Osteoarthritis cartilage collection

Osteoarthritis cartilage samples were obtained from 12 patients (aged 55 to 65 years) who underwent total knee arthroplasty for clinically and radiologically diagnosed OA. The patients were excluded if they presented with a history of rheumatic arthropathies or infection in the knee. All tissues were obtained with fully informed consent and prior institutional ethical approval. All samples were harvested from the major load-bearing areas on the medial tibial plateau (MTP) and lateral tibial plateau (LTP).

Isolation of chondrocytes

Articular cartilage was obtained from the major load-bearing areas of the LTP from an OA knee, as much of the cartilage tissue in the force area was removed so that more chondrocytes could be obtained after digestion. The cartilage was harvested and subjected to sequential trypsin/collagenase digestion in order to isolate the chondrocytes, as previously described.⁹ Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, New York), penicillin/streptomycin (50 000 U/50 mg), and L-glutamine (4.5 mM), was used to expand the chondrocytes. The cells were cultured under standard conditions. To avoid interference caused by dedifferentiated chondrocytes, we used two to three passage cells.

Overexpression of Ob-Rb in the chondrocytes

The green fluorescent protein (GFP)-expressing lentiviral particles were produced by cotransfection with lentiviral plasmids, psPAX2 and pMD2.G (Shanghai Genechem, Shanghai, China). The Ob-Rb-expressing lentiviral particles were produced by the cotransfecting plasmids pCDH-Ob-Rb, psPAX2, and pMD2.G (Shanghai Genechem). The cells at passage 2 with 60% to 70% confluence were used for transduction. After grouping, the cells were transduced with lenti-GFP or lenti-Ob-Rb at a multiplicity of infection (MOI) of 50 for ten hours. After transduction, the cells were exposed to 4 μg/ml puromycin (Sigma-Aldrich, St. Louis, Missouri) for one day in order to obtain stable transduction. The cells transfected with the only GFP-expressing lentiviral vector were used as controls. Cells were used for further experiments after amplification.

Autophagic flux measured by flow cytometry

The monomeric red fluorescent protein (mRFP)-enhanced green fluorescent protein (eGFP)-LC3 lentiviral vectors (mRFP-eGFP-LC3) were provided by Shanghai Genechem. The lentivirus was transfected into chondrocytes according to the manufacturer's protocol at a MOI of 50 for six hours. After grouping, lentiviral-transfected chondrocytes were used for subsequent experiments. After completing the corresponding experiment, each group of cells was digested with trypsin to prepare a cell suspension. The mRFP and eGFP signals were analyzed by flow cytometry (BD FACSCalibur; BD Biosciences, Franklin Lakes, New Jersey). Autophagic flux of chondrocytes was measured according to a protocol described by Gump and Thorburn.¹⁴ The autophagic flux was calculated by quantifying the ratio of mRFP to eGFP by Flowing Software (created by Perttu Terho, University of Turku, Finland; http://flowingsoftware.btk.fi).

Immunohistochemical analysis

Knee cartilage from the major load-bearing areas on the MTP and LTP was subjected to immunohistochemical analysis (IHC) using antimammalian target of rapamycin (mTOR) antibodies (Abcam, Cambridge, United Kingdom). Briefly, the cartilage was deparaffinized and rehydrated and then subjected to antigen retrieval by incubating the tissues in sodium citrate buffer (0.01 M, pH 6.0) at 95°C for ten minutes. The tissue sections were exposed to hydrogen peroxide (3% H₂O₂) for five minutes to quench the endogenous peroxidase and were then blocked in 30% horse serum for 30 minutes. The slides containing the tissue sections were incubated overnight at 4°C with primary mTOR antibodies (1:150 dilution). Nonimmune mouse immunoglobulin G (IgG) was used as a negative control. After the tissues were washed with 1 × tris-buffered saline containing 0.1% Tween-20 (TBST), the slides were then incubated with biotinylated secondary antibodies (antigoat IgG; Santa Cruz Biotechnology, Santa Cruz, California) and detected using an avidin-biotin complex (ABC) kit (Vector Laboratories, Burlingame, California).

