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Department of Orthopaedic Surgery, Xiaoshan Chinese Medical Hospital, ChinaDepartment of Orthopaedic Surgery, Affiliated Jiangnan Hospital of Zhejiang Chinese Medical University, ChinaZhejiang Chinese Medical University, ChinaInstitute of Orthopaedics and Traumatology of Zhejiang Province, China
Zhejiang Chinese Medical University, ChinaInstitute of Orthopaedics and Traumatology of Zhejiang Province, ChinaDepartment of Orthopaedic Surgery, the First Affiliated Hospital of Zhejiang Chinese Medical University, China
Zhejiang Chinese Medical University, ChinaInstitute of Orthopaedics and Traumatology of Zhejiang Province, ChinaDepartment of Orthopaedic Surgery, the First Affiliated Hospital of Zhejiang Chinese Medical University, China
Leptin has been found highly expressed in human osteoarthritis. We aimed to explore the possible effects and mechanisms of leptin on the apoptosis and autophagy of chondrocytes during osteoarthritis pathogenesis.
Methods
Gene expression profile from osteoarthritis affected and preserved cartilage were downloaded from NCBI's Gene Expression Omnibus database (GSE57218). Lysyl oxidase-like 3 (LOXL3) mRNA expression in cartilage tissues and leptin concentration in joint synovial fluid (SF) was measured in samples from 45 osteoarthritis patients and 25 healthy donors by real-time PCR and radioimmunoassay, respectively. Rat osteoarthritis model was induced by anterior cruciate ligament transection (ACLT). The expression of apoptosis regulators and autophagy markers were detected by Western blot. Cell survival and cell apoptosis were identified by CCK-8 and flow cytometry, respectively.
Results
Re-analysis on GSE57218 indicated that LOXL3 mRNA was upregulated in osteoarthritis affected cartilage. LOXL3 mRNA was upregulated in osteoarthritis patients, which was positively correlated with SF leptin concentration. Similar results were obtained in rat osteoarthritis model. Moreover, ACLT surgery led to a significant increase in the protein levels of cleaved caspase 3, and a notable decrease in the protein levels of Bcl-2, LC3 II/LC3 I and Beclin1. Silencing of LOXL3 in ACLT and leptin treated primary chondrocytes significantly inhibited cell apoptosis, and promoted cell proliferation and autophagy. Moreover, overexpression of LOXL3 remarkably inhibited autophagy of chondrocytes via activating mTORC1.
Conclusions
LOXL3, a downstream of leptin, stimulated the apoptosis, but inhibited the autophagy of chondrocytes. LOXL3 is a potential therapy target for osteoarthritis.
Osteoarthritis is the most common form of bone and joint disease, which is characterized by the degeneration and loss of joint cartilage and underlying bone. Risk factors of osteoarthritis include older age, obesity, previous joint injury, joint deformity, and inherited factors
. Chondrocytes as the single cellular component in cartilage play key roles in the degeneration of cartilage. During pathology of osteoarthritis, chondrocytes show an increase in apoptosis, cytokine production and matrix degeneration
Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes.
. Recently, autophagy, a self-degradative process, has been reported to protect human chondrocytes from stresses and may relate to pathogenesis of osteoarthritis
. The ubiquitous expression suggests that leptin have various physiological roles. Recently, several investigations have provided evidence to link leptin to cell apoptosis
Differential expression of leptin and leptin's receptor isoform (Ob-Rb) mRNA between advanced and minimally affected osteoarthritic cartilage; effect on cartilage metabolism.
, was overexpressed in osteoarthritis cartilage. Additionally, LOXL3 mRNA expression was positively correlated with the leptin levels in synovial fluid (SF), which was further confirmed in a rat osteoarthritis model. Experiment of primary cultured chondrocytes suggested LOXL3 was upregulated by leptin exposure and played an important role in the apoptosis and autophagy of chondrocytes. Collectively, LOXL3 is a potential therapy target for osteoarthritis.
