The aim of the current study was to explore the effects of dexamethasone (DXM) on autophagy and senescence in chondrocytes. Collagen II and aggrecan were examined in normal chondrocytes isolated from Sprague-Dawley rats. Following stimulation with DXM, LysoTracker Red staining, monodansylcadaverine (MDC) staining, green fluorescent protein-red fluorescent protein-light chain 3 (LC3) and western blotting were used to detect autophagy levels in the chondrocytes. Mechanistic target of rapamycin (mTOR) pathway-associated molecules were investigated by western blotting. Cell senescence was analyzed by senescence-associated (SA)-β-galactosidase (β-gal) staining. A dose-dependent increase in the number of autophagic vacuoles was observed in the DXM-treated chondrocytes, as demonstrated by LysoTracker Red and MDC staining. A dose-dependent increase in autophagosome formation was observed in the DXM-treated chondrocytes. Expression of LC3-II and beclin-1 was increased by DXM, in particular in the cells treated with DXM for 4 days. However, P62 expression was reduced as a result of treatment. SA-β-gal staining indicated that DXM increased cell senescence. Notably, DXM-induced cell senescence was exacerbated by the autophagic inhibitor 3-MA. Autophagy induced by DXM protected chondrocytes from senescence, and it is suggested that the mTOR pathway may be involved in the activation of DXM-induced autophagy.
Osteoarthritis (OA) is a common disease, and its incidence rate in people over 25 years old is 14%, with ~400,000 patients diagnosed with OA per year in the Catalonia region of Spain (
Autophagy, a self-protective mechanism, has been suggested to maintain cellular homeostasis by removing protein aggregates and damaged organelles by the fusion of autophagosomes and lysosomes. Previous studies have demonstrated that autophagy is a protective process preventing against chondrocyte apoptosis and cartilage degeneration (
Previous studies have demonstrated that the ability of joint tissues to restore the articular surface is reduced with age; patients with intra-articular fractures of the knee >50 years have a greater risk of developing OA than younger patients (
A previous study demonstrated that autophagy is a protective mechanism in human chondrocytes with mitochondrial dysfunction (
All procedures involving Sprague-Dawley rats were performed under the approval and guidance of the Animal Care and Use Committee at Southern Medical University (Guangzhou, China).
A total of 34 3-month-old Sprague-Dawley male rats, ranging in weight between 300 and 340 g, were obtained from the Laboratory Animal Center of Southern Medical University. These rats were anesthetized by 10% chloral hydrate and sacrificed by cervical dislocation, and the articular cartilage was separated from the femoral condyles and tibial plateaus under a microscope. Cartilage slices were incubated with trypsin (0.5 mg/ml) (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 30 mins at 37°C. Subsequent to removal of trypsin, the cartilage slices were incubated with 0.1% collagenase, type II (Sigma-Aldrich; Merck Millipore) in Dulbecco's modified Eagle's medium (DMEM; Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% fetal calf serum (FCS; Life Technologies; Thermo Fisher Scientific, Inc.) for 4 h at 37°C with shaking. The isolated chondrocytes were recovered and plated in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. The chondrocytes were incubated at 37°C in a humidified gas mixture containing 5% CO2 balanced with air. In the current study, all cells used were second-generation chondrocytes. DXM (Sigma-Aldrich; Merck Millipore) was added to the chondrocytes at various concentrations (0, 0.1, 1, 25 and 50
3-MA and DXM were added to chondrocytes at various concentrations (0
Chondrocytes were fixed with 4% paraformaldehyde for 30 min at 37°C. Chondrocytes were stained with 1% Alcian Blue 8GX (Sigma-Aldrich; Merck Millipore) dissolved in glacial acetic acid for 30 min at room temperature. The sections were counterstained with 0.1% nuclear read dissolved in 5% aluminium sulfate for 20 sec followed by routine dehydration. The stained sections and cells were washed three times with phosphate-buffered saline (PBS) and observed by light microscopy.
Chondrocytes (3×105 cells/well) were fixed with various concentrations of DXM for 4 days at 37°C in 24-well plates and rinsed with DMEM three times. LysoTracker Red culture medium (66 mM; 1 ml) was added to each well and then the cells were cultured for 30 min at 37°C. Subsequent to washing with PBS three times, the chondrocytes were observed using an inverted fluorescence microscope.
