A total of 90 6-week-old male Wistar rats, weighing 160–230 g, were obtained from the Centre of Laboratory Animals of Harbin Medical University (Harbin, China). The rats were given free access to food and water and were caged individually in a controlled temperature (21–22°C) in 50–60% humidity, with an artificial light cycle (12-h light/dark). All rats were acclimated for 5 days and randomized into the following three groups (n=10/group): vehicle (control), DXM, and icariin (DXM group treated with icariin). The vehicle group received normal feed for 12 weeks; the DXM group were injected intramuscularly with 5 mg/kg body weight DXM (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) three times a week for 12 weeks; rats in the icariin group were dosed orally for 12 weeks with icariin (100 mg/kg/day; Sigma-Aldrich; Merck KGaA) combined with DXM for 12 weeks. The animal protocol was approved by the Committee on the Ethics of Animal Experiments of Harbin Medical University.

Articular chondrocytes were isolated from the knee joints of rats in the vehicle group as previously described (17), with some alterations. Articular cartilage tissues were cut into small pieces (<1 mm³) and digested with 0.2% trypsin and 0.2% type II collagenase for 30 min and 2 h, respectively. The released cells were maintained in Dulbecco's modified Eagle's medium/F12 supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of CO₂ in air. When the cells reached ~90% confluence, they were treated with various concentrations of DXM (0, 10, 50 and 100 µM) for 48 h at 37°C. For miR-206 inhibitor treatment, cells were transfected with anti-miR-206 inhibitor (200 nM; Riboxx GmbH, Radebeul, Germany) using GeneCellin transfection reagent (BioCellChallenge, Toulon, France), according to manufacturer's instructions. A total of 48 h after transfection, cells were harvested and used for further experiments. The sequence of the miR-206 inhibitor was 5′-CCACACACUUCCUUACAUUCCA-3′.

RT-qPCR was performed as previously reported (18), with some changes. Briefly, the cartilage tissues were crushed under liquid nitrogen conditions and total RNA extraction was performed with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to manufacturer's instructions. Samples (1 µg RNA) were reverse-transcribed using a Reverse Transcription System for real-time PCR (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's instructions. Synthesized cDNA was used in qPCR experiments using SsoFast™ EvaGreen Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). qPCR was performed with the following primers: alkaline phosphatase (ALP) sense, 5′-GCCCTCTCCAAGACATATA-3′ and antisense, 5′-CCATGATCACGTCGATATCC-3′; TRAP sense, 5′-AGATCTCCAAGCGCTGGAAC-3′ and antisense, 5′-AGGTAGCCGTTGGGGACCTT-3′; osteocalcin sense, 5′-ATGAGAGCCCTCACACTCCTC-3′ and antisense, 5′-CTAGACCGGGCCGTAGAAGCG-3′; cathepsin K sense, 5′-GGGAGACATGACCAGCGAAG-3′ and antisense, 5′-CTGAAAGCCCAACAGGAACC-3′; collagen type I (Col-1) sense, 5′-TCCTGCCGATGTCGCTATC-3′ and antisense, 5′-CCATGTAGGCTACGCTGTTCTTG-3′; matrix metalloproteinase (MMP)-13 sense, 5′-CCCTGGAGCCCTGATGTTT-3′ and antisense, 5′-CTCTGGTGTTTTGGGGTGCT-3′; transforming growth factor (TGF)-β1 sense, 5′-CCAAGGAGACGGAATACAGG-3′ and antisense, 5′-GTGTTGGTTGTAGAGGGCAAG-3′; miR-612 sense, 5′-CAGGGCTTCTGAGCTCCTTA-3′ and antisense, 5′-TGAGAGTCCTGTCCTGGCTG-3′; miR-206 sense, 5′-GATTCGCCAAAGGAAATAGC-3′ and antisense, 5′-GTTACAAGGTCATCCAAGAC-3′; miR-28-5p sense, 5′-GTGCACTGTCACGGGTTTTC-3′ and antisense, 5′-CCTCTGCAGCCTGGTGAC-3′; miR-714 sense, 5′-CTGCAAGGGTGGAGGTGTAG-3′ and antisense, 5′-AGGCAGTGGTCTAAACTCGC-3′; miR-7a sense, 5′-TGTTGGCCTAGTTCTGTGTGG-3′ and antisense, 5′-GGCAGACTGTGATTTGTTGTCG-3′; miR-365 sense, 5′-AAATGAGGGACTTTCAGGGGC-3′ and antisense, 5′-AACAATAAGGATTTTTAGGGGCATT-3′; and GAPDH sense, 5′-GTCGGTGTGAAC GGATTTG-3′ and antisense, 5′-CTTGCGTGGGTAGAGTCAT-3′. qPCR amplification was performed with an initial denaturation at 95°C for 5 min, followed by 27 cycles of 95°C for 1 min, 65°C for 1 min and 72°C for 1 min, with a final extension step of 72°C for 10 min. Results were analyzed with Opticon Monitor software 3.1 (Bio-Rad Laboratories, Inc.). Specificity was determined by 1% agarose gel electrophoretic analysis of the reaction products. GAPDH was used as an internal standard. Data were analyzed using the 2^−ΔΔCq method, as previously described (19).