Materials and methods
Cartilage and joint SF samples
This study was approved by the Ethics Committee for Human Studies, Department of Orthopaedic Surgery, Xiaoshan Chinese Medical Hospital (Hangzhou, China). Specimens of human cartilage and joint SF were obtained from 45 osteoarthritis patients (ages 53–77 years). and 25 healthy donors (ages 26–61 years) with no history of joint disease after written informed consent was obtained at Xiaoshan Chinese Medical Hospital. The osteoarthritis patients enrolled in this study was classified by Kellgren–Lawrence grading scale
: 14 patients with stage I, 18 with stage II and 13 with stage III. Specimens of osteoarthritis and normal cartilage were received at the time of total knee replacement surgery and autopsy, respectively. The collected cartilage samples were snap-frozen in liquid nitrogen and stored at −80°C until RNA isolation. SF samples were collected at arthroscopy and centrifuged to remove cells and particulate matter as described previously
Synovial proinflammatory cytokines and their correlation with matrix metalloproteinase-3 expression in Behçet's disease. Does interleukin-1β play a major role in Behçet's synovitis?.
Ten-week-old male Wistar rats weighing approximately 200 g were obtained from Shanghai Center of Experimental Animals (Shanghai, China). Anterior cruciate ligament transection (ACLT) was performed to induce osteoarthritis. Eighteen rats were randomly divided into three groups (n = 6 per group): sham-operated group, ACLT+vehicle group and ACLT+ rapamycin (RAPA) group. After anesthetized, the right knee joints of rats were shaved and disinfected with 70% ethanol. In sham-operated group, the joint space was opened following subluxation of the patella and closing of the wound. In ACLT+vehicle group and ACLT+RAPA group, ACLT was performed as previously described
. Rats in ACLT+vehicle group and ACLT+RAPA group received intraperitoneal injections of 0.3 ml DMSO vehicle (0.4% in PBS) and rapamycin (LC Laboratories, Woburn, MA, USA; 1 mg/kg body weight) daily for 10 weeks, respectively. Ten weeks after the surgery, the rats were euthanized. Synovial lavage fluid (SF) were collected and centrifuged to remove cells as described previously
. Briefly, after removing the skin overlying the knee and dissecting the knee ligament, the knee joint cavity was washed twice by injecting and immediately aspirating 100 μL of PBS containing 4 mM ethylene-diamine tetraacetic acid (EDTA). The resulting SF was combined, centrifuged and stored at −20°C for further analysis. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital, School of Medicine, Zhejiang University (Hangzhou, China).
Histological analysis
The knee joints were collected, fixed in 4% paraformaldehyde (PFA), and decalcified with 10% EDTA at 4°C for 2 weeks. After dehydration by immersed in an increasing concentration of ethanol, specimens were embedded in paraffin. Paraffin sections (5 μm) were cut, deparaffinized and stained with Safranin-O-fast green.
Leptin radioimmunoassay
Leptin levels were measured with leptin radioimmunoassay kit (Linco Research, St. Charles, MO, USA) following the manufacturer's instructions.
RNA extraction and real-time PCR
Total RNA was isolated from tissues and cells using TRIzol reagent (Life Technologies, Grand Island, NY, USA) and reverse transcribed by using cDNA synthesis kit (Life Technologies) according to the manufacturer's instructions. Quantitative real-time RT-PCR was performed using the SYBR Green PCR kit (Fisher Scientific, Rockford, IL, USA) on ABI7300 (Applied Biosystem, Foster City, CA, USA) PCR instrument. GAPDH was used as an internal control and the relative gene expression was calculated using the ΔΔCt method. The primers used were list in Table S1.
Lentivirus production
The rat LOXL3 gene was synthesized by Genewiz (Beijing, China) and cloned into the lentiviral vector pLvx-AcGFP-C1 (Clontech, Palo Alto, CA, USA) to make construct pLvx-AcGFP-C1/LOXL3. The construct was verified by sequencing. Lentiviruses were produced as described
. pLvx-AcGFP-C1 was used to generate mock viruses for control (NC).
Silencing of LOXL3 by small interfering RNA
One siRNA (GGUGGUUAUCAACCCAAAU, siLOXL3) targeting rat LOXL3 mRNA (XM_008763023.1) and a non-specific scramble siRNA (siNC) were synthesized. Primary chondrocytes were transfected with siRNAs by using Lipofectamine 2000 (Invitrogen) according to the manufacture's instruction. Following assays were conducted at 48 h after treatment.
