UTI was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); antibodies for caspase-3 (cat. no. 14220S), B-cell lymphoma (Bcl)-2 (cat. no. 15071S), induced myeloid leukemia cell differentiation protein Mcl-1 (Mcl-1; cat. no. 94296S), Bcl-extra large (Bcl-xl; cat. no. 2762S), Bcl-associated protein X (Bax; cat. no. 2772S), activating transcription factor 4 (ATF4; cat. no. 11815S), GAPDH (cat. no. 8884S), horseradish peroxidase-linked secondary anti-rabbit (cat. no. 7074) and anti-mouse (cat. no. 7076) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). C/EBP homologous protein (CHOP; cat. no. ab11419), phospho-Akt (cat. no. ab38449), total Akt (cat. no. ab85683), phospho-phosphoinositide 3-kinase (PI3K; cat. no. ab182651), total-PI3K (cat. no. ab32089), phospho-mammalian target of rapamycin (mTOR; ab109268) and total-mTOR (cat. no. ab134903) were purchased from Abcam (Cambridge, UK). The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit was purchased from BD Bioscience (Franklin Lakes, NJ, USA). The Caspase-3 Activity Colorimetric Assay Kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). LY294002 and wortmannin, which were used at a concentration of 20 µM, were purchased from Selleck Chemicals (Houston, TX, USA). Cells were pretreated with LY294002 and wortmannin for 1 h at 37°C prior to experimentation. All other chemicals were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rapamycin was purchased from Sigma-Aldrich (Merck KGaA), which was kept as 1 mM stock at −80°C and used at 20 µM. Cells were pretreated with rapamycin for 1 h at 37°C prior to experimentation.

MSCs were isolated from the bone marrow of two male 2-week-old Sprague-Dawley rats (weighing 60–80 g) as described earlier (8). The rats were maintained under the following conditions: Temperature, 22±2°C; humidity, 55±5%; 12-h light/dark cycle; free access to food and water. All studies were performed under the approval of the Institutional Animal Care and Use Committee of Ningbo Medical Center Lihuili Eastern Hospital (Ningbo, China). Briefly, MSCs were flushed from femurs and tibias with 10 ml Dulbecco's-modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 1% penicillin/streptomycin (HyClone; GE Healthcare, Logan, UT, USA). The cells were centrifuged at 300 × g for 5 min at 4°C. The pellets were resuspended in 6 ml DMEM with 10% fetal bovine serum (HyClone; GE Healthcare) and 1% penicillin/streptomycin and plated in a plastic flask at 37°C in a humidified atmosphere containing 5% CO₂. After 3 days, the medium was replaced, and the cells were washed with PBS. The medium was replaced every 3 days. At 8 days after seeding, the cells became >70% confluent. Subsequently, the cells were collected from the dishes and expanded at a 1:2 dilution. All subsequent experiments were performed using MSCs at passages 3–5.

For the identification of the MSC immunophenotypic characteristics, the cells were collected, washed with PBS (HyClone; GE Healthcare) and labeled with the following antibodies: Phycoerythrin (PE)-labeled anti-cluster of differentiation 45 (CD45; cat. no. 553091; BD Pharmingen; BD Biosciences), anti-CD105 (cat. no. 611314; BD Pharmingen; BD Biosciences), anti-CD90 (cat. no. 55595; BD Pharmingen; BD Biosciences) and FITC-labeled-CD73 (cat. no. 561254; BD Pharmingen; BD Biosciences). Subsequently, the cells were detected by flow cytometry and analyzed using FACSDiva software (version 6.1.3; BD Biosciences).

MSCs (4×10⁵ cells/well) were seeded in 6-well plates. When cells reached ~70% confluence, MSCs were transfected with 200 pM negative control siRNA (5′-ACGGAACAGCGCACCGAGGCGAA-3′), siRNA against CHOP (5′-CCAGGAAACGGAAACAGAGTT-3′) or ATF4 (5′-TCCCTCCATGTGTAAAGGA-3′; Genepharm Inc., Sunnyvale, CA, USA) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 24 h post-transfection, cells were harvested and the effects of knockdown were investigated.

The harvested MSCs were cultured in 6-well (1×10⁵ cells/well) or 96-well plates (1×10⁴ cells/well) (Corning Incorporated, Corning, NY, USA) for subsequent experiments. To induce MSC apoptosis by exposing them to H/SD conditions, the normal medium was replaced with glucose- and serum-free medium. Subsequently, the MSCs were placed in an oxygen-free incubator for 12 h. MSCs cultured under normal conditions were used as controls.

Cell viability was assayed using the MTT assay as described previously (9). Briefly, cells were seeded in 96-well plates with a density of 2×10⁴/well. When reaching ~80% confluence, the cells were exposed to H/SD treatment with or without UTI (100, 200 and 400 U/ml). After 12 h, 25 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to the cultured media, and the cells were incubated for another 4 h. The medium was then removed, and 200 µl dimethyl sufoxide was added to each well. The absorbance of the solutions was measured using a microplate reader (BioTek China, Beijing, China) at 495 nm. The relative cell viability was measured by comparison with cells cultured under normal conditions. The calculated cell viability percentages from the three parallel experiments were averaged for each set of experimental conditions.

Cellular apoptosis was detected using an Annexin V/propidium iodide (PI) Apoptosis Detection kit (BD Pharmingen; BD Biosciences) according to the manufacturer's protocol. Briefly, 1×10⁵ cells were harvested and washed in ice-cold PBS, then cells were resuspended in 300 µl binding buffer and incubated with 5 µl Annexin V-FITC solution for 30 min in the dark at 4°C, followed by further incubation with 5 µl PI for 5 min at room temperature. Apoptotic cells were then determined using a flow cytometer (FACSCalibur; BD Biosciences). The results were analyzed by the FlowJo 10.4 (FlowJo LLC, Ashland, OR USA).