Western blotting was performed as previously reported (20). Briefly, proteins were extracted by lysing cells in buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na₃VO₄, 1 mM phenyl methylsulfonyl fluoride, 25 mg/ml leupeptin and 25 mg/ml aprotinin). Protein concentration was determined using a bicinchoninic acid protein assay (Bio-Rad Laboratories, Inc.). Subsequently, proteins (20 µg) were separated by 10% SDS-PAGE mini-gel and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA) for 60 min at 100 V. Following incubation in blocking buffer (Tris-buffered saline containing 150 mM NaCl, 50 nM Tris and 0.05% Tween-20; pH 7.5) for 1 h at room temperature, the membrane was hybridized in blocking buffer with specific primary antibodies against cathepsin K (sc-48353; 1:100), B-cell lymphoma 2 (Bcl-2; sc-56015; 1:50), Bcl-2-associated X protein (Bax; sc-70407; 1:200), caspase-3 (sc-271759; 1:200), caspase-9 (sc-8355; 1:200) and β-actin (sc-70319; 1:200) overnight at 4°C. Subsequently, the membrane was incubated with secondary antibodies labeled with horseradish peroxidase (sc-516102; 1:200) for 1 h at room temperature, followed by detection with an enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA). All antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). β-actin was used as the protein loading control. Band intensities were quantified by immunoblot densitometry using Image-Pro Plus 4.5 (Media Cybernetics, Inc., Rockville, MD, USA).

Anesthetized rats were placed in a euthanasia chamber. Directly after aspiration, blood was transferred to plain tubes. Serum was centrifuged at 2,000 × g for 1 min at 4°C, and stored at −80°C until analysis. Serum levels of CTX-II and deoxypyridinoline (DPD) were measured using a rat ELISA assay (60-2700; Immutopics, Inc., San Clemente, CA, USA), as previously described (21).

Cartilage tissues were dissected along the axial plane into pieces of 10×5×7 mm with a thin layer of subchondral bone and were fixed in 4% formaldehyde for over 24 h at room temperature. Following decalcification in 10% EDTA solution for over 2 weeks, the samples were embedded in paraffin. The specimens were cut into 4-µm sections and stained with safranin-O for 5 min at room temperature. To evaluate the volume of cartilage formation, the area occupied by chondrocytes and cartilage matrix stained with safranin-O was quantified using an image analysis system (ImageJ version 1.43u; National Institutes of Health, Bethesda, MD, USA).

The potential binding sites between cathepsin K and miR-206 were predicted using TargetScan (targetscan.org/mamm_31/). A luciferase assay was used to validate cathepsin K as a target of miR-206. Articular chondrocytes (~7.5×10⁴ cells/well) were seeded in 12-well plates, then transfected with the wild-type, mutant cathepsin K 3′ untranslated region (3′UTR) or the vector alone (co-transfected using pGL3-control) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's instructions. After 48 h, cells were lysed, then the firefly and Renilla luciferase activities were measured using the dual luciferase reporter assay kit (Promega, Madison, WI, USA) on a luminometer (Orion II Microplate Luminometer; Berthold Detection Systems GmbH, Pforzheim, Germany). To check the specificity of the miR-206 effect, chondrocytes were co-transfected with wild-type cathepsin K 3′UTR or miR-206 before luciferase activities were measured. Control cells were transfected with the scramble sequence. Renilla reporter luciferase activity was normalized to the firefly luciferase activity.

Cell viability was assessed by LIVE/DEAD assay as previously reported (22). Briefly, cells were stained using a LIVE/DEAD stain kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. This kit contains two fluorescent dyes, calcein-AM to stain living cells green and ethidium homodimer-1 (Ethd-1) to stain dead cells red. Following staining, samples were observed through an epifluorescent microscope (Carl Zeiss AG, Oberkochen, Germany) at a magnification of ×200. For each well, at least five different fields were examined, and a minimum of 1,000 cells were counted to determine the fraction of calcein-AM-positive cells vs. Ethd-1-positive cells.

In addition, to evaluate apoptotic activity of chondrocytes, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) staining was performed, as previously described (23). Frozen cartilage sections were fixed with 4% methanol-free formaldehyde solution in PBS for 10 min at room temperature, then washed with PBS three times. The DNA fragments were labeled with fluorescein-12-dUTP in terminal deoxynucleotidyl transferase incubation buffer (Promega) in a humidified chamber (37°C for 60 min) to avoid exposure to light. The reactions were stopped by transferring the slides to SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) for 15 min and washing with PBS to remove unincorporated fluorescein-12-dUTP. The slides were then counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA, USA) for 5 min at room temperature to provide a blue background. The green fluorescence of apoptotic cells (fluorescein-12-dUTP) may be detected using a fluorescence microscope at 520 nm.