Isolation, primary culture and treatment of chondrocytes
Cartilage tissues (about 0.5 cm × 0.5 cm) were collected from knee joints of osteoarthritis rat model, finely minced into small pieces (<1 mm), and digested with 0.4% collagenase solution (Sigma, St. Louis, MO, USA) at 37°C for 5 h. After centrifugation, the isolated chondrocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and antibiotics (Invitrogen). Chondrocytes were maintained in monolayer. Chondrocyte were seeded onto 96-well or 6-well plates, and treated with leptin (Sigma), rapamycin (10 ng/ml), siRNA or lentivirus.
Western blot
Cell lysates were prepared from cultured cells with RIPA buffer (50 mM Tris–HCl, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholic acid and 0.05% SDS). Equal amounts of protein were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were probed with primary antibodies following with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime, Shanghai, China). Blots were detected by enhanced chemiluminescence system (Fisher Scientific). The sources of the primary antibodies were as follows: Anti-LOXL3 and anti-Bcl-2 from Santa Cruz Biotech. (Santa Cruz, CA, USA). Antibodies against cleaved caspase 3 and LC3 were purchased from Abcam (Cambridge, MA, USA). Antibodies against p-p70S6K, p70S6K, p-AKT (S473), AKT and GAPDH were purchased from Cell Signaling (Danvers, MA, USA).
Cell proliferation analysis
Cell proliferation was determined by Cell Counting Kit-8 Assay (CCK-8, Beyotime) at 24, 48 and 72 h after leptin treatment. CCK-8 solution was added to culture medium and incubated for additional 1 h. Absorbance was detected at a wavelength of 450 nm by a microplate reader (BioRad, Richmond, CA, USA). Five wells were measured for each treatment and all experiments were performed in triplicate.
Apoptosis analysis
Cells were double stained with Annexin V-fluorescein isothiocyanate (FITC) and PI, and analyzed by flow cytometry (FACScalibur, BD Biosciences, Franklin Lakes, NJ, USA). Annexin V-FITC-positive and PI-negative cells were counted as apoptotic cells.
Statistical analyses
All statistical calculations were performed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). All data are presented as means ± 95% confidence interval (CI). Differences between groups were determined by one-way ANOVA test. The relationships between two factors were assessed by correlation analysis. P value less than 0.05 was considered statistically significant.
Results
LOXL3 mRNA level was positively correlated with SF concentration of leptin
Sato T et al. reported that LOXL3 was highly expressed in damaged osteoarthritis cartilage. Here, we re-analyzed expression data from NCBI Gene Expression Omnibus database (GSE57218)
and found that LOXL3 expression was significantly higher in osteoarthritis cartilage than in healthy cartilage [Fig. 1(A), P < 0.0001]. Real-time PCR analysis on cartilage tissues from 45 osteoarthritis patients and 25 healthy donors also demonstrated the overexpression of LOXL3 mRNA in osteoarthritis cartilage tissues [Fig. 1(B), P < 0.0001].
Fig. 1LOXL3 mRNA was positively correlated with leptin concentration. (A) LOXL3 expression was significantly higher in osteoarthritis cartilage (n = 33) than in healthy cartilage (n = 7) from GSE57218 dataset. (B) LOXL3 mRNA level of cartilage from osteoarthritis patients (n = 45) and health volunteers (n = 25) was evaluated by real-time PCR. (C) Leptin concentrations in joint SF from osteoarthritis patients (n = 45) and health volunteers (n = 25) were measured by using leptin radioimmunoassay kit. (D) Pearson correlation analysis between LOXL3 and leptin (P < 0.0001, n = 45).
The SF levels of leptin, an adipokine involved in pathogenesis of osteoarthritis, were then measured. As shown in Fig. 1(C), the SF levels of leptin were notably higher in osteoarthritis patients than in healthy donors (healthy: 6.54 ± 0.99; osteoarthritis: 11.91 ± 0.49, P < 0.0001). Pearson correlation analysis revealed a positive correlation between LOXL3 and leptin levels in osteoarthritis patients [Fig. 1(D), r = 0.6982, P < 0.0001].