Immunofluorescence

To immunostain the cells or tissues (frozen sections), the samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes at room temperature. After the cells were washed three times in PBS/0.1% bovine serum albumin (BSA) for five minutes, they were permeabilized using 0.2% Triton (T9284; Sigma-Aldrich) in PBS for 20 minutes and then washed in PBS/0.1% BSA. Primary antibodies against Ob-Rb (Abcam) were diluted in PBS/0.1% BSA and incubated overnight at 4°C. After the samples were washed, the cells or frozen sections were incubated with a fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (1:500; Abcam), a goat antimouse IgG secondary antibody (1:500; Abcam), and 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for one hour at room temperature. Fluorescence images were obtained using a NikonEclipse TE2000-U inverted fluorescent microscope (Nikon, Tokyo, Japan).

Effects of leptin on chondrocyte proliferation

The chondrocytes were seeded in 96-well plates at a density of 10⁴ cells/ml. Cell viability was measured by the Cell Counting Kit-8 (CCK-8) reagent (Dojindo Molecular Technologies, Inc., Rockville, Maryland). After treatment for the indicated times, the culture medium was changed to fresh medium with 10% CCK-8 reagent. After incubation for two to four hours, the absorbance at 450 nm was measured by a microplate reader. Each treatment group had three replicates.

Cell cycle analysis

The cell cycle assay was performed using a cell cycle and apoptosis kit (Beyotime Institute of Biotechnology, Shanghai, China). Cells (1 × 106 cells per sample) were collected and passed through a 40 μm nylon membrane (Corning Inc., Corning, New York). Cold 75% alcohol (1 ml) was added to the cells for 24 hours at 4°C, and the cells were then resuspended in 250 μl of propidium iodide (PI) solution/0.1% Triton X-100/RNase A (Beyotime Institute of Biotechnology), incubated at room temperature for 30 to 45 minutes, and analyzed by flow cytometry (BDBiosciences, San Jose, California) for cell cycle analyses.

Senescence-associated β-galactosidase staining

Briefly, the cells were washed twice with PBS, fixed in the wells using 1 ml fixing solution per well, and incubated at room temperature for 15 minutes. Following washes with PBS, the cells were incubated in a freshly prepared senescence-associated β-galactosidase (SA-β-gal) staining solution at 37°C, without CO₂ and protected from light, for 24 hours (Cell Signaling Technology, Danvers, Massachusetts). Senescent cells were determined by counting the number of blue-stained cells under a BX40 Olympus microscope (Olympus America, Miami, Florida) equipped with a Nikon N2000 camera (Nikon, Melville, New York). The total number of cells and the number of β-gal-positive cells were determined for five fields of view (200× magnification) per sample. The total number of cells was independently quantitated, and the percentage of senescent cells was calculated accordingly.

Cell signalling studies

The chondrocyte cultures were treated with 200 ng/ml leptin (598-LP-05M; R&D Systems, Minneapolis, Minnesota), rapamycin (Sigma-Aldrich), or AZD (Sigma-Aldrich).¹⁵ The cells were cultured in DMEM supplemented with 5% FBS, penicillin/streptomycin (50 000 U/50 mg), and L-glutamine (4.5 mM). After 48 hours of treatment, p-S6 kinase (S6K), p53, p21, and LC3-II (all Abcam) were detected using Western blot analysis. At 48 hours after treatment, cell senescence was evaluated using a commercial staining kit. SA-β-gal-positive cells were counted in five visual fields using light microscopy (200× magnification).

Western blot analysis

All cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing a cocktail of protease and phosphatase inhibitors (Beyotime Institute of Biotechnology) at 4°C for one hour. The cell lysates were centrifuged at 13 000 × g and 4°C for 20 minutes, and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein were heated to 100°C for five minutes, separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, California). The membranes were blocked with TBST containing 5% nonfat dried milk for one hour and incubated with primary antibodies overnight at 4°C. The membranes were washed three times with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies for one hour at room temperature. After three additional washes with TBST, the signal intensity was quantified with a ChemiDoc-It Imaging System (UVP, Upland, California) attached to a BioChemi HR camera (UVP). To measure protein abundance, the grey values of the blots in the scanned images were measured using ImageJ Plus software (National Institutes of Health, Bethesda, Maryland). Before comparisons were made, the grey value of each target protein was normalized to the value for β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Safranin-O and Fast Green staining

