Abstract
It has been demonstrated that oral administration of N-acetylglucosamine (GlcNAc) alleviates the symptoms of osteoarthritis (OA). The aim of the present study was to elucidate the molecular mechanisms for the chondroprotective action of GlcNAc in OA. Biomarkers for type II collagen degradation and synthesis were evaluated, as were histopathological changes, using a rat anterior cruciate ligament transection (ACLT)-induced OA model. Changes in the expression of genes in the cartilage were assessed via DNA microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results indicated that ACLT induced histopathological changes of articular cartilage, whereas oral administration of GlcNAc (1,000 mg/kg/day for 28 days) significantly suppressed these changes. Additionally, GlcNAc significantly decreased levels of a type II collagen degradation marker in sera compared with that in the ACLT group, although there were no significant changes in the levels of a type II collagen synthesis marker. Furthermore, DNA microarray and reverse transcription-quantitative polymerase chain reaction results demonstrated that GlcNAc treatment downregulated the expression of periostin, which is likely involved in the degradation of cartilage, whereas GlcNAc upregulated the expression of lipocalin 2, which is involved in the regulation of chondrocyte proliferation and differentiation. In conclusion, the results of the present study suggest that GlcNAc is able to suppress the histopathological changes induced by OA and exhibits a chondroprotective action by inhibiting type II collagen degradation in the articular cartilage, possibly via modulation of the expression of inflammatory and chondroprotective molecules, including periostin and lipocalin 2.
Keywords: N-acetylglucosamine, dietary supplement, cartilage metabolism, biomarker, osteoarthritis
Introduction
Knee osteoarthritis (OA) is the most prevalent disease of the joints. The prevalence of knee OA is estimated at 9.6% in men and 18% in women aged 60 years or older (1). OA is characterized by a progressive degradation of cartilage and the symptoms of joint pain and severe impaired mobility of the knee as the knee is a weight-bearing joint (2). An imbalance between the synthesis and degradation of articular cartilage leads to joint pain, stiffness and severely impaired joint mobility (3). Pharmacological treatments for knee OA typically utilize analgesic agents, such as nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors, which are aimed at controlling pain and limiting loss of function. However, previous studies have suggested that the benefits of these pharmaceutical agents are often insufficient and have an associated risk of adverse reactions (4,5). Therefore, the development of alternative strategies for the treatment of knee OA is necessary.
Glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are naturally occurring amino monosaccharides and are the key components of glycosaminoglycans contained in articular cartilage, such as chondroitin sulfate and hyaluronan; due to their crucial role in glycosaminoglycan formation in cartilage (6,7), GlcN and GlcNAc have been widely used as dietary supplements or alternative treatments for the management of OA (8). Furthermore, clinical studies and a meta-analysis investigating the effect of GlcN on OA have demonstrated that glucosamine sulfate or hydrochloride has potential benefits for relieving pain associated with OA (9–11). Additionally, it has previously been demonstrated that oral administration of GlcNAc alleviated symptoms in patients with OA (12,13).
It has previously been demonstrated that GlcN is incorporated into cells via glucose transporters (14) and used by synovial cells and chondrocytes to produce hyaluronic acid via the upregulation of hyaluronic acid-synthesizing enzymes (8,15). GlcN has also been suggested to exert anti-inflammatory actions by inhibiting nitric oxide production, and downregulating cyclooxygenase-2 and the expression of pro-inflammatory cytokines (16,17). Based on these biochemical and pharmacological findings, it has been suggested that the administration of GlcN recovers cartilage metabolism by inhibiting degradation and stimulating the synthesis of proteoglycans (18). However, the underlying molecular mechanisms of the chondroprotective action of GlcNAc in patients with OA remain to be elucidated.
In the present study, serum biomarkers for type II collagen degradation (type II collagen cleavage neoepitope; C2C) and synthesis (carboxyterminal propeptide of type II procollagen, PIICP) were evaluated in addition to histopathological changes in a rat anterior cruciate ligament transection (ACLT)-induced OA model. The ACLT model has been widely used to analyze histological and biochemical changes that occur during the progression of OA (19). Changes in gene expression in the cartilage were also investigated using a DNA microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
Materials and methods
Animal model
All procedures were carried out at I Tech Lab (Kaizu, Japan) according to the institutional guidelines for animal experiments based on the Guidelines for Proper Conduct of Animal Experiments, Act on Welfare and Management of Animals, and standards for the care and management of experimental animals. The study protocol was approved by the Institutional Animal Care and Use Committee at I Tech Lab. In the present study, a total of 37 male Slc:SD rats (8-weeks-old, initially weighing 307.5±17.5 g; Japan SLC, Inc., Hamamatsu, Japan) were used. All animals were housed in a room maintained at 23±3°C with 62±5% humidity and a 12-h light/dark cycle. Sterile food (FR-2; Funabashi Farm Co., Ltd., Chiba, Japan) and water were freely available. Rats were acclimated to the environmental conditions for 1 week prior to the experiment. Rats, with the exception of 3 heavy and 4 lightweight rats, were randomly divided into three groups based on body weight: Sham-operated group (sham; n=10), ACLT group without GlcNAc (ACLT; n=10) and ACLT group with GlcNAc (GlcNAc; n=10).
