Patients were enrolled at Guizhou Osteological Hospital (Guiyang, China) between May 2015 and May 2016. Cartilage tissue was obtained from 30 OA patients receiving knee joint replacement. The diagnosis of OA was based on the America OA diagnosis standards (2008) (15). Disease history, clinical examinations and x-ray scans were referred to in order to diagnose OA. The age of the patients ranged from 56–85 years with an average of 68 years, including 12 males and 18 females. In addition, cartilage tissue was collected from 10 sex- and age-matched control subjects, without OA diagnoses. Informed written consent was obtained from each participant. Experiments were approved by the Ethics Committee of Guizhou Osteological Hospital (Guiyang, China).

Cartilage tissues were fixed in 4% paraformaldehyde and embedded in paraffin wax. Subsequently, the specimens were sliced (20 µm), and xylene dewaxing and dehydration in graded alcohol were performed. Antigen retrieval was conducted in sodium citrate buffer (10 mM) at room temperature for 2 days and the slices were blocked in 5% horse serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at room temperature for 2 h. Sections were incubated with a Beclin 1 primary antibody (1:100 dilution; cat. no. ab62557; Abcam, Shanghai, China) at 4°C overnight. After incubation with the donkey anti-rabbit IgG H&L horseradish peroxidase-conjugated secondary antibody (1:200; cat. no. ab98488; Abcam) at room temperature for 2 h, sections were incubated with 3,3′-diaminobenzidine substrate (1 mg/ml) at room temperature for 5 min. Hematoxylin (Beyotime Institute of Biotechnology, Haimen, China) was applied to stain the nuclei for 1 min at room temperature. Beclin 1 positive staining (brown staining) was localized to the cytoplasm based upon the negative control (incubated without primary antibody). The images were taken under a light microscope (BX51; Olympus Corporation, Tokyo, Japan).

Total RNA was extracted using TRIzol^® reagent according to the manufacturer's protocol (cat. no. 15596026; Thermo Fisher Scientific, Inc., Shanghai, China) and the concentration and purity of the RNA were determined using a Nanodrop 2000 spectrophotometer. RNA (2 µl) was used to synthesize cDNA using a one-step reverse transcription kit according to the manufacturer's protocol (Takara Biotechnology Co., Ltd., Dalian, China). Primers were synthesized by Shanghai Sangong Pharmaceutical Co., Ltd. (Shanghai, China). Primer sequences were as follows: Forward, 5′-TATTGAAACTCCTCGCCAGGA-3′ and reverse, 5′-GGCATAACGCATCTGGTTTT-3′ for BECN1; and forward, 5′-AGCCACATCGCTCAGACA-3′ and reverse, 5′-TCTCCTGGGAGGCATAGACC-3′ for GAPDH. Cycling conditions were as follows: An initial predenaturation step at 95°C for 5 min, followed by 37 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 13 sec and extension at 72°C for 30 sec. PCR products were detected by 2% agarose gel electrophoresis with ethidium bromide staining and the density of the bands was analyzed using ImageJ Software v1.48u (National Institutes of Health, Bethesda, MD, USA).

Cartilage tissue was cut into sections in a sterile environment and digested using 0.25% trypsin for 30 min and 0.2% collagenase for 2 h. The mixture was centrifuged at 1,896 × g for 10 min to collect the chondrocytes. Cells were resuspended in Dulbecco's Modified Eagle medium/F-12 supplemented with 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO₂. When cells reached 80% confluence, they were divided into two groups: the Beclin 1 overexpression group and the control group. The Beclin 1 overexpression group was transfected with the pcDNA^™ 3.1-BECN1 (https://www.ncbi.nlm.nih.gov/gene/8678) plasmid (1 µl) using Lipofectamine^™ 2000 (Shanghai Jima Industrial Co., Ltd., Shanghai, China). The control group was transfected with an empty vector (pcDNA^™ 3.1; Shanghai Jima Industrial Co., Ltd.). After 24, 48, 72 and 96 h transfection at 37°C, cells were collected for subsequent experiments.

Chondrocytes in the logarithmic growth phase were seeded in a 96-well culture plate. Following transfection, 20 µl MTT reagent (5 mg/ml) was added to the 200 µl culture medium in each well. After a 4 h incubation at 37°C, the medium was removed, and 150 µl dimethyl sulfoxide was added into each well to dissolve the precipitation. The absorbance was measured at a wavelength of 560 nm using an automated microplate reader (IMark Group, Inc, MD, USA). A blank control (media without cells) was set to subtract the background for comparisons.