Function analysis of leptin and LOXL3 in an experimental osteoarthritis rat model
To further explore the relationship of LOXL3 and leptin in osteoarthritis, we then established a rat osteoarthritis model by ACLT
. Eighteen rats were randomly divided into three groups: sham-operation (sham), ACLT+vehicle and ACLT+RAPA. Ten weeks after surgery, histological examinations were performed by using Safranin-O staining [Fig. 2(A)]. Specimens from sham group were evenly stained with Safranin-O. ACLT+vehicle group showed significant cartilage degeneration with loss of surface lamina and depletion of chondrocytes. Injection of rapamycin (RAPA, a well-known autophagy inducer) attenuated the severity of the osteoarthritis-like changes, which was consistent with a previous study
Fig. 2Function analysis of LOXL3 in a rat osteoarthritis model. Eighteen rats were randomly divided into three groups: Sham: sham-operation, ACLT+vehicle: ACLT model injected with vehicle and ACLT+RAPA: ACLT model injected with rapamycin. Ten weeks after surgery, the cartilage and joint SF samples were collected. (A) After fixation, decalcification and embedding, sections were cut and stained with Safranin-O-fast green. Scale bar: 100 μm. (B) LOXL3 mRNA level in rat cartilage were evaluated by real-time PCR (n = 6). (C) SF leptin concentrations were assessed by radioimmunoassay (n = 6). (D) Positive correlation between LOXL3 and leptin as determined by Spearman correlation analysis (P < 0.0001, n = 18). (E, F) The expression of cleaved caspase 3, Bcl-2, LC3 and Beclin1 were measured by western blot. The left panel showed representative western blot and the right panel showed the quantification of western blot (n = 3). *P < 0.05, **P < 0.01 vs sham group; #P < 0.05, ##P < 0.01 vs ACLT+ vehicle group.
LOXL3 mRNA levels and leptin concentration were then measured in cartilage and joint SF samples, respectively. ACLT significantly increased LOXL3 mRNA levels and leptin concentration, which was attenuated by injection of RAPA [Fig. 2(B)]. A positive correlation was also observed between LOXL3 and leptin levels [Fig. 2(C), r = 0.7679, P = 0.0157]. These data suggested that higher leptin and LOXL3 levels were associated with the severity of osteoarthritis-like changes.
The protein levels of LOXL3, and important regulators in apoptosis (cleaved caspase 3 and Bcl-2) and autophagy markers (LC3 II/LC3 I and Beclin1) were then determined [Fig. 2(E) and (F)]. ACLT led to a notable increase in the expression of LOXL3 and cleaved caspase 3, and a significant decrease in the expression of Bcl-2, LC3 II/LC3 I and Beclin1. RAPA treatment on osteoarthritis model decreased LOXL3 expression and apoptosis, but increased the autophagy. These data indicated that higher leptin and LOXL3 levels were associated with increased apoptosis and decreased autophagy.
Cytokines, such as TNF-α, IL-1β, IL-6 and IL-8 have also been shown to be implicated in OA
. Here, ACLT surgery significantly elevated the SF concentration of cytokines and MMPs, which was partially reversed by RAPA treatment (Supplemental Fig. 2).
Leptin induced LOXL3 expression in primary cultured chondrocytes
In order to further explore the function of LOXL3 and leptin in osteoarthritis, chondrocytes were isolated from rat knee joint with sham-operation. IHC staining for collagen II
, markers of chondrocytes, demonstrated that primary chondrocytes have been isolated (Supplementary Fig. S1).
Chondrocytes isolated from rat knee joint with ACLT surgery were then treated with series dosages of leptin (from 5 ng/ml to 20 ng/ml). LOXL3 protein level of chondrocytes derived from rat joint with ACLT surgery was higher than that from sham-operated group. Leptin exposure led to a more notable increase in LOXL3 expression in a dose-dependent manner [Fig. 3(A)].
Fig. 3Silencing of LOXL3 promoted the proliferation and inhibited the apoptosis of chondrocytes. (A) Primary cultured chondrocytes were treated with a serial dosages of leptin for 48 h. LOXL3 protein level was detected by western blot (n = 3). (B) Primary chondrocytes isolated from rat knee joint with ACLT surgery were divided into three groups: Control: ACLT+leptin (20 ng/mL), siNC: ACLT+leptin (20 ng/mL) + siNC, and siLOXL3: ACLT+leptin (20 ng/mL) + siLOXL3. The chondrocytes viability as tested by CCK-8 (n = 3). (C) Detection of cell apoptosis by Annexin V-PI staining and flow cytometry analysis (n = 3). (D) Western blot analysis of LOXL3 and apoptosis associated protein expression. **P < 0.01, ***P < 0.001, vs siNC group (n = 3).