Caspase-3 activity was evaluated as previously reported (24). Assays were performed in 96-well microtiter plates by incubating 20 µg cell lysate in 100 µl reaction buffer (1% NP-40, 20 mM Tris-HCl pH 7.5, 137 mM NaCl and 10% glycerol) containing 5 µM caspase-3 substrate (Ac-DEVD-pNA; Sigma-Aldrich; Merck KGaA). Lysates were incubated at 37°C for 2 h. Subsequently, the absorbance was measured at 405 nm with a spectrophotometer.

All experiments were conducted at least three times. Data were presented as the mean ± standard error of the mean. Statistical analyses were performed using PRISM version 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). The differences among groups were analyzed by one-way analysis of variance, followed by Tukey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

The expression of biochemical markers of bone turnover in the three experimental groups, including TRAP as a bone resorption marker, and ALP and osteocalcin as bone formation markers, were first determined using RT-qPCR. Compared with the vehicle control group, the DXM group demonstrated significantly increased expression of ALP and TRAP; however, icariin treatment significantly eliminated the enhancing effect of DXM on the expression of ALP and TRAP compared with the DXM group (Fig. 1A and B). In contrast, icariin significantly stimulated osteocalcin expression following DXM treatment compared with the levels observed in the DXM treatment group (Fig. 1C). Additionally, it was also determined that icariin abolished the promotion effect of DXM on cathepsin K expression, an enzyme expressed by osteoclasts in response to bone resorption, at the mRNA and protein expression levels (Fig. 1D and E).

The present study assessed the effects of icariin on articular cartilage in the three groups. As demonstrated in Fig. 2A, a small volume of cartilage formation was observed in the DXM group by safranin-O staining, relative to the control group; however, continuous administration of icariin enhanced the volume of cartilage formation. Similarly, administration of icariin to DXM-treated rats significantly elevated the reduced cartilage area (occupied by chondrocytes and cartilage matrix dyed with safranin-O; Fig. 2B). More importantly, icariin significantly reversed the DXM-induced increase in serum concentrations of DPD and CTX-II, the commonly used serum markers of cartilage metabolism, in experimental rats (Fig. 2C and D). Furthermore, the expression of Col-1, MMP-13 and TGF-β in the three experimental groups, which are cartilage metabolism-related genes, were determined. Icariin significantly reduced the elevated expression of Col-1 and MMP-13 induced by DXM, but promoted TGF-β expression (Fig. 2E–G).

The results in Fig. 2 demonstrated the beneficial effect of icariin on cartilage metabolism in DXM-treated rats, thus it was speculated whether icariin affected articular cartilage of experimental rats at the miRNA level. The relative expression of the miRNA in articular cartilage of all experimental rats were presented as a heatmap (Fig. 3A). The deeper the color, the higher the expression. A total of five miRNA were further determined to be significantly upregulated in the icariin group, compared with the reduced levels in the DXM-treated group, including miR-612, -206, -28-5p, -7a and -365 (Fig. 3B-G). In view of the role of miR-206 as a key regulator during osteoblast differentiation (25), miR-206 was selected as the candidate for the following experiments.

To determine whether miR-206 regulated cathepsin K through the predicted binding sites in its 3′UTR (Fig. 4A), two luciferase constructs were employed by incorporating wild-type or mutant 3′UTR of cathepsin K, which expressed luciferase unless repressed by the incorporated 3′UTR. As demonstrated in Fig. 4B, co-transfection with the pMIR-REPORT construct containing mutant cathepsin K 3′UTR and PLemiR-206 did not exhibit a significant difference compared with the control; however, co-transfection with the luciferase construct containing wild-type cathepsin K 3′UTR and PLemiR-206 resulted in an ~70% decline in luciferase activity compared with the control. As demonstrated in Fig. 4C and D, compared with the control group, miR-206 overexpression significantly decreased the expression of cathepsin K; however, the inhibition of miR-206 elevated the level of cathepsin K, both at the mRNA and protein expression levels.

Finally, the present study assessed the effects of icariin on chondrocytes in the presence of DXM at the indicated concentrations (Fig. 5). As demonstrated in Fig. 5A, the addition of DXM (100 µM) to chondrocytes resulted in a maximum reduction in cell viability compared with the control cells; however, this effect was reversed significantly following treatment with icariin (100 µM). As demonstrated in Fig. 5B and C, icariin (100 µM) significantly suppressed DXM-induced elevation of chondrocyte apoptosis, accompanied by an increase in the level of Bcl-2, and decreases in the levels of Bax, caspase-3 and caspase-9 (Fig. 5D). RT-qPCR further indicated that icariin (100 µM) abolished the maximal inhibition effect of DXM (100 µM) on miR-206 expression in chondrocytes (Fig. 5E). Additionally, Fig. 5F and G demonstrated that the dose-dependent addition of DXM significantly elevated the expression of cathepsin K, which peaked at a dose of 100 µM. This level was significantly decreased following icariin stimulation (100 µM).