The knee OA cartilage samples (n = 24) from the major load-bearing areas in the medial and lateral cartilage of the tibial plateau were cut into two to three pieces by bone saw. The specimens were fixed with 4% paraformaldehyde, decalcified with 10% ethylenediaminetetraacetic acid (EDTA; pH 7.4), and embedded in paraffin, followed by dehydration with serial ethanol and clearance with xylene. Sections 5 μm thick were then cut, dewaxed, and hydrated with serial ethanol, then stained with Safranin O and Fast Green for histological grading, as described by Mankin et al.¹⁶ The Mankin scoring system contains four indicators: structure (seven grades), cell (four grades), Safranin O staining (five grades), and tidemark (two grades).

Two blinded observers (ZL and GL) scored for histological grading using the Mankin scoring system.

Statistical analysis

For detecting Ob-Rb-mRNA expression, the reverse transcriptase polymerase chain reaction (RT-PCR) experiment was repeated three times. We used the OA 3, OA 5, OA 8, and OA 11 specimens from which to harvest and culture chondrocytes from the LTP cartilage. The results from the in vitro experiments were analyzed using one-way analysis of variance (ANOVA) or Student’s t-tests if only two conditions were being compared. All data were analyzed using Prism v. 5.0b software (GraphPad Software, San Diego, California). p-values ⩽ 0.05 were considered statistically significant. The results are expressed as the mean and standard deviation.

Degenerative cartilage has a high expression of Ob-Rb

Leptin bioactivity is mediated by specific leptin receptors. At least six types of leptin receptors have been found in mammalian cells. However, the long isoform of Ob-Rb is the functional receptor and plays a major role in signal transduction.

Although 40% of the elderly population have symptoms of knee cartilage degeneration, individuals’ cartilage tissue degeneration is usually uneven. Using the characteristics of uneven degeneration in OA cartilage, we conducted a self-controlled study that compared the medial cartilage (degenerated cartilage) with the lateral cartilage (as non-OA-affected region) from the tibial plateau in the same OA patients to assess whether Ob-Rb is highly expressed in degenerated cartilage tissue. We collected medial and lateral cartilage samples of the tibial plateau from 12 OA patients (four male, eight female). The details of the patients, including sex, age, and body mass index (BMI), are presented in Table I. In addition, the degree of degeneration of the medial and lateral cartilage of the tibial plateau was assessed using Safranin O and Fast Green staining (Fig. 1a). In all 12 cases, medial cartilage wear was more severe than lateral cartilage wear (Fig. 1b). Immunofluorescence results showed that the expression of Ob-Rb in the medial cartilage of the tibial plateau was high (Fig. 1c). The results from RT-PCR also confirmed that the expression of Ob-Rb in the medial cartilage of the 12 OA patients was high (Fig. 1d).

Fig. 1

Degenerative cartilage with high expression of the long form of the leptin receptor (Ob-Rb). a) Analysis of the degree of lateral and medial tibial cartilage degeneration by using Safranin O and Fast Green staining (scale bar: 20 μm). b) Chart showing histological grading as scored using the Mankin scoring system.¹⁶ Error bars represent the mean and standard deviation. c) The results of immunofluorescence revealed distribution of Ob-Rb-positive cells in medial and lateral cartilage samples of the tibial plateau (scale bar: 20 μm). d) Chart showing medial and lateral cartilage samples of the tibial plateau were collected from 12 osteoarthritis patients. Ob-Rb messenger RNA (mRNA) expression was detected in medial and lateral cartilage samples of the tibial plateau by reverse transcriptase polymerase chain reaction (RT-PCR). Relative Ob-Rb mRNA expression of each paired group in the medial cartilage sample was normalized to the expression in the lateral cartilage samples. *p < 0.05 was considered statistically significant.