Monodansylcadaverine is a specific
Chondrocytes incubated with various concentrations of DXM (0, 0.1, 1, 25 and 50
Chondrocytes were treated with 3-MA and DXM at various concentrations (0
Prior to treatment with glucocorticoids, chondrocytes were transfected with monomeric (m)RFP-GFP-LC3 when the confluence was 50–70% using RFP-GFP-LC3 adenoviral vectors (HanBio Technology Co., Ltd., Shanghai, China). The multiplicity of infection was 100. The chondrocytes were incubated with the adenovirus in DMEM with no serum for 2 h at 37°C. The transfected chondrocytes were incubated with 10% DMEM supplemented with fetal bovine serum overnight prior to glucocorticoid treatment to eliminate the effect of starvation on an autophagic level. Following treatment with different doses of glucocorticoids for 4 days, autophagosomes and autolysomes in chondrocytes were observed under a confocal microscope (SP8; Leica Microsystems GmbH, Wetzlar, Germany).
Statistical analyses were performed using SPSS statistical software, version 16 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to analyze the differences between groups. Tukey's significance test was used to detect differences between two groups. P<0.05 was considered to indicate a statistically significant difference.
Chondrocytes were stained with toluidine blue. The extracellular matrices of the chondrocytes were stained blue and the nuclei were stained dark blue, which is consistent with typical chondrocyte characteristics (
Chondrocytes were incubated with different concentrations of DXM for 4 days and stained with LysoTracker and MDC to observe autophagic vacuoles. Compared with the control group, only a small number of chondrocytes displayed LysoTracker-positive staining (
The results of LysoTracker staining were similar to that of MDC staining. In the control group, only certain cells were stained positive for MDC (
Autophagy is a recycling process including the maturation of autophagosomes, fusion of autophagosomes and lysosomes, autolysosome formation and degradation. The total process is termed autophagic flux.
To determine the role of DXM in regulating chondrocyte autophagy, western blotting was conducted in order to observe the expression levels of LC3, beclin-1 and P62. When chondrocytes were incubated with different concentrations of DXM for 2 days, no significant differences in the expression levels of LC3-II/β-actin in the different groups were observed. However, after 4 days, the expression levels of LC3-II/β-actin were significantly increased with increasing concentrations of DXM, and after 6 days, the expression levels of LC3-II/β-actin were reduced at the highest concentration of DXM (
The mechanism of activating autophagy is predominantly associated with mTOR-dependent and mTOR-independent pathways, with p70S6K and 4EBP1 involved as downstream effectors in mTOR-dependent pathways. In the 1 and 25
When the cells become senescent, β-gal-positive staining occurs. The proportion of β-gal-positive chondrocytes significantly increased with time upon treatment with 25
In the current study, it was investigated whether DXM is able to inhibit chondrocyte autophagy through an mTOR-dependent pathway and induce senescence. Senescence activated by DXM was observed to be inhibited by autophagy, and it was identified that chondrocyte senescence increased with the inhibition of autophagy.
DXM is widely used to relieve a variety of symptoms caused by OA, however long-term and repeated treatment often results in complications (
Autophagy is not only similar to apoptosis but also has a protective effect on chondrocytes in OA (
mTOR is one of the components of mTOR complex 1 (mTORC1) and serves as a key switch in regulating autophagy, thus the autophagic signal pathway can be divided into mTOR-dependent and mTOR-independent pathways (
DXM can reduce cell growth and inhibit cell activity. Poulsen
The association between autophagy and senescence remains controversial. Kamalakannan
In conclusion, the current study identified that DXM can inhibit chondrocyte autophagy through an mTOR-dependent pathway and induce senescence. Autophagy may therefore serve as a protective process against senescence.
The present study was supported by a grant from the WenZhou Science and Technology Research Project (grant no. Y20140582).
(A) Chondrocytes were stained with alcian blue staining. (B)The LysoTracker Red staining of chondrocytes treated with dexamethasone at different concentrations for 4 days. A dose-dependent increase in the intensity of LysoTracker Red staining in chondrocytes stimulated with dexamethasone was observed. Scale bar, 100
The MDC staining, flow cytometry analysis and RFP-GFP-LC3 assay of chondrocytes treated with DXM. MDC staining of chondrcytes treated with different concentrations of DXM: (A) 0
The expression of autophagy-associated protein and mechanistic target of rapamycin pathway-associated proteins in chondrocytes treated with dexamethasone. (A, C and E) The expression of beclin-1, P62 and LC3 in chondrocyte analyzed by western blotting. (B, D and F) The corresponding optical densities of LC3-II/β-actin were analyzed. (G) The expression of P-p70S6 K and P-4EBP1 in chondrocytes analyzed by western blotting. (H and I) The corresponding optical densities of P-p70S6K/p70S6K and P-4EBP1/4EBP1 were analyzed. Data are presented as the mean ± standard deviation; n=3; *P<0.05 vs. control; **P<0.01 vs. control. LC3, light chain 3; P-, phosphorylated.
β-gal staining of the chondrocytes treated with DXM and/or 3-MA. (A–D) β-gal staining of the chondrocytes treated with 25