In total, 5-ml blood samples were collected from healthy individuals (2 males, aged 25 and 27 years), and PBMCs were isolated using lymphocyte separation medium (MP Biomedicals, LLC, Santa Ana, CA, USA). The PBMCs were subsequently washed twice with phosphate-buffered saline. In total, 2×10⁶ PBMCs were maintained in 10% 1640 culture medium (Invitrogen, Carlsbad, CA, USA) and stimulated with 100 ng/ml flagellin (IMG2205; Novus Biologicals, LLC, Littleton, CO, USA), the TLR-5 agonist. The present study was approved by the Ethics Committee of the West China Hospital of Sichuan University (Chengdu, China). Written informed consent was obtained from all the individuals prior to their participation.

At 4 h after stimulation, the flagellin-treated and untreated control PBMCs were washed three times to remove any remaining flagellin. The total RNA was extracted using a RNeasy mini kit (Qiagen, Dusseldorf, Germany), and a ReverTra Ace reverse transcription kit (FSQ-101; Toyobo Co., Ltd., Kagoshima, Japan) was used to synthesize cDNA following the removal of genomic DNA by DNaseI (RT411; Tiangen Biotech Co., Ltd., Beijing, China). The conditions for the reverse transcription reaction were as follows: 65°C for 5 min, followed by 37°C for 15 min and 98°C for 5 min.

Quantitative PCR was performed using SYBR Green RealMasterMix (FP202; Tiangen Biotech Co., Ltd.). The quantitative PCR assay was performed in an iCycler iQTM Optical Module (Beckman Coulter, Inc., Fullerton, CA, USA) under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and elongation at 72°C for 30 sec. A melting curve was constructed between temperatures of 55–95°C, in 0.5°C increments and 10-sec intervals. The primers used for quantitative PCR are shown in Table I, and glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Untreated PMBCs were used as a negative control, and all experiments were performed in triplicate.

Detection of secretory molecules using an antibody chip array. At 4 h after stimulation with flagellin, the cell supernatants were collected from the treated and untreated PBMCs. A RayBio® Human Antibody Array (C series; RayBiotech, Inc., Norcross, GA, USA) was applied for the screening of the secretory proteins, according to the manufacturer's instructions.

Data obtained from the quantitative PCR assay were analyzed using Bio-Rad iQ5 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The results are expressed as the mean ± standard error of the mean, and were statistically analyzed by a χ² test using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 and P<0.001 were considered to indicate a statistically significant difference. The figures were constructed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Expression levels of cytokines were analyzed using quantitative PCR, in order to determine the candidate molecules that were important in the TLR-5-mediated biological functions of PBMCs.

In the detection of cytokine expression, several important molecules were found to be highly expressed in the flagellin-stimulated PBMCs, which were IL-12 (P<0.001), tumor necrosis factor-α (P<0.001) and transforming growth factor (TGF)-β, as well as IL-2 and IFN-β (P<0.05; Fig. 1). By contrast, the expression levels of IL-1β (P<0.001) and IFN-γ-induced protein (IP)-10 (P<0.05) were inhibited upon TLR-5 activation, while the expression levels of IL-6 and IL-15 were found to be unchanged. Notably, the expression levels of IL-8 (P<0.05), IFN-γ (P<0.001) and nuclear factor-κB (P<0.001) were markedly inhibited following stimulation with the TLR-5 ligand (Fig. 1). These results were in contrast to a former study, which demonstrated that increased expression levels of IL-8 and IFN-γ upon pathogen stimulation were important in the immune response against foreign invaders.

In the study, the expression levels of a number of important chemokines from the chemokine (C-C motif) ligand (CCL) and chemokine (C-X-C) ligand (CXCL) families were detected. The results demonstrated that flagellin stimulation significantly increased the expression levels of CCL19 (P<0.001), CCL20 (P<0.001), CCL26 (P<0.001), CCL28 (P<0.001), CXCL6 (P<0.001) and CXCL12 (P<0.001) when compared with the untreated cells (Fig. 2). However, inhibited expression was observed for CCL5 (P<0.05), CCL8 (P<0.001) and CCL17 (P<0.001), and the expression levels of CCL22 and CXCL2 were shown to remain at the same level as the control cells (Fig. 2).

A number of molecules are known to play an important role in tumor initiation. The molecules selected for the study included the antitumor genes, P53 and phosphatase and tensin homolog (PTEN), the oncogenes, c-Myc and proliferating cell nuclear antigen (PCNA), as well as a number of important kinase signaling molecules, including β-catenin (CTNNB), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase kinase 1 and phosphorylated signal transducer and activator of transcription (pSTAT3). The results indicated that the expression levels of P53 (P<0.001) and PTEN (P<0.001) were highly induced by flagellin, while c-Myc expression was inhibited (P<0.05; Fig. 3). However, the expression levels of PCNA and vascular endothelial growth factor were not significantly influenced by TLR-5 activation. With regard to the kinase signaling molecules, induced expression was observed for MAPK and pSTAT3, while CTNNB and JNK expression levels were inhibited (Fig. 3).

Supernatants of the TLR-5-stimulated and unstimulated PBMCs were collected, and the detection of the secreted molecules was performed using a RayBio® antibody chip array. In total, 20 important cytokines and chemokines were included in the array. The results indicated that only four molecules produced a positive response. Among these molecules, the secretion of monocyte chemoattractant protein (MCP)-1 (P<0.05), MCP-3 (P<0.05) and macrophage inflammatory protein-1α were significantly inhibited (Fig. 4). However, the secretion of IL-8 was identified in the supernatant, which contradicted with the results from the quantitative PCR that indicated that IL-8 expression was inhibited by flagellin stimulation. An explanation for this phenomenon is that changes in gene expression are not always consistent with those in protein secretion.