Chondrocytes in logarithmic growth phase (3×10³) were seeded. After transfection for 48 h, cells were collected for Annexin V/propidium iodide staining (Beyotime Institute of Biotechnology) and detected within 1 h using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed using FlowJo v10 windows software (Tree Star, Inc., Ashland, OR, USA).

Chondrocytes in the logarithmic growth phase (3×10³) were seeded. After transfection for 48 h, cells were stained with Hoechst (Beyotime Institute of Biotechnology) for 5 min at room temperature, and the images were captured using a fluorescence microscope (DMI4000B; Leica Microsystems GmbH, Wetzlar, Germany).

Chondrocytes in the logarithmic growth phase (3×10³) were seeded. After transfection for 48 h, cells were collected and lysed using radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology) for 5 min on ice. Protein concentration was quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). An equal amount of protein (20 µg) was loaded onto 10% gels prior to SDS-PAGE. After electrophoresis, proteins were transferred onto nitrocellulose membranes. Non-specific protein binding was blocked with 4% skimmed milk for 2 h at room temperature. The membranes were incubated at 4°C overnight with primary antibodies against Beclin 1 (1:1,000; cat. no. ab62557; Abcam), Bcl-2 associated X (Bax; 1:1,000; cat. no. ab32503; Abcam), B-cell lymphoma (Bcl-2; 1:1,000; cat. no. ab32124; Abcam), PI3K (1:1,000; cat. no. ab86714; Abcam), protein kinase B (Akt; 1:1,000; cat. no. ab8805; Abcam), phosphorylated (p)-Akt (1:1,000; cat. no. ab8932; Abcam), mammalian target of rapamycin (mTOR; 1:1,000; cat. no. ab2732; Abcam), p-mTOR (1:1,000; cat. no. ab109268; Abcam), MMP1 (1:1,000; cat. no. ab52631; Abcam), MMP3 (1:1,000; cat. no. ab53015; Abcam), MMP13 (1:1,000; cat. no. ab39012; Abcam) and GAPDH (1:1,000; cat. no. AG019; Beyotime Institute of Biotechnology). After washing with PBST (PBS containing 0.1% Tween-20), membranes were probed with anti-mouse or anti-rabbit horseradish peroxidase secondary antibodies (1:100; cat. nos. ab131368 and ab191866; Abcam) at room temperature for 2 h. The signal was detected using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences, Chalfont, UK) and images were captured using the ChemiDoc^™ XRS instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The densities of the blots were quantified using Quantity One software v4.6.0 (Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± standard deviation. Statistical analyses of the data were performed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Beclin 1 expression was reduced in cartilage tissues from patients with OA when compared with the 10 sex- and age-matched control subjects, as determined by immunohistochemistry (Fig. 1A), sqPCR (Fig. 1B) and western blotting (Fig. 1C and D).

As Beclin 1 expression in the cartilage tissues of OA patients was reduced, it was investigated whether Beclin 1 overexpression altered cell viability. Beclin 1 was overexpressed at the mRNA (Fig. 2A) and protein (Fig. 2B) levels compared with the control. Compared with the control, the optical density was significantly enhanced after 48, 72 and 96 h transfection with the Beclin 1-containing plasmid (Fig. 2C), which reached a peak at 72 h.

Apoptosis was reduced following transfection with the Beclin 1-containing plasmid compared with the control, as demonstrated by Hoechst staining (Fig. 3A) and Annexin V/propidium iodide double staining and flow cytometric analysis (Fig. 3B). The early apoptotic rates in the control and Beclin 1 plasmid groups were 12.46±0.31 and 2.89±0.28%. The late apoptotic rates in the control and Beclin 1 plasmid groups were 3.38±0.31 and 2.18±0.20%. Additionally, the expression levels of apoptotic-associated proteins were measured. The expression level of the anti-apoptotic protein, Bcl-2, was significantly enhanced, whereas expression of the pro-apoptotic Bax protein was significantly reduced following Beclin1 overexpression compared with the control (P<0.01; Fig. 3C and D).

Expression levels of proteins of the PI3K/Akt/mTOR signaling pathway were compared between the two groups. Beclin 1 overexpression markedly reduced the expression levels of PI3K, p-Akt and p-mTOR compared with the control, whereas the expression levels of total Akt and mTOR were not significantly affected (Fig. 4A and B).

The expression levels of MMPs were also detected after Beclin 1 overexpression. As demonstrated in Fig. 5A and B, MMP1, MMP3 and MMP13 were significantly reduced after Beclin1 overexpression compared with the control.