High doses of leptin induce cell cycle arrest and senescence in chondrocytes

Although we previously observed leptin-induced cell senescence in CPCs from knee cartilage in Sprague Dawley rats, whether this phenomenon exists in human knee chondrocytes needs to be verified. The physiological concentration of leptin in humans is approximately 10 pg/ml;¹⁷ however, in vitro and human body conditions are different. We therefore treated the chondrocytes with the following doses of leptin: 0 ng/ml as control; 10 ng/ml as a physiological dose; and 100 ng/ml and 200 ng/ml as high doses. We explored the effects of different doses of leptin (0 ng/ml, 10 ng/ml, 100 ng/ml, and 200 ng/ml) on chondrocyte proliferation using the CCK-8 reagent and cell cycle analyses. Treating the cells with high doses of leptin resulted in less proliferation than that observed when the cells were treated with the control or physiological doses, and leptin treatment induced cell cycle arrest in the chondrocytes by inhibiting the G1/S cycle and decreased the cell proliferation rate by reducing the (S+G2)% (Figs 2a and 2b). Cell cycle arrest generally leads to quiescence or senescence.¹⁸ Treating the cells with 100 ng/ml and 200 ng/ml leptin resulted in a higher percentage of SA-β-gal-positive chondrocytes than that observed in the cells treated with the control or physiological dose of leptin (Fig. 2c). The high doses of leptin therefore induced senescence in the chondrocytes. High doses of leptin induce senescence by p53/p21 pathway activation in chondrocytes (Figs 2d and 2e).

Fig. 2

High-dose leptin causes chondrocyte senescence. a) Histograms showed chondrocyte cell cycle analysis after different doses of leptin treatment for two days. Compared with vehicle and 10 ng/ml doses of leptin treatment, 100 ng/ml and 200 ng/ml leptin causes chondrocyte cell cycle arrest at phase G1 and decreases the cell proliferation rate by reducing the (G2+S)%. b) Chart showing the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, Maryland) analysis results of cell viability after different doses of leptin treatment. c) Chart showing that high-dose leptin dramatically induces chondrocyte senescence. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. d) The expression of senescence markers p53 and p21 dramatically increased in chondrocytes when treated by high-dose leptin. e) Chart showing senescence cells (senescence-associated β-galactosidase (SA-β-gal)-staining positive cells) increased by high-dose leptin. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant.

After overexpression of Ob-Rb, the physiological dose of leptin induced cell senescence in chondrocytes

The lateral cartilage of the tibial plateau, as a non OA-affected region, has a low expression of Ob-Rb (Fig. 1a). After performing polymerase chain reaction (PCR) to verify the effect of Ob-Rb overexpression by lenti-Ob-Rb (Fig. 3a), the Ob-Rb-overexpressing chondrocytes and controls were treated with different doses of leptin for two days. The results showed that a physiological dose of leptin activated the p53/p21 pathway in the chondrocytes (Figs 3b and 3c). SA-β-gal staining showed that after overexpression of Ob-Rb, the level of SA-β-gal-positive cells significantly increased in the chondrocytes (Fig. 3d). These results showed that the physiological dose of leptin induced cell senescence in Ob-Rb-overexpressing chondrocytes. This finding indicates that a high expression of Ob-Rb in cartilage may cause degeneration by mediating leptin pathway activation.

Fig. 3

After overexpression of the long form of the leptin receptor (Ob-Rb), the physiological dose of leptin induced cell senescence in chondrocytes. a) Chart showing that after lenti-Ob-Rb, expression of Ob-Rb messenger RNA (mRNA) in chondrocytes derived from the lateral tibial plateau cartilage was detected by real-time polymerase chain reaction (PCR). Relative Ob-Rb mRNA expression of each group was normalized to the expression of GAPDH mRNA. Error bars indicate the mean and standard deviation. b) Chart showing that after Ob-Rb overexpression, the Ob-Rb-overexpressed chondrocytes and control were treated with different doses of leptin for two days. Expression of p53/p21 was then detected and statistically analyzed. c) Chart showing statistical analysis of the expression of p53 and p21. Relative protein abundance of each blot was normalized to the grey value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Error bars indicate the mean and standard deviation. d) Chart showing that after Ob-Rb overexpression, the Ob-Rb-overexpressed chondrocytes and control were treated with different doses of leptin for two days. The senescence-associated β-galactosidase (SA-β-gal)-staining positive cells were counted and statistically analyzed in these samples. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant. †p < 0.01.