OA was surgically induced in the knee joint. Each rat was anesthetized via intramuscular injection of 0.8 ml/kg each of ketamine (Ketalar 500 mg; Daiichi Sankyo Propharma Co., Ltd., Tokyo, Japan) and xylazine (Selactar 2%; Bayer Yakuhin, Ltd., Osaka, Japan). The surgical area was shaved and disinfected with povidone iodine, and the right knee joint was subsequently exposed by dissecting the skin on the inside of the patella along the patellar tendon. The patella was dislocated laterally, the knee was placed in full flexion, and the anterior cruciate ligament was subsequently transected. A positive anterior drawer test ensured complete transection of the ligament. Following surgery, the joint surface was washed with sterile saline and disinfected with 4 ml of antibiotics solution (250 mg/ml ampicillin sodium salt). The knee joint and patella were then returned to their original position. The capsule and skin were subsequently sutured using Vicryl 4–0 absorbable sutures (Ethicon, Somerville, NJ, USA) and monofilament 4–0 nylon threads (Ethicon), respectively. For rats in the sham group, the right knee joint was exposed and washed with saline, following which the incisions were sutured.
GlcNAc was supplied by Yaizu Suisankagaku Industry Co., Ltd. (Yaizu, Japan). On days 1–28 post-surgery, 1,000 mg/kg GlcNAc (200 mg/ml GlcNAc solution) dissolved in sterile water was orally administered to rats in the GlcNAc group daily. An equivalent volume of sterile water was orally administered to rats in the sham and ACLT groups daily.
Blood samples (1 ml/rat) were harvested from the carotid artery at 1, 14 and 28 days post-surgery. Sera were prepared by centrifugation at 1,700 × g for 15 min at 4°C and stored in aliquots at −80°C. Rats were sacrificed at 29 days post-surgery for histopathological and genetic analyses.
Enzyme-linked immunosorbent assay (ELISA) of serum biomarkers
Serum samples obtained on days 1, 14 and 28 post-surgery were analyzed for each biomarker. The biomarkers investigated were C2C and PIICP. C2C was measured using a competitive inhibition ELISA (Collagen Type II Cleavage ELISA kit; IBEX Technologies, Inc., Montreal, QC, Canada). PIICP was assessed using an ELISA kit for Procollagen II C-terminal Propeptide (USCN Life Sciences, Inc., Wuhan, China).
Histopathological evaluation
Histopathological evaluation was performed on sagittal sections of cartilage in the weight-bearing area of the femoral condyle and medial tibia plateau from 4 rats per group. Knee joint samples were dissected, fixed in 10% formalin for 24 h at 20–25°C, decalcified using 10% ethylenediaminetetraacetic acid solution for 2 weeks, and embedded in paraffin. Sections with a thickness of 5 µm were stained with 0.05% toluidine blue (pH 4.1), and the severity of OA lesions was graded on a scale of 0–13, using the modified Mankin scoring system (18), with a combined score of structure (0–6 points), matrix staining (0–4 points) and cellular abnormalities (0–3 points).
RNA isolation
RNA was prepared as described previously (20). Briefly, total RNA was extracted from the harvested the sections of cartilage in the weight-bearing area of the medial tibia plateau using an RNAiso plus reagent (Takara Bio Inc., Otsu, Japan) and the RNase-Free DNase Set (Qiagen, Inc., Valencia, CA, USA). The extracted RNA was further purified using an RNeasy MinElute Cleanup kit (Qiagen, Inc.). The quantity and purity of total RNA were evaluated photometrically at 260, 280 and 320 nm using a spectrophotometer (Ultrospec 2000; GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The ratio of A260/280 was confirmed to be >2.0, and total RNA was used for the following experiments.
