Interleukin‑10 promotes proliferation and migration, and inhibits tendon differentiation via the JAK/Stat3 pathway in tendon‑derived stem cells in vitro

Abstract

Tendon repair follows a slow course of early inflammatory, proliferative and remodeling phases, which commonly results in the failure and loss of normal biomechanical properties. Previous studies have demonstrated that tendon‑derived stem cells (TDSCs) are vital healing cells and that mRNA expression of anti‑inflammatory cytokine interleukin (IL)‑10 is significantly upregulated at the late inflammatory phase. To explore how IL‑10 may impact tendon healing, the present study investigated the in vitro effects of IL‑10 on TDSCs isolated from rat Achilles tendons. Cellular activities of TDSCs and the expression levels of tendon cell markers were measured treatment with IL‑10 and subsequent performance of wound healing assays, reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The results demonstrated that IL‑10 treatment markedly increased the proliferative capacity of TDSCs. In addition, IL‑10 significantly enhanced cell migration when compared with the control cells. Furthermore, IL‑10 treatment significantly activated the JAK/Stat3 signaling pathway and inhibited the protein expression of tendon cell markers, including scleraxis and tenomodulin. Notably, IL‑10 treatment also reduced the gene expression levels of type 1 collagen, type 3 collagen, lumican and fibromodulin in TDSCs. These findings indicated that IL‑10 enhanced cell proliferation and migration, and inhibited tenogenic differentiation in TDSCs in vitro. Reducing the negative effects whilst enhancing the positive effects of IL‑10 may be a potential therapeutic target in tendon repair.

Keywords: interleukin‑10; tendon; tendon‑derived stem cells; injury.

Figure 1.

IL-10 promotes cell proliferation and leads to G2 phase activation in TDSCs. TDSCs were treated with the indicated concentrations of IL-10 for (A) 1, (B) 3 and (C) 5 days. Cell proliferation was subsequently assessed with a Cell Counting Kit-8 assay. (D) TDSCs were treated with 10 ng/ml IL-10 for 3 days and subjected to cell cycle analysis by flow cytometry. (E) The percentage of TDSCs in the G1 phase decreased, whereas the percentage in the G2/M phase increased. Data are expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control cells. IL-10, interleukin-10; TDSCs, tendon-derived stem cells; P, population.

Figure 2.

IL-10 promotes TDSC migration (A) Representative time-lapse migration images of control and IL-10-treated cells from the wound healing assay. Images were acquired 0, 12 and 24 h after scratching. Original magnification, ×100. (B) The migration rate was measured by quantifying the total area of the entire strip lacking cells. The relative migration rate of recovered area at 12 and 24 h was calculated from three independent experiments. Data are expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control cells. IL-10, interleukin-10; TDSCs, tendon-derived stem cells.

Figure 3.

IL-10 inhibits spontaneous tenogenic differentiation. Tendon-derived stem cells were treated with the indicated concentrations of IL-10 for 3 days and subjected to quantitative polymerase chain reaction to detect the gene expression of (A) Scx and (B) Col1. (C) Representative images of the morphological alterations in control and IL-10-treated TDSCs (magnification, ×100). Data are expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control cells. IL-10, interleukin-10; Col1, collagen type 1; Scx, scleraxis.

Figure 4.

IL-10 alters the mRNA and protein expression levels tendon-associated molecules in TDSCs. (A) The mRNA expression Tnmd, Col3, Mkx, Egr1, Fmod and Lum was altered by IL-10 treatment. (B) Fluorescent staining of Col1 expression following treatment with IL-10 for 3 days. (C) Rat TDSCs were treated with the indicated concentrations of IL-10 for 3 days and subjected to western blot analysis for Scx, Col1, Tnmd and Col3 protein expression. (D) Densitometric analysis of the western blotting results. Data are expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control cells. IL-10, interleukin-10; TDSCs, tendon-derived stem cells; Tnmd, tenomodulin; Col3, collagen type 3; Mkx, mohawk; Egr1, early growth response gene 1; Fmod, fibromodulin; Lum, lumican; Dcn, decorin; Bgn, biglycan.

Figure 5.

IL-10 activates Stat3 but has no effect on Akt expression in TDSCs. (A) Rat TDSCs were treated with the indicated concentrations of IL-10 and were subjected to western blot analysis to detect p-Stat3, Stat3, p-Akt and Akt protein expression. (B) TDSCs were treated with the indicated concentrations of IL-10 with or without the Stat3 inhibitor WP1066 and subjected to western blot analysis for Col1, Tnmd and Col3 protein expression. Data are expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. 10 ng/ml IL-10. IL-10, interleukin-10; TDSCs, tendon-derived stem cells; p-, phosphorylated; Stat3, Signal transducer and activator of transcription 3; Akt, protein kinase B; Tnmd, tenomodulin; Col3, collagen type 3.