Silencing of LOXL3 promoted the proliferation and inhibited the apoptosis of chondrocytes
To study the effects of LOXL3 in the biological behavior of chondrocytes, primary chondrocytes isolated from rat knee joint with ACLT surgery were treated with leptin (20 ng/ml) and siLOXL3 or siNC. As shown in Fig. 3(D), siLOXL3 efficiently suppressed LOXL3 expression (76.8%).
The proliferation of chondrocytes was measured by CCK-8 assay depicted in Fig. 3(B). LOXL3 knockdown notably increased the proliferation of chondrocytes.
We then assessed the apoptosis of chondrocytes by flow cytometry analysis [Fig. 3(C)]. The apoptotic percentage was decreased from 21.3% ± 1.1% (siNC-transfected cells) to 11.6% ± 2.2% (siLOXL3-transfected cells). Next, we detected the expression of important regulator in apoptosis and autophagy by western blot [Fig. 3(D)]. In chondrocytes transfected with siNC and control chondrocytes, we observed high-level of an apoptosis marker (cleaved capase3), and low-level of an anti-apoptosis protein (Bcl-2). In contrast, in chondrocytes transfected with siLOXL3, the protein level of apoptosis marker was significantly reduced, and the expression of anti-apoptosis protein was remarkably increased. These data demonstrated siLOXL3 inhibited the apoptosis of chondrocytes.
LOXL3 inhibited the autophagy of chondrocytes via activating mTOR
Beclin1 expression and the ratio of LC3 II/LC3 I was significantly higher in siLOXL3-transfeted cells than in siNC-transfected cells [Fig. 4(A)]. To further investigate the effects of LOXL3 on chondrocyte autophagy, LOXL3 lentivirus was produced to infect primary chondrocytes isolated from rat knee joint with ACLT surgery. LOXL3 lentivirus infection significantly elevated its expression (Supplemental Fig. 3). LOXL3 overexpression significantly decreased the levels of autophagy markers [Fig. 4(B)], while reduced the phosphorylation of p70S6K, a downstream target of mTORC1 [Fig. 4(C)]. Such effects were abolished by the exposure of rapamycin, the most commonly used inhibitor of mTOR. No obvious change in the phosphorylation of AKT (S473), a downstream target of mTORC2, was observed after LOX3 lentivirus and/or rapamycin treatment. These data suggested that LOXL3 might suppress chondrocytes autophagy through mTORC1-dependent signaling.
Fig. 4LOXL3 inhibited the autophagy of chondrocytes via activating mTORC1. (A)Western blot analysis of Beclin1 and LC3 (n = 3). Control: ACLT+leptin (20 ng/mL), siNC: ACLT+leptin (20 ng/mL) + siNC, and siLOXL3: ACLT+leptin (20 ng/mL) + siLOXL3. **P < 0.01 vs siNC group. (B, C) Primary chondrocytes isolated from rat knee joint with ACLT surgery were divided into four groups: NC: cells treated with NC virus, LOXL3: cells treated with LOXL3 virus, RAPA: cells treated with rapamycin, and LOXL3+RAPA: cells treated with LOXL3 virus and rapamycin. Western blot analysis of Beclin1, LC3, p-p70S6K, p70S6K, p-AKT and AKT. *P < 0.05, **P < 0.01 vs ACLT+NC group; #P < 0.05, ##P < 0.01 vs ACLT+LOXL3 group (n = 3).
Differential expression of leptin and leptin's receptor isoform (Ob-Rb) mRNA between advanced and minimally affected osteoarthritic cartilage; effect on cartilage metabolism.
. Cell death of chondrocytes, a key step of osteoarthritis, is a combination between apoptosis and autophagy. Increasing evidence implied that leptin can regulate the apoptosis
of various cell types. Here, we try to discover the role of leptin on chondrocyte apoptosis and autophagy during osteoarthritis pathogenesis.