DNA microarray analysis
Total RNA (200 ng) was used to generate cDNA and Cy3-labeled cRNA using a Low Input Quick-Amp Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. The labeled cRNA was used to photometrically determine the quantity and dye-incorporation ratio with an Ultrospec 2000 spectrophotometer, and hybridized to a Whole Rat Genome Microarray (Ver 3.0; cat. no. G4847B; Agilent Technologies, Inc.) at 65°C for 17 h. The array was scanned with GenePix 4000B (Molecular Devices, LLC, Sunnyvale, CA, USA), and the resulting image was processed using GenePix Pro 6.0 Software (Molecular Devices, LLC). The features were manually examined, and spots of poor quality were flagged and filtered out according to the method of Shintani et al (21) and Chae et al (22). The signal data for the features (spots of array) were then imported into GeneSpring 12.6 (Agilent Technologies, Inc.), and further analyzed with the software. The signal data from the arrays were normalized with the 75th percentile method (23), and baseline transformation was performed with the median of the control samples. Quality control was performed in order to filter out signal data with standard errors >0.2.
To analyze the differentially expressed genes, the fold change (FC) vs. the sham samples was calculated, and genes with an FC >1.5 or <0.75 were extracted for the ACLT group. To analyze the effects of GlcNAc, the genes that were shifted toward the baseline levels (the sham group) in the GlcNAc group compared with the ACLT group were selected.
RT-qPCR
RT-qPCR was performed to confirm the expression of 41 target genes selected on the basis of the DNA microarray analysis following the method of Shintani et al (21) and Chae et al (22). cDNA was synthesized from 1 µg total RNA from the cartilage using PrimeScript Reverse Transcriptase (Takara Bio, Inc.), oligo (dT) primer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and dNTP (Promega Corporation, Madison, WI, USA). qPCR was performed with a Mx3000P qPCR System (Agilent Technologies, Inc.) using a SYBR Premix Ex Taq kit (Takara Bio, Inc.), ROX (Takara Bio, Inc.) and specific primer sets designed using Primer-BLAST program (The sequences of the primers are available upon request) (24). PCR conditions were: 95°C for 15 sec, followed by 35 cycles of 95°C for 10 sec and 63°C for 30 sec. In each run, a standard curve was generated by a serially diluted known GAPDH amplicon to calculate the cDNA copy number of the genes, as described previously (20). The quantity of the mRNA of interest was expressed as its ratio against that of a suitable reference gene, peptidylprolyl isomerase A (25).
Statistical analyses
Values are expressed as the mean ± standard deviation or mean + standard deviation. Student's unpaired t-test was used to assess the intergroup differences in histopathological evaluation and serum biomarkers. Student's paired t-test was used to assess the chronologic changes in serum biomarkers. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using EZR version 1.31 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) (26), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria, version 3.22).
Results
Effect of GlcNAc administration on the gross morphology and the histopathological changes in articular cartilage
Images of the articular cartilage from rats in the three groups are shown in Fig. 1. In the sham group, no macroscopic abnormalities were detected on the articular surfaces of femoral condyles (Fig. 1A) and tibial plateau (Fig. 1D). However, the rats of the ACLT group exhibited marked erosive changes on the surfaces of these sites (Fig. 1B and E). The degenerative changes were markedly suppressed in the GlcNAc group (Fig. 1C and F).
Figure 1.
Macroscopic changes of femoral condyles and the tibial plateau at 29 days following ACLT surgery. Femurs from the (A) sham, (B) ACLT and (C) GlcNAc groups. Tibias from the (D) sham, (E) ACLT and (F) GlcNAc groups. Sham-operated joints showed no macroscopic changes, whereas ACLT induced erosive changes on the joint surfaces (indicated by large arrowheads) and GlcNAc administration substantially suppressed these degenerative changes (indicated by small arrowheads). ACLT, anterior cruciate ligament transection; sham, sham-operated; GlcNAc, N-acetylglucosamine.
The histopathological changes in articular cartilage were also assessed, and representative images are shown in Fig. 2. When compared with cartilage from the sham group (Fig. 2A and E), cartilage from rats in the ACLT group (Fig. 2B and F) displayed histopathological changes including surface depletion, a marked reduction in the number of chondrocytes and reduced toluidine blue staining. Furthermore, treatment with GlcNAc markedly suppressed these structural abnormalities (Fig. 2C and G). These structural changes were further evaluated using the modified Mankin scoring system. The total score was significantly higher in the femoral condyles and tibial plateau of the ACLT group compared with the sham group (both P<0.01). Treatment with GlcNAc markedly reduced this effect in the femoral condyles (Fig. 2D) and significantly reduced the effect in the tibial plateau (P<0.05 vs. the ACLT group; Fig. 2H).
