Dulbecco’s modified Eagle’s medium (DMEM)/F12, trypsin, fetal bovine serum (FBS) and type-II collagenase were purchased from Gibco Life Technologies (Carlsbad, CA, USA); penicillin-streptomycin was from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA); lipopolysaccharides (Escherichia coli serotype 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, MO, USA); Annexin V: FITC Apoptosis Detection kit was from BD Biosciences (San Diego, CA, USA); the Cell Death Detection ELISA^PLUS Assay kit was from Roche Diagnostics (Mannheim, Germany); anti-c-FLIP rabbit monoclonal antibody (#8510) was from Cell Signaling Technology, Inc. (Danvers, MA, USA); rabbit anti-GAPDH polyclonal antibody (bs-2188R) and goat anti-rabbit IgG polyclonal antibody (bs-0295G) were purchased from Bioss Company (Beijing, China); caspase 8 and 3 Activity Assay kits were purchased from Beyotime Institute of Biotechnology (Haimen, China).

hUCMSCs were isolated, as previously described (25). Umbilical cord tissues (15–20 cm) from three full-term healthy babies delivered by caesarean section at the First Affiliated Hospital of PLA General Hospital (Beijing, China), were thoroughly rinsed with phosphate-buffered saline (PBS) and cut into 1-mm³ samples, following removal of the umbilical vessels and external membrane. The tissues were placed in culture flasks (Corning, Tewksbury, MA, USA) at a distance of 0.5 cm with DMEM/F12, supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂. The medium was replaced slowly and gradually every 3 days, to ensure that fixation of the tissue. When cells in the tissue samples reached 80–85% confluence, the tissues were removed and the cells were digested with trypsin-EDTA (Gibco Life Technologies) and transferred to T-75 culture flasks for propagation and culture. Passage 3 cells were stored for use in subsequent studies. The protocol of the current study was approved by the ethics committee of the First Affiliated Hospital of PLA General Hospital (Beijing, China).

hUCMSCs were inoculated in 96-well plates at a density of 2×10³ cells/well for 24 h, and the medium was replaced with media containing different concentrations of LPS (0, 0.01, 0.1, 1, 10, 20, 30, 40 or 50 μg/ml) for continuous culturing. One plate from each concentration group was sampled at each time point (12, 24 and 48 h) and 20 μl/well of 5 mg/ml MTT was added. Following a 4-h incubation, the supernatant was removed from the plate and dimethyl sulfoxide (200 μl/well) was added. The samples were shaken for 5 min, the MTT formazan product was measured using a microplate reader (Synergy 2; BioTek Instruments, Ltd., Winooski, VT, USA) and the absorbance was measured at 490 nm.

hUCMSCs were cultured in 6-well plates and either treated with LPS (0, 1, 40 or 50 μg/ml) for 24 h, or pretreated with LPS (0, 0.1, 1, 10 or 20 μg/ml) for 12 h with subsequent exposure to 50 μg/ml LPS for 24 h. Following treatment, cells were harvested using trypsin-EDTA and collected by centrifugation at 900 × g for 5 min at room temperature. Cells were washed, resuspended in PBS, and labeled with Annexin V and propidium iodide (PI; from the BD Apoptosis Detection kit) for 20 min. Analyses were performed by flow cytometry (FC500; Beckman Coulter, Brea, CA, USA) using FC500 MPL CXP2.1 software.

hUCMSCs were cultured in 6-well plates. Following treatment, cultured cells were washed twice with PBS and treated with 4% paraformaldehyde (Guge Biological Technology Co., Ltd., Wuhan, China) for 10 min. After three washes with PBS, the cells were stained with 5 μg/ml Hoechst 33258 (Sigma-Aldrich) for 10 min. The morphology of the nuclei of the hUCMSCs was then analyzed under an inverted phase contrast microscope (DMI6000B; Leica Microsystems GmbH, Wetzlar, Germany).

