum-deprived cells compared with the control group (P<0.05). This effect was reversed by treatment with TGF-β1 (10 ng), which indicates that TGF-β1 inhibits apoptosis induced by serum deprivation.

Bcl-2 and Bax are two important apoptosis-associated proteins, located on the outer membrane of mitochondria. Bcl-2 is an anti-apoptotic protein and Bax is a pro-apoptotic protein. They participate in maintaining mitochondrial integrity and regulating mitochondrial-dependent apoptosis (31). In the present study, TGF-β1 was found to inhibit the activation of caspase-9, a key molecular in mitochondrial-dependent apoptosis, and thus the protein expression levels of Bcl-2 and Bax were examined. The mRNA expression levels of Bcl-2 and Bax were examined using qPCR. The expression of Bcl-2 was reduced and the expression of Bax was increased in serum-deprived PASMCs compared with the control cells, while treatment with TGF-β1 (10 ng) reversed these trends (Fig. 3; n=3; P<0.05). These results indicated that TGF-β1 inhibits apoptosis by upregulating the expression of Bcl-2 and downregulating the expression of Bax, thus increasing the ratio of Bcl-2/Bax.

The PI3K/Akt pathway is one of the most important survival pathways, and has been reported to always be activated in PASMCs during PAH (32). TGF-β1 (10 ng) was found to significantly promote the phosphorylation of Akt compared with the control group (Fig. 4; P<0.05). LY294002 (10 µM) effectively blocked the activation of the PI3K/Akt pathway (Fig. 4A; n=3; P<0.05). In addition, the pro-survival effects of TGF-β1 on PASMCs were weakened following the blocking of the PI3K/Akt pathway (Fig. 4B and C; n=3; P<0.05). Caspase-3 and caspase-9 activity was not inhibited by TGF-β1 when the PI3K/Akt pathway was blocked (Fig. 4D and E; n=3; P<0.05). These results indicate that TGF-β1 inhibits the apoptosis of PASMCs via the PI3K/Akt pathway.