Figure 2.
Histopathological evaluation of articular cartilage in a rat OA model. Knee joints were dissected at 29 days following ACLT surgery. Sagittal sections of cartilage in the weight-bearing area of the femoral condyle and the medial tibia plateau were stained with toluidine blue. Femurs from the (A) sham, (B) ACLT and (C) GlcNAc groups, and (D) the severity of OA lesions in femurs. Tibias from the (E) sham, (F) ACLT and (G) GlcNAc groups, and (H) the severity of OA lesions in tibias. ACLT induced surface depletion, a marked reduction in the number of chondrocytes and a reduction in toluidine blue staining of the cartilage from femurs and tibias (indicated by arrowheads). The severity of OA lesions was graded on a scale of 0–13 using the modified Mankin scoring system. Data are presented as the mean ± standard deviation (n=4 per group). *P<0.05 and **P<0.01 as indicated. OA, osteoarthritis; ACLT, anterior cruciate ligament transection; sham, sham-operated; GlcNAc, N-acetylglucosamine.
Evaluation of biomarkers
The effect of GlcNAc administration on cartilage metabolism was assessed using biomarkers for type II collagen degradation and synthesis. Serum C2C levels were significantly decreased from the baseline (day 1) on days 14 and 28 post-surgery in the ACLT and GlcNAc groups (Fig. 3A). Furthermore, on day 28 the C2C level was significantly lower in the GlcNAc group compared with the ACLT group (P<0.05; Fig. 3A). No significant differences in PIICP levels were observed between the sham, ACLT and GlcNAc groups. However, the PIICP levels in the sham and GlcNAc groups were demonstrated to be significantly decreased from the baseline on day 28 (P<0.05; Fig. 3B). Consequently, the C2C/PIICP ratio in the GlcNAc group was significantly decreased from the baseline on days 14 and 28 post-surgery (Fig. 3C). Together, these observations suggest that the administration of GlcNAc suppresses the degradation of type II collagen in the articular cartilage of ACLT rats.
Figure 3.
Changes in the biomarkers for type II collagen during GlcNAc treatment for rats with surgically induced ACLT. Changes in (A) C2C, (B) PIICP and (C) the C2C/PIICP ratio from the baseline were calculated for sham (open circle), ACLT (filled square) and GlcNAc (filled triangle) groups on days 14 and 28 post-surgery. Baseline levels on day 1 post-surgery were as follows: C2C, 621.09±139.20, 764.27±50.18 and 881.21±91.97 ng/ml in the sham, ACLT and GlcNAc groups, respectively; PIICP, 8,449.32±1,873.34, 8,698.22±1,977.0 and 8,081.33±765.5 ng/ml in the sham, ACLT and GlcNAc groups, respectively; C2C/PIICP, 77.84±29.46, 92.39±22.82 and 109.91±15.08 in the sham, ACLT and GlcNAc groups, respectively. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. day 1; #P<0.05 as indicated. GlcNAc, N-acetylglucosamine; ACLT, anterior cruciate ligament transection; C2C, type II collagen cleavage neoepitope; PIICP, carboxyterminal propeptide of type II procollagen; sham, sham-operated.
DNA microarray analysis
To elucidate the mechanism for the changes of histopathological findings and biomarkers, the effect of GlcNAc administration was investigated further using microarray analysis. The results indicated that 74 genes were upregulated by ACLT (>1.5-fold) compared with the sham group and downregulated in the GlcNAc group compared with the ACLT group (Table I). By contrast, 18 genes were downregulated in the ACLT group (<0.75-fold) compared with the sham group and upregulated in the GlcNAc group compared with the ACLT group (Table II). Pathway analysis indicated that the majority of the modulated genes are associated with synthesis of the components of extracellular matrix in articular cartilage: Tenascin, collagen, laminin and thrombospondin (data not shown).
Table I.
