Oxymatrine protects articular chondrocytes from IL-1β-induced damage through autophagy activation via AKT/mTOR signaling pathway inhibition

Abstract

Background: Osteoarthritis (OA) is a common degenerative joint disease characterized by persistent articular cartilage degeneration and synovitis. Oxymatrine (OMT) is a quinzolazine alkaloid extracted from the traditional Chinese medicine, matrine, and possesses anti-inflammatory properties that may help regulate the pathogenesis of OA; however, its mechanism has not been elucidated. This study aimed to investigate the effects of OMT on interleukin-1β (IL-1β)-induced damage and the potential mechanisms of action.

Methods: Chondrocytes were isolated from Sprague-Dawley rats. Toluidine blue and Collagen II immunofluorescence staining were used to determine the purity of the chondrocytes. Thereafter, the chondrocytes were subjected to IL-1β stimulation, both in the presence and absence of OMT, or the autophagy inhibitor 3-methyladenine (3-MA). Cell viability was assessed using the MTT assay and SYTOX Green staining. Additionally, flow cytometry was used to determine cell apoptosis rate and reactive oxygen species (ROS) levels. The protein levels of AKT, mTOR, LC3, P62, matrix metalloproteinase-13, and collagen II were quantitatively analyzed using western blotting. Immunofluorescence was used to assess LC3 expression.

Results: OMT alleviated IL-1β-induced damage in chondrocytes, by increasing the survival rate, reducing the apoptosis rates of chondrocytes, and preventing the degradation of the cartilage matrix. In addition, OMT decreased the ROS levels and inhibited the AKT/mTOR signaling pathway while promoting autophagy in IL-1β treated chondrocytes. However, the effectiveness of OMT in improving chondrocyte viability under IL-1β treatment was limited when autophagy was inhibited by 3-MA.

Conclusions: OMT decreases oxidative stress and inhibits the AKT/mTOR signaling pathway to enhance autophagy, thus inhibiting IL-1β-induced damage. Therefore, OMT may be a novel and effective therapeutic agent for the clinical treatment of OA.

Keywords: AKT/mTOR pathway; Autophagy; Chondrocytes; Oxymatrine; interleukin-1β.

Fig. 1

Morphological observations and identification of rat chondrocytes. (a) Toluidine blue-stained chondrocytes are observed under an inverted-phase contrast microscope. (b) Collagen II immunofluorescence-stained cultured chondrocytes are observed under fluorescence microscope. Scale bar = 50 μm

Fig. 2

OMT improves IL-1β treated chondrocyte viability. (a) The cell viability of chondrocytes treated with different concentrations of OMT for 24 h as detected with the MTT assay. (b) The cell viability of chondrocytes treated with different concentrations of IL-1β for 24 h as detected with the MTT assay. (c) The effect of OMT on cell viability of chondrocytes treated with IL-1β for 24 h as detected with the MTT assay. Data are presented as the mean ± SD, n = 9 **P < 0.01, *P < 0.05 versus Con group; ^##P < 0.01, ^#P < 0.05 versus IL-1β group

Fig. 3

OMT mitigates IL-1β-induced injury in chondrocytes. (a, b) The cell apoptosis rates of chondrocytes as detected with Annexin V-FITC/PI assay using flow cytometry. (c, d) The cell damage rate of chondrocytes as detected with SYTOX Green staining. (e–g) The protein levels of collagen II and MMP-13 as detected with Western blot. Data are presented as the mean ± SD. A, B: n = 3/group; D–G: n = 3/group. **P < 0.01, *P < 0.05 versus Con group; ^##P < 0.01, ^#P < 0.05 versus IL-1β group

Fig. 4

OMT decreases ROS levels in chondrocytes treated with IL‑1β. (a, b) The ROS level as assessed with the DCFH-DA method using flow cytometry. Data are presented as the mean ± SD, n = 3/group. **P < 0.01, *P < 0.05 versus Con group; ^##P < 0.01, ^#P < 0.05 versus IL-1β group

Fig. 5

OMT decreases the AKT/mTOR signaling pathway and increases autophagy in chondrocytes treated with IL‑1β. (a–e) Western blot analysis and quantitative analysis of p-AKT, AKT, p-mTOR, mTOR, p62, and LC3 in chondrocytes. (f) LC3 expression is detected using the immunofluorescence assay. Data are presented as the mean ± SD, n = 3/group. **P < 0.01, *P < 0.05 versus Con group; ^##P < 0.01, ^#P < 0.05 versus IL-1β group

Fig. 6

Role of autophagy in OMT protection. (a) The cell viability of chondrocytes detected by MTT assay. (b-f) Western blot analysis and quantitative analysis of MMP-13, collagen II, p62, and LC3. Data are presented as the mean ± SD. A: n = 9/group; B-F: n = 3/group. ^△P < 0.05; ^△△P < 0.01