All aspects of the research were approved by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University (Guangzhou, China). Female Sprague-Dawley rats (n=2; 6-weeks-old; 170–200 g) were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, China).

TDSCs were isolated from the Achilles tendons of Sprague-Dawley rats as previously reported (18,19). Briefly, rats were anesthetized via an intramuscular injection of pentobarbital (30 mg/kg) and were subsequently sacrificed. Following this, the Achilles tendons were dissected and incubated in 600 U/ml (3 mg/ml) type I collagenase (cat. no. C0130; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and PBS for 2 h at 37°C with gentle shaking. The dissociated cells were plated at a density of 140 cells/cm² in 100 mm dishes and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) for 8–10 days at 5% CO₂ and 37°C. TDSCs at passage 3 or 4 were used in the subsequent experiments. The stem cell characteristics of TDSCs, including the proliferation, clonogenicity and multi-lineage differentiation potential, were confirmed prior to use in subsequent experiments using standard assays, including colony-forming unit fibroblast assays, Oil red O or Alizarin red staining and Alcian blue staining, as described previously (19).

To perform the cell proliferation assay, TDSCs were plated at a density of 10³ cells/well in a 96-well plate cultured in DMEM containing 10% FBS and allowed to adhere overnight at 5% CO₂ and 37°C. Following this, DMEM containing 10% FBS with 0.1, 1, 10 and 100 ng/ml rat IL-10 (cat. no. 400-19; PeproTech, Inc., Rocky Hill, NJ, USA) was added to TDSCs, which were then cultured for 1, 3 or 5 days at 37°C. Untreated cells cultured for 1, 3 or 5 days at 37°C were treated as the control. TDSC proliferation was subsequently determined using a Cell Counting Kit-8 assay (CCK-8; cat. no. KL640; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to previously published protocol (20,21).

Based on the results of aforementioned cell proliferation assays, TDSCs that were either untreated or treated with IL-10 (10 ng/ml) cultured in DMEM containing 10% FBS for 3 days were washed once in PBS and fixed with 500 µl cold 70% ethanol in PBS for 2 h at 4°C. TDSCs were centrifuged at 800 × g at 4°C for 5 min and washed again in PBS, then resuspended in 100 µl RNase A (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) and incubated at 37°C for 30 min. Following this, TDSCs were incubated with 400 µl propidium iodide (PI; Nanjing KeyGen Biotech Co., Ltd.) at 4°C for 30 min, and analyzed with a flow cytometer (FACScan; BD Biosciences, San Jose, CA, USA) and FlowJo 7.6.1 software (FlowJo LLC, Ashland, OR, USA).

Cell migration was determined with a wound-healing assay, as previously described (22). TDSCs were grown to 100% confluence in 6-well plates and treated with IL-10 (10 ng/ml) or DMEM containing 2% FBS at 37°C. A sterile P200 pipette tip was used to create a scratch across the cell monolayer. Cultures were subsequently washed once with 1 ml growth medium to remove the damaged and detached cells. Following medium replacement, TDSCs were cultured for 24 h at 37°C. Cell cultures were examined with a phase-contrast microscope (Olympus Corporation, Tokyo, Japan), and images of the entire strip, including three from the same scratch fields, were acquired at 0, 12 and 24 h after cells were scratched. The recovered gap area of the entire strip was measured at different time points using ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA). The migration rate was based on the calculated recovered gap area and was expressed as a percentage.

To demonstrate that IL-10 exerts its functions through the janus kinase (JAK)/Stat3 signaling pathway, TDSCs were plated at a density of 4×10⁵/well on a 60 mm dish, cultured in DMEM containing 10% FBS and allowed to adhere overnight at 5% CO₂ and 37°C. TDSCs were subsequently cultured in DMEM containing 10% FBS, supplemented with or without 10 ng/ml IL-10, and with or without 5 µM Stat3 inhibitor (WP1066; cat. no. S2796; Selleck Chemicals, Shanghai, China) for 3 days at 37°C. Following the above treatments, cells were collected and used in further experiments.