High doses of leptin inhibit autophagy by activating the mTOR signalling pathway in chondrocytes

Western blot analysis revealed that high doses of leptin significantly decreased the levels of autophagy, as measured by flow cytometry (Fig. 4a), while significantly reducing LC3-II expression and increasing p62 expression (Figs 4b and 4c). The lysosomes were dyed with LysoTracker (Beyotime Institute of Biotechnology), and the results revealed that high doses of leptin caused lysosome accumulation, which means that high doses of leptin can inhibit autophagy (Fig. 4d).

Fig. 4

High-dose leptin inhibits autophagy in chondrocytes. a) High-dose leptin decreased autophagic flux in chondrocytes. b) Expression of p62 and LC3B-I/II were detected by Western blot analysis. c) Chart showing that high-dose leptin significantly increases expression of p62 and LC3B-I/II expression, which are markers for cell autophagy. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. d) In chondrocytes, lysosome was increased when treated with high-dose leptin (scale bar: 50 μm). *p < 0.05 was considered statistically significant. mRFP, monomeric red fluorescent protein; eGFP, enhanced green fluorescent protein.

The targets of rapamycin (mTOR) play an important role in regulating autophagy in mammals.¹⁹ Immunohistochemistry analysis of the mTOR antibody showed that the areas of cartilage that had degenerated the most also had higher mTOR expression than other areas in the same OA patient (Fig. 5a). We next examined S6K (a downstream target of mTOR signalling) expression.²⁰ Western blot analysis showed that high doses of leptin significantly increased the level of phosphorylated S6K (Figs 5b and 5c).

Fig. 5

High-dose leptin induces senescence and inhibits autophagy by activating the mammalian target of rapamycin (mTOR) pathway. a) Expression and distribution of mTOR in medial tibial plateau (MTP) and lateral tibial plateau (LTP) cartilage areas were detected. b) In chondrocytes treated by high-dose leptin, increased expression of mTOR, S6 kinase (S6K), pS6K, and Beclin1 was detected. c) Chart showing statistical analyses of expression of mTOR, S6K, and pS6K. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant.

Blocking the mTOR signalling pathway can restore autophagy and partially reverse senescence induced by leptin in chondrocytes

To assess the effect of mTOR on the inhibition of autophagy by leptin in chondrocytes, we measured the protein expression of Beclin1, S6K, and pS6K in the presence and absence of rapamycin and AZD8055. However, the leptin-mediated increase in Beclin1 was abolished once mTOR signalling was inhibited by rapamycin or AZD8055, which was demonstrated by diminished levels of phospho-S6K protein (Figs 6a and 6b). Western blot analysis showed that rapamycin or AZD8055 partially reversed the increased expression of p53 and p21 that was induced by leptin in chondrocytes (Figs 6c and 6d). Reduced autophagy levels in the chondrocytes by leptin treatment can be restored by rapamycin or AZD8055 (Fig. 6e). The proportion of SA-β-gal-positive chondrocytes induced by leptin was reduced when rapamycin or AZD8055 was present (Fig. 6f). These results suggested that leptin may inhibit autophagy by activating the mTOR pathway in chondrocytes.

Fig. 6

Mammalian target of rapamycin (mTOR) inhibitors, rapamycin and AZD8055 can attenuate effects of high-dose leptin on chondrocytes. a) Expression of mTOR, Beclin1, S6 kinase (S6K), and pS6K were detected by rapamycin (Rapa) + 200 ng/ml-leptin and AZD8055 + 200 ng/ml-leptin. b) Chart showing that Rapa and AZD8055 attenuated the effects of high-dose leptin on expression of mTOR, Beclin1, S6K, and pS6K. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. c) Expression of p53 and p21 was detected by Rapa + 200 ng/ml-leptin and AZD8055 + 200 ng/ml-leptin. d) Chart showing that mTOR inhibitors (Rapa and AZD8055) attenuated the effects of high-dose leptin on expression of p53 and p21, which are markers of cell senescence. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. e) mTOR inhibitors partially attenuated the effects of high-dose leptin on chondrocytes’ autophagic flux. f) Chart showing that mTOR inhibitors (Rapa and AZD8055) partially attenuated chondrocyte senescence by high-dose leptin induction. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant. mRFP, monomeric red fluorescent protein; eGFP, enhanced green fluorescent protein.

Inhibition of the mTOR pathway can restore autophagy and partially reverse leptin-induced senescence in chondrocytes.