Cells were collected and lysed in radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology). Equal amounts of lysates (20 µg) were resolved by SDS-PAGE on 12% gels and transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were incubated with primary antibodies at a dilution of 1:1,000 overnight at 4°C. Subsequently, the membranes were washed three times with TBS with Tween20 and incubated for 1 h at room temperature with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; anti-mouse cat. no. 7076, or anti-rabbit cat. no. 7074; Cell Signaling Technology, Inc.). The signals were visualized using an enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.). The density of the protein bands was analyzed using ImageJ 1.41 software (National Institutes of Health, Bethesda, MD, USA).

The caspase-3 activity was assayed using the Caspase-3 Activity Colorimetric Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In brief, cellular lysates (150 µg) were incubated with 50 µl reaction buffer containing 10 mM dithiothreitol for 1 h at 37°C in the substrate DEVD-pNA (200 µM) in a total volume of 100 µl. The pNA light emission following cleavage from the substrate was quantified using a microplate reader (BioTek China) at 405 nm.

Statistical analysis was performed using SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Experiments were repeated three times and data are expressed as the mean ± standard deviation. Differences among groups were tested by one-way analysis of variance, followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

MSC characteristics were confirmed by flow cytometric analysis, which showed that the cells were negative for CD45, and positive for CD90, CD103 and CD73 markers (data not shown). To investigate the protective effects of UTI, cell viability was initially examined. As shown in Fig. 1A, UTI exhibited little cytotoxicity on MSCs under normal culture conditions. Cells were treated with increasing concentrations of UTI (100, 200 and 400 U/ml) when exposed to H/SD for 12 h (Fig. 1B). The data revealed that H/SD reduced cell viability and UTI protected MSCs against H/SD injury in a dose-dependent manner.

To further analyze the cytoprotective effects of UTI, the apoptosis rate of the MSCs was assayed by Annexin V/PI staining and flow cytometry. As indicated in Fig. 2A and B, treatment with UTI reduced the apoptosis induced by H/SD in a dose-dependent manner. Subsequently, the levels of cleaved caspase-3 were evaluated by western blotting. Cleaved caspase-3 was notably inhibited by UTI in a dose-dependent manner (Fig. 2C). Furthermore, caspase-3 activity assays revealed that activation of caspase-3 induced by H/SD was also decreased by UTI in a dose-dependent manner (Fig. 2D). Taken together, these findings demonstrated that UTI protects MSCs against apoptosis under H/SD conditions.

The process of apoptosis is regulated by Bcl-2 family proteins, which can be divided into anti-apoptotic and pro-apoptotic members (10). Therefore, whether Bcl-2 family members were affected by UTI was investigated. As indicated in Fig. 3, anti-apoptotic Bcl-2, Mcl-1 and Bcl-xl were decreased, while pro-apoptotic Bax was upregulated in MSCs exposed to H/SD conditions. Treatment with UTI reversed the effects of H/SD on Bcl-2 family proteins (Fig. 3). These findings suggested that UTI exerts a protective effect against H/SD-induced apoptosis via modulation of Bcl-2 family proteins in MSCs.

Previous studies indicated that ATF4 and CHOP are vital players in ER stress that are involved in apoptosis induced by H/SD (11). To determine whether ER stress is involved in the protective effect of UTI, ATF4 and CHOP expression levels were examined by western blotting. As shown in Fig. 4A, H/SD induced the upregulation of ATF4/CHOP, and treatment with UTI reversed the effect of H/SD on ATF4/CHOP expression levels. To further confirm the role of ER stress in H/SD-induced apoptosis of MSCs, siRNA was performed to knock down CHOP and ATF4 expression levels (Fig. 4B). As indicated in Fig. 4C, silencing of CHOP or ATF4 protects MSCs from apoptosis induced by H/SD. Taken together, these results suggested that UTI protects MSCs from H/SD-induced apoptosis, at least partially, by inhibiting ER stress.

The PI3K/Akt/mTOR signaling pathway serves a critical role in cell survival (9). Thus, to examine whether the PI3K/Akt/mTOR pathway is also involved in the anti-apoptotic effects of UTI, its activation was investigated by western blotting. As shown in Fig. 5A, H/SD treatment reduced expression levels of phospho-Akt, phospho-PI3K and phospho-mTOR compared with the control, whereas these effects were reversed by UTI treatment. To verify that the PI3K/Akt/mTOR signaling pathway was involved in UTI-mediated anti-apoptotic effects, two PI3K inhibitors, wortmannin and LY294002, were used. As shown in Fig. 5B, H/SD inhibited the expression of phospho-Akt, which can be activated by UTI treatment. However, both wortmannin (20 µM) and LY294002 (20 µM) fully inhibited the upregulation of phospho-Akt induced by UTI (Fig. 5B). Furthermore, the apoptotic cells were increased after wortmannin and LY294002 treatment compared with the UTI group under H/SD conditions (Fig. 5C). Additionally, rapamycin, an mTOR inhibitor (20 µM), was applied to investigate the role of mTOR in the protective effects of UTI. As indicated in Fig. 5D, rapamycin successfully inhibited the phosphorylation of mTOR. Additionally, the apoptotic cells increased following rapamycin treatment compared with the UTI group under H/SD conditions (Fig. 5E). Taken together, these findings further confirmed that UTI exerts protective effects via activation of the PI3K/Akt/mTOR pathway.