hUCMSCs, with or without LPS stimulation, were rinsed twice with ice-cold PBS and lysed with ice-cold lysis buffer [1% Triton X-100, 20 mmol/l HEPES (pH 7.5), 1 mmol/l each of EDTA, DTT and PMSF, and 1 mg/ml each of leupeptin, aprotinin and pepstatin; Macgene (Beijing) Biotechnology Ltd., Beijing, China] for 30 min. Cell and nuclear lysates were centrifuged at 12,000 × g for 10 min at 4°C, and the supernatants were mixed with 5X SDS sample buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), boiled for 5 min and separated on 15% SDS-PAGE gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Following electrophoresis, proteins were transferred to nitrocellulose blotting membranes by electrophoretic transfer. Non-specific binding was blocked with 5% non-fat milk for 1 h. The membrane (Merck Millipore, Darmstadt, Germany) was then rinsed with TBST and incubated overnight at 4°C with the primary antibody (c-FLIP, 1:1,000 or GAPDH, 1:1,500). It was then washed in TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. After washing in TBST, bands were visualized using enhanced chemoluminescence and exposed to radiography film (Kodak, Tokyo, Japan).

The targeting sequence of human c-FLIP is GGAGCAGGGACAAGTTACA (26). Double-stranded, siRNA for c-FLIP was chemically synthesized by GeneChem, Inc. (Shanghai, China). The transfection of siRNA was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. In brief, cells were harvested using 0.25% trypsin, 1 mM EDTA in PBS without Ca²⁺ and Mg²⁺, and plated in 6-well plates at 10⁵ cells/cm². Next, 5 μl siRNA (100 lM) solution was mixed with 125 μl serum-free culture medium for 5 min at room temperature. During this incubation period, 5 μl Lipofectamine 2000 was diluted in 125 μl serum-free culture medium. These two mixtures were combined, mixed gently, and incubated for 20 min at room temperature to allow complex formation. The resulting 250 μl siRNA-Lipofectamine mixture was then added to the cells to produce a final volume of 2.5 ml/well. Forty-eight hours after infection, cells were washed and then incubated with LPS prior to use. Knockdown efficiencies were routinely monitored by western blotting.

Caspase 3 and caspase 8 activity levels were measured with the Caspase-Glo Assay System (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. Internucleosomal degradation of genomic DNA was detected using the Cell Death Detection ELISA^PLUS assay. hUCMSCs that were either untreated or treated with LPS, with or without transfection of siRNA, were washed twice with cold PBS and lysed on ice in a cold lysis buffer. Cell lysates were centrifuged at 12,000 × g for 10 min in order to precipitate cellular debris. The assay was performed in a 96-well plate, according to the manufacturer’s instructions, using the microplate reader, and the absorbance was measured at 405 nm.

All data are expressed as the mean ± standard deviation. Significance was analyzed using SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA). Statistical analysis was performed by one-way analysis of variance, followed by Bonferroni multiple-comparison test from >3 independent experiments. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the cytotoxicity of LPS, hUCMSCs were incubated with various concentrations of LPS for 12, 24 and 48 h, and cell viability was examined using an MTT assay. As shown in Fig. 1A, exposure of hUCMSCs to 40 or 50 μg/ml LPS resulted in a significant reduction in cell viability compared with a dose of 0 μg/ml, for all three time periods, while the cell viability was increased following treatment with low concentrations of LPS. Flow cytometric analysis using Annexin V/PI staining, further confirmed that cells exposed to 40 and 50 μg/ml LPS for 24 h exhibited a significant increase in apoptosis (Fig. 1B and C; P<0.01). By contrast, LPS (0.01–30 μg/ml) produced little or no cytotoxicity in hUCMSCs, indicating that LPS may exert antithetical effects, depending upon its concentration; low doses of LPS appeared to enhance cell viability while higher doses exhibited cytotoxicity in hUCMSCs. The current data indicates that LPS induces apoptosis in hUCMSCs in a dose-dependent manner.