Firstly, re-analysis of public available NCBI GEO database demonstrated that LOXL3 was significantly increased in osteoarthritis affected cartilage [Fig. 1(A)], which was consistent with the finding of a previous study
. We then confirmed the overexpression of LOXL3 by real-time PCR analysis on osteoarthritis patients and healthy donors. LOXL3 consists of a copper-binding domain, conserved residues for lysyl-tyrosyl quinone (LTQ), a cytokine receptor-like (CRL) domain and four scavenger receptor cysteine-rich (SRCR) domains
, little studies have been carried out on the functions and mechanisms of LOXL3 in osteoarthritis pathogenesis. Here, a strong positive correlation was found between cartilage LOXL3 mRNA level and leptin SF concentration by using human samples [Fig. 1(C)] and rat osteoarthritis model (Fig. 2). To further confirm the relationship of LOXL3 and leptin, we treated isolated chondrocyte model with a series dosages of leptin [Fig. 3(A)]. Leptin exposure upregulated the expression of LOXL3 in a dose-dependent manner. These data suggested that LOXL3 may play a role in leptin-associated osteoarthritis pathogenesis.
Chondrocyte apoptosis is one of the most important factors involved in the pathological condition of osteoarthritis. In primary culture chondrocytes isolated from rat osteoarthritis model, siLOXL3 treatment caused a significantly reduction in apoptotic percentages and cleaved Caspase 3 expression, and a notable increase in cell survival and Bcl-2 expression (Fig. 3). Although further research is needed to explore the detailed mechanisms, we supposed that leptin could stimulate the expression of LOXL3, thus inducing chondrocytes apoptosis and osteoarthritis.
It has been reported that autophagy can protect human chondrocytes from stresses and may relate to pathogenesis of osteoarthritis
. In the present study, decreased cell autophagy (characterized by reduced LC3 II/LC3 I and Beclin1) was observed in LOXL3 higher expression group by using a rat osteoarthritis model [Fig. 2(E)]. In primary culture chondrocytes isolated from rat osteoarthritis model, the expression of LC3II and Beclin1 was significantly increased by LOXL3 knockdown [Fig. 4(A)] and reduced by ectopic expression of LOXL3 [Fig. 4(B)]. Furthermore, LOXL3 lentivirus infection increased the phosphorylation level of a major negative regulator of autophagy, mTOR
. Combined treatment with rapamycin (the most commonly used inhibitor of mTOR) significantly reversed the effects of LOXL3 lentivirus on the phosphorylation of p70S6K and the expression of autophagy markers. These data suggested that LOXL3 might suppress chondrocytes autophagy through mTORC1-dependent signaling. Here, considering that LOXL3 expression was elevated by leptin, we supposed that leptin could inhibited chondrocytes autophagy by rising the expression of LOXL3 and activation of mTORC1. A number of studies have been performed to explore the effects of leptin on autophagy. Leptin inhibited autophagy of human CD4+CD25-conventional T cells
indicated that leptin might exert its function on autophagy in a tissue and cell-specific manner.
Conclusions
In summary, increased leptin in osteoarthritis patients significantly stimulated LOXL3 expression. The increased LOXL3 expression induced chondrocyte apoptosis, activated mTORC1 and inhibited chondrocyte autophagy. LOXL3 might be considered as a promising therapeutic target during osteoarthritis therapy.
Author contributions
Conceived and designed the experiments: ZMH and PJT. Performed the experiments: ZMH, JHL and SHD. Analyzed the data: ZMH, SHD, LWX and LGH. Wrote the paper: ZMH and PJT.
Competing interests
The authors declare that they have no competing interests.
Acknowledgment
The work was supported by the Science Technology Program of Zhejiang Province (2013C33096), the Key Medical Disease Program of Hangzhou City (20120533Q39 and 2013B51), the Key Science Technology Program of Xiaoshan District (2012234), the TCM Foundation for Distinguished Young Talents of Zhejiang Province (2012ZQ023 and 2012ZQ024) and the Medical Disease Program of Zhejiang Province (2012KYB169 and 2013KYB226).
Appendix A. Supplementary data
The following is the supplementary data related to this article:
Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes.
Differential expression of leptin and leptin's receptor isoform (Ob-Rb) mRNA between advanced and minimally affected osteoarthritic cartilage; effect on cartilage metabolism.
Synovial proinflammatory cytokines and their correlation with matrix metalloproteinase-3 expression in Behçet's disease. Does interleukin-1β play a major role in Behçet's synovitis?.
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