To investigate the effect of IL-10 on spontaneous tenogenic differentiation, spontaneously differentiated tenocytes were used as the control group, in which TDSCs were treated with basic culture medium without IL-10 for 3 days. Following the treatment of cells with 0, 0.1, 1, 10 or 100 ng/ml IL-10 for 3 days, total RNA was isolated from TDSCs with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Following this, total RNA was reverse-transcribed into cDNA using the PrimeScript^™ RT Master Mix kit (cat. no. RR036A; Takara Biotechnology Co., Ltd., Dalian, China). Reactions were incubated in a LightCycler^® 480 system (Roche Diagnostics, Basel, Switzerland) for 15 min at 37°C, followed by 5 sec at 85°C and then cooling to 4°C. The resulting cDNA was subjected to qPCR in a LightCycler^® 480 system with SYBR green reagent (cat. no. AK8307; Takara Biotechnology Co., Ltd.). The thermocycling conditions used were as follows: Pre-denaturation at 95°C for 30 sec; followed by 40 cycles of denaturation at 95°C for 5 sec, and annealing and extension at 60°C for 30 sec. The mean Cq value was calculated from triplicate reactions. The relative expression levels of scleraxis (Scx), Col1, tenomodulin (Tnmd), collagen type 3 (Col3), mohawk (Mkx), early growth response gene 1 (Egr1), fibromodulin (Fmod), lumican (Lum), decorin (Dcn) and biglycan (Bgn) were calculated based on a standard curve and normalized to GAPDH (23). The primer sequences used are listed in Table I.

Following the aforementioned treatments, Col1 immunofluorescence staining was performed on the cell monolayer in a two-step procedure. Specifically, Cells were cultured until a 40–60% confluence was reached prior to immunofluorescence staining. Following this, cells were fixed in 4% paraformaldehyde at room temperature for 10 min, lysed using 0.5% Triton X-100 and then blocked with 5% bovine serum albumin (cat. no. 36101ES25; Shanghai Yusheng Biotechnology Co., Ltd., Shanghai, China) at room temperature for 30 min. Cells were then incubated overnight at 4°C with primary rabbit anti-Col1 antibodies (1:100; cat. no. 14695-1-AP; ProteinTech Group, Inc., Chicago, IL, USA) and subsequently incubated with tetramethylrhodamine-tagged goat anti-rabbit IgG secondary antibodies (1:100; cat. no. HA1016; Hangzhou HuaAn Biotechnology Co., Ltd., Hangzhou, China) for 2 h at room temperature. Following this, cells were incubated with 4′,6-diamidino-2-phenylindole at room temperature for 5 min prior to being photographed using a fluorescence microscope (Olympus BX51; magnification, ×100; Olympus Corporation, Tokyo, Japan).

TDSCs were collected and radioimmunoprecipitation assay lysis buffer was used to extract total cell protein. Protein concentration in the samples was measured using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). Protein samples (25 µg) were separated via 10% SDS-PAGE, electrotransferred onto polyvinylidene fluoride membranes and then blocked using 5% bovine serum albumin (cat. no. 36101ES25; Shanghai Yusheng Biotechnology Co., Ltd.) at room temperature 1 h. Following this, membranes were incubated with primary antibodies [rabbit polyclonal antibodies against Scx (1:500; cat. no. ab58655; Abcam, Cambridge, MA, USA), Tnmd (1:500; cat. no. ab203676; Abcam), Col1 (1:1,000; cat. no. 14695-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), Col3 (1:1,000; cat. no. 13548-1-AP; ProteinTech Group, Inc.), Stat3 (1:1,000; 10253-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-Stat3 (1:1,000; Tyr705; cat. no. AF3295; Affinity Biosciences, Cincinnati, OH, USA), protein kinase B (Akt; 1:500; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-Akt (1:500; cat. no. 66444-1-Ig; ProteinTech Group, Inc.) and GAPDH [1:5,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.)] overnight at 4°C. Membranes were subsequently incubated with horseradish peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) secondary antibodies (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) for 2 h at room temperature. Proteins were visualized with the electrochemiluminescence kit (cat. no. P0018; Beyotime Institute of Biotechnology, Shanghai, China) and images were captured using the Tanon imaging system (Tanon-5200; Tanon Science and Technology Co., Ltd., Shanghai, China).

Results were analyzed using the SPSS version 20 (IBM Corp., Armonk, NY, USA) and expressed as the mean ± standard error of the mean. Data represents at least three replicates for each experimental condition. A Student's t-test, or one-way analysis of variance followed by Dunnett's post-hoc test was used to identify statistical differences. P<0.05 was considered to indicate a statistically significant difference.

