A549 type II alveolar epithelial cell line was obtained from American Type Culture Collection (ATCC; Rockville, Maryland, USA). Cells were grown in RMPI 1640 medium with 10% fetal bovine serum (FBS) (Gibco Life Technologies, Carlsbad, CA, USA), 1% 100 U/ml penicillin, and 1% 100 mg/ml streptomycin sulfate. Cells were incubated in a humidified incubator with 5% CO₂ at 37°C. MP strain M129 was cultured in SP-4 broth medium at 37°C until the medium turned to a peach yellow color. M. pneumoniae were dislodged with a dish scraper and suspended in sterile saline. The resulting M. pneumoniae mixture was passed through a 25-gauge needle ten times. A fraction of the M. pneumoniae was serially diluted and plated onto pleuropneumonia-like organism (PPLO) blood agar, after which the CFU counts were determined as previously described. M. pneumoniae M129 grew slowly, yielding extremely small colonies after culturing in PPLO blood agar for 7 days at 37°C (11). Plates were then overlaid with blood agar, and colonies were visible as hemolytic plaques after 2 days. The original stock was diluted to 1×10⁸ cells/50 µl aliquot and stored at −80°C. All subsequent experiments were performed with aliquots from the same frozen stock (12).

A549 cells were cultured in RPMI 1640 medium supplemented with 10% FBS in a 5% CO₂ at 37°C. After reaching 70% confluence, cells were serum-starved for 24 h to ensure that cell apoptosis and related signaling pathways that are potentially activated by serum are returned to basal levels before exposure to M. pneumoniae at the indicated multiplicity of infection or to various inhibitors or transfection with small interfering RNA (siRNA). The siRNA against caspase-3 and negative control (NC) were chemically synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China). Samples in the control group were exposed to sterile phosphate-buffered saline (PBS).

Total RNA was extracted from the cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using miScript Reverse Transcription kit (Qiagen N.V., Venlo, The Netherlands). Primers for caspase-3 and GAPDH were synthesized by GenePharma Co., Ltd. PCR runs were performed in triplicate. Primers for the target genes are presented in Table I.

Cells were collected and washed twice with cold PBS solution to remove floating cells before analysis using the Annexin V-APC Apoptosis Detection kit (Nanjing KeyGen BioTech Co., Ltd.,. Nanjing, China). Apoptosis was monitored using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). TUNEL detection kit (Roche Diagnostics, Ltd., Shanghai, China) was used to detect cell apoptosis following the manufacturer's instructions. Cells were counterstained with DAPI and examined with a fluorescence microscope.

The Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay was used for cell proliferation analysis following the manufacturer's instructions. Cells treated with M. pneumoniae were seeded at a density of 5×10³ cells per well in 96-well plates and exposed for varying incubation periods (0, 24, 48, 72 h). The absorbance was measured at 450 nm (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The effects of M. pneumoniae treatment on secretion of cytokines into culture medium by alveolar epithelial cells were determined using ELISA assays (R&D Systems, Inc., Minneapolis, MN, USA) or MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead multiplex assay (Millipore, Billerica, MA, USA) according to manufacturer's instructions.

Extracted proteins were resolved via SDS-PAGE and analyzed via western blotting. Antibodies used for western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA). Following incubation with horseradish peroxidase-coupled secondary anti-mouse/rabbit (Beyotime Institute of Biotechnology, Jiangsu, China), protein bands were visualized with ECL Blotting Detection Reagents (Millipore).

Student's t-test was used to analyze the differences between groups. Data were presented as means ± SD. Statistical analysis was performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered statistically significant. All experiments were independently performed at least three times.

After treatment with M. pneumoniae, apoptosis in A549 cells was detected using flow cytometry and TUNEL assay. Flow cytometry results showed that apoptosis rates at 0, 12, and 24 h were 8.3, 23.5, and 45.3%, respectively after treatment with M. pneumonia, respectively (Fig. 1A). However, apoptosis rates in cells treated for 12 and 24 h were significantly higher than those in the 0 h group (P<0.05). Similarly, results of the TUNEL assay showed that A549 cells exposed to M. pneumoniae at 12 and 24 h showed considerably higher apoptosis rates compared to those in the 0 h group (Fig. 1B).

M. pneumoniae influenced the apoptosis rates of A549 cells. Hence, we explored the changes in the expression of proteins involved in apoptosis. mRNA levels of caspase-3 and Bcl-2 were measured via real-time PCR. Results showed that transcriptional expression of caspase-3 increased with prolonged exposure to M. pneumoniae, with expression peaking at 24 h incubation (P<0.05; Fig. 2A). However, mRNA levels of Bcl-2 showed opposite expression patterns (Fig. 2B). In addition, results of real-time PCR were validated by western blotting, which showed similar changes in caspase-3 and Bcl-2 expression (Fig. 2C).

CCK-8 assay was performed to determine the effect of M. pneumoniae infection on A549 cell proliferation. OD values at 450 nm in the CCK-8 assay revealed marked differences in the proliferation of A549 cells treated with M. pneumoniae at three different treatment periods (P<0.05; Fig. 3A). A549 cells showed the lowest cell proliferation after treatment for 24 h. M. pneumoniae exposure also resulted in downregulation of JNK protein levels and upregulation of phosphorylated JNK levels (Fig. 3B).

Proliferation and apoptosis of alveolar epithelial cells were found to be closely associated with IL secretion. In this study, the concentrations of IL-4, IL-6, and IL-13 in the culture supernatant of alveolar epithelial cells were observed to increase with increasing incubation periods after stimulation by M. pneumoniae, and significant differences were observed among groups incubated for 0, 12, and 24 h (P<0.05; Fig. 4). By contrast, IL-10 levels decreased with prolonged exposure to M. pneumonia and showed significant differences among the three treatment periods (P<0.05; Fig. 4C).

In this study, we used siRNA interference to inhibit protein expression of caspase-3 in alveolar epithelial cells and further explored the mechanisms underlying M. pneumoniae-induced cell apoptosis. After siRNA interference, mRNA levels of caspase-3 in siRNA group treated for 24 h were found to be lower than that in the NC group (Fig. 5A). The siRNA group and NC group showed significant differences in apoptosis rates after stimulation with M. pneumoniae for 24 h (P<0.05; Fig. 5B). Results of the TUNEL assay showed that treatment with siRNA targeting caspase-3 suppressed M. pneumoniae-induced apoptosis in A549 cells (Fig. 5C). IL-6 and IL-10 levels also showed similar changes in expression upon M. pneumoniae treatment as Fig. 4. M. pneumoniae infection produces an opposite effect on the secretion of IL-6 and IL-10 by alveolar epithelial cells. But when transferred with siRNA of caspase-3, IL-6 and IL-10 secreted by cells infected M. pneumoniae showed the opposite changes, IL-6 decreases and IL-10 increases. These results also suggested that caspase-3 siRNA increased the level of IL-10 and reduced the level of IL-6 of alveolar epithelial cells. This may be due to the effect of siRNA of caspase-3 on cell apoptosis, and further affect the secretion of alveolar epithelial cells (P<0.05; Fig. 5D). Inhibition of caspase-3 expression abolished the effects of M. pneumoniae infection on alveolar epithelial cells and induced corresponding changes in Bcl-2 protein levels (Fig. 5E).