An increasing number of studies have reported that low-dose LPS pretreatment results in cytoprotection in several cell types (16–19). Fig. 1A also indicates that low doses of LPS may promote hUCMSC viability. In order to explore the possible cytoprotective effects of low doses of LPS, prior to administration of a high-dose of LPS (50 μg/ml), LPS pretreatment was performed in hUCMSC cultures by incubation with a range of doses (0, 0.1, 1, 10 and 20 μg/ml) of LPS for 12 h. The apoptosis induced by LPS at 50 μg/ml was inhibited by pretreatment with 0.1 and 1 μg/ml LPS (particularly with 1 μg/ml; P<0.01). However, pretreatment with 10 and 20 μg/ml LPS did not significantly alter the apoptosis induced by treatment with high-dose LPS (Fig. 2A). As indicated by the cell death detection ELISA^PLUS assay, the severity of DNA fragmentation in hUCMSCs was consistent with the flow cytometry results (Fig. 2B). In addition, the apoptotic nuclear condensation in hUCMSCs was detected by Hoechst 33258 staining. In addition, as shown by Hoechst 33258 staining, cells treated with 50 μg/ml LPS presented apoptotic phenotypes, with clear chromatin conden sation, and cellular and nuclear shrinkage (Fig. 3A and B). LPS (1 μg/ml) pretreatment markedly reduced 50 μg/ml LPS-induced hUCMSC cell apoptosis (Fig. 3C). These results indicated that early exposure to low-dose LPS may protect hUCMSCs from the apoptotic consequences of subsequent lethal LPS insults, and that this effect is dependent upon the concentration of LPS used. The most effective cytoprotective effect was observed using 1 μg/ml LPS, which had not effect on the induction of apoptosis in hUCMSCs. Therefore, this dose of LPS was selected for LPS pretreatment in the subsequent experiments.

c-FLIP has been demonstrated to be involved in the prevention of apoptosis in a number of systems (22,27). In order to explore whether pretreatment with low concentrations (0, 0.1, 1, 10 or 20 μg/ml) of LPS modulates expression of c-FLIP in hUCMSCs, and to assess the response to high-dose LPS, the expression levels of c-FLIP were measured by western blot analysis. Following pretreatment with LPS for 12 h, the expression of c-FLIP was markedly increased in the hUCMSCs that had been pretreated with 0.1 and 1 μg/ml LPS. Statistical analysis indicated that pretreatment with 0.1 and 1 μg/ml LPS increased the expression level of c-FLIP to 1.63±0.11 and 2.36±0.15 times that of the control (P<0.01), respectively. However, pretreatment with 10 and 20 μg/ml LPS did not significantly enhance c-FLIP expression levels (Fig. 4A).

Following exposure of hUCMSCs to 50 μg/ml LPS for 24 h, the level of c-FLIP expression was markedly reduced, to 0.69±0.09-fold that of the control (P<0.01; Fig. 4B). However, the inhibition of the expression of c-FLIP induced by 50 μg/ml LPS, was significantly reduced by pretreatment with 1 μg/ml LPS for 12 h (P<0.01). This suggested that LPS pretreatment promotes the expression of c-FLIP and also prevents the reduction of expression induced by high-dose LPS treatment. It was thus inferred that the cytoprotective effects of pretreatment with low doses of LPS in hUCMSCs may be associated with overexpression of c-FLIP.

LPS has been used to induce a model of caspase-dependent apoptosis (26,28). As shown in Fig. 5, 50 μg/ml LPS induced apoptosis, and enhanced the activity of caspase 8 and 3 in hUCMSCs, and these effects were inhibited by the caspase inhibitor Z-VAD-fmk. This indicates that LPS-mediated hUCMSC apoptosis is also caspase-dependent. c-FLIP has been shown to block the apoptotic pathway by interacting with caspase 8 at the death-inducing signaling complex, through DED-DED interactions (22,29). The present study aimed to determine whether the upregulation of c-FLIP induced by 1 μg/ml LPS inhibits LPS-induced apoptosis in hUCMSCs that have been pretreated with low-dose LPS. Following treatment with 50 μg/ml LPS for 24 h, the activity of caspase 8 and 3 in LPS-pretreated hUCMSCs was significantly reduced compared with that in the group without LPS pretreatment (Fig. 5C and D; P<0.01). In addition, following siRNA c-FLIP inhibition, caspase 8 and 3 activity, and the level of apoptosis in hUCMSCs subjected to high-dose LPS exposure, were significantly elevated (P<0.01), and this effect was reduced by pretreatment with 1 μg/ml LPS for 12 h prior to exposure to the high-dose LPS. This result confirmed that LPS pretreatment prevents caspase-dependent apoptosis induced by high-dose LPS in hUCMSCs, via the upregulation of c-FLIP.