The effect of IL-10 on cell proliferation was initially examined. TDSCs were treated with 0, 0.1, 1, 10 or 100 ng/ml IL-10 for 1, 3 or 5 days, and cell proliferation was determined with a CCK-8 assay. The results revealed a statistically significant increase in cell proliferation in the IL-10-treated cells compared with the controls. At day 1, there was no significant difference between any groups (Fig. 1A). However, the optical density values of TDSCs treated with 10 and 100 ng/ml IL-10 were significantly higher after 3 days compared with the control (P<0.05; Fig. 1B). At day 5, all experimental groups exhibited a significant increase in cell proliferation compared with the control group (Fig. 1C). Based on these results, it was concluded that a 3 day incubation with 10 ng/ml IL-10 would be the condition used to induce the desired effects in the subsequent experiments.

IL-10 altered the cell cycle of TDSCs. Following incubation of TDSCs with 10 ng/ml IL-10 for 3 days, cell cycle progression was analyzed by flow cytometry. The results demonstrated that the G1 phase was activated and an increased number of cells were in the G2/M phase in IL-10-treated cells, compared with the control (Fig. 1D). In the control and 10 ng/ml IL-10-treated TDSCs, the percentages of cells in G1 phase was 80.7±0.78 and 74.2±0.95%, respectively (Fig. 1E), indicating that IL-10 accelerated G1 phase cell cycle division, compared with the controls (P<0.05). By contrast, the percentage of control and 10 ng/ml IL-10-treated TDSCs in the G2/M phase was 16.3±0.71 and 21.9±1.12%, respectively (P<0.05; Fig. 1E). However, the percentage of TDSCs in the S phase with and without IL-10 treatment was almost identical.

To examine whether IL-10 promotes the repair capacity of TDSCs, an in vitro wound-healing assay was performed. A confluent monolayer of TDSCs was wounded and treated with or without 10 ng/ml IL-10 for 24 h. Microscopy indicated that wound closure in IL-10-treated TDSCs appeared to be significantly greater at 12 and 24 h compared with the control cells (Fig. 2A). Quantitative analyses indicated the cells in the IL-10-treated group had a quicker migration velocity, compared with that in the control group at the two different time points. The mean relative gap closure in cells treated with 0 and 10 ng/ml IL-10 was 53.35±5.54 and 79.18±4.65% after 12 h, and 66.31±4.50 and 92.10±1.04% after 24 h, respectively (Fig. 2B; P<0.01).

Following the treatment of cells with 0, 0.1, 1, 10 or 100 ng/ml IL-10 for 3 days, the gene expression levels of tendon cell markers, Scx and Col1, were analyzed. The results demonstrated that a significant dose-dependent reduction in the mRNA expression levels of Scx (Fig. 3A) and Col1 (Fig. 3B) occurred. Notably, cells treated with 10 or 100 ng/ml IL-10 were suppressed to a larger extent when compared with the control cells (P<0.01). Furthermore, it was revealed that cells in the IL-10-treated group exhibited long spindle-like shapes and orderly arrangement, while those in control group were ovoid in shape (Fig. 3C).

RT-qPCR analysis revealed that IL-10 significantly downregulated the gene expression levels of Tnmd, Col3 and Egr1, Fmod and Lum; however MKx expression was significantly upregulated compared with the control (Fig. 4A). Additionally, no distinctions were observed in the gene expression of Dcn and Bgn between the control and IL-10-treated cells (Fig. 4A). Immunofluorescence analysis demonstrated that the cytoplasm of the control group contained more Col1 than that of the IL-10-treated group (Fig. 4B). At the protein level, western blot analysis consistently revealed significant reductions in the expression levels of Col1 (P<0.01), Scx (P<0.05), Tnmd (P<0.01) and Col3 (P<0.01) in TDSCs incubated with 10 ng/ml IL-10, compared with the control (Fig. 4C and D).

In order to investigate the molecular pathways responsible for the effects of IL-10, the response of TDSCs to IL-10 (10 ng/ml) was examined. It was demonstrated that Stat3 was phosphorylated to a significantly higher degree following IL-10 treatment, compared with the control group, although the PI3K/Akt signaling pathway was not activated (Fig. 5A). These results demonstrated that TDSCs responded directly to IL-10 stimulation. The effect of IL-10 on the gene expression of tendon cell markers, Col1, Col3 and Tnmd, was significantly suppressed by treatment of TDSCs with the Stat3 inhibitor, WP1066, which confirmed that IL-10 inhibited spontaneous tenogenic differentiation of TDSCs by activating the JAK/Stat3 pathway (Fig. 5B).