List of genes upregulated by ACLT (>1.5-fold) compared with the sham group and downregulated by GlcNAc administration compared with the ACLT group.
| Fold-change | ||
|---|---|---|
| Gene symbol | ACLT/sham | GlcNAc/ACLT |
| Tnn | 7.009 | 0.584 |
| LOC685106 | 3.782 | 0.255 |
| Postn | 3.311 | 0.364 |
| Crabp2 | 2.933 | 0.714 |
| Fbn2 | 2.688 | 0.628 |
| RGD1565772 | 2.594 | 0.642 |
| Sorcs2 | 2.572 | 0.612 |
| RGD1311501 | 2.215 | 0.575 |
| Wisp2 | 2.179 | 0.718 |
| Vwa1 | 2.088 | 0.687 |
| N/A (Q40555_TOBAC (Q40555) Peroxidase, partial (5%) [TC599198]) | 2.055 | 0.590 |
| LOC100363229 | 2.040 | 0.709 |
| Qprt | 2.034 | 0.524 |
| Smoc2 | 1.994 | 0.696 |
| Scx | 1.899 | 0.599 |
| Col18a1 | 1.881 | 0.697 |
| H19 | 1.879 | 0.596 |
| Nanos3 | 1.864 | 0.615 |
| Prrx2 | 1.845 | 0.636 |
| Apln | 1.838 | 0.649 |
| Mmp2 | 1.831 | 0.746 |
| Nnmt | 1.812 | 0.641 |
| LOC500846 | 1.786 | 0.708 |
| Tmem119 | 1.755 | 0.679 |
| Srpx2 | 1.745 | 0.716 |
| Slc6a8 | 1.742 | 0.687 |
| Col12a1 | 1.740 | 0.650 |
| Vegfa | 1.737 | 0.680 |
| Cacnb3 | 1.725 | 0.604 |
| RGD1305689 | 1.712 | 0.626 |
| Timp1 | 1.708 | 0.732 |
| A2m | 1.708 | 0.600 |
| Sp1 | 1.683 | 0.531 |
| Tnc | 1.675 | 0.725 |
| Cyb561 | 1.674 | 0.587 |
| Mmp14 | 1.670 | 0.695 |
| Col1a1 | 1.667 | 0.714 |
| Aspn | 1.647 | 0.499 |
| Igsf10 | 1.643 | 0.717 |
| Pvalb | 1.640 | 0.137 |
| Actg2 | 1.637 | 0.558 |
| Dchs1 | 1.630 | 0.739 |
| Aifm2 | 1.628 | 0.683 |
| Col5a2 | 1.627 | 0.688 |
| Col8a1 | 1.626 | 0.511 |
| LOC100361571 | 1.622 | 0.688 |
| Pdpn | 1.619 | 0.737 |
| Hoxa10 | 1.612 | 0.599 |
| Mxra7 | 1.610 | 0.572 |
| Dapk3 | 1.610 | 0.664 |
| Wtip | 1.608 | 0.700 |
| Eps8l2 | 1.596 | 0.656 |
| Cpxm2 | 1.594 | 0.512 |
| Ano1 | 1.593 | 0.700 |
| Vkorc1 | 1.591 | 0.747 |
| Plat | 1.590 | 0.746 |
| Thbs4 | 1.590 | 0.733 |
| Mmp13 | 1.575 | 0.734 |
| Lama5 | 1.573 | 0.559 |
| C1qtnf6 | 1.570 | 0.704 |
| Sertad4 | 1.565 | 0.639 |
| Rcn3 | 1.562 | 0.700 |
| Fibin | 1.555 | 0.610 |
| Adamts1 | 1.552 | 0.741 |
| Alkbh | 1.543 | 0.383 |
| Rftn2 | 1.542 | 0.674 |
| Mcts2 | 1.537 | 0.704 |
| LOC287167 | 1.523 | 0.717 |
| Fxyd6 | 1.521 | 0.462 |
| Pmepa1 | 1.515 | 0.728 |
| LOC679958 | 1.512 | 0.682 |
| Mmp16 | 1.509 | 0.663 |
| Cyp26b1 | 1.509 | 0.731 |
| Ppp1r12a | 1.507 | 0.462 |
ACLT, anterior cruciate ligament transection; GlcNAc, N-acetylglucosamine; sham, sham-operated.
Table II.
List of genes downregulated by ACLT (<0.75-fold) compared with the sham group and upregulated by GlcNAc administration compared with the ACLT group.
| Fold-change | ||
|---|---|---|
| Gene symbol | ACLT/sham | GlcNAc/ACLT |
| Usp25 | 0.245 | 3.446 |
| Inpp5f | 0.345 | 2.191 |
| Try5 | 0.370 | 1.991 |
| Dnajc22 | 0.391 | 2.947 |
| Lrrc28 | 0.399 | 1.553 |
| Lcn2 | 0.425 | 2.772 |
| Nip30 | 0.448 | 2.238 |
| Atad2 | 0.352 | 2.347 |
| Prss2l | 0.531 | 2.524 |
| Shfm1 | 0.544 | 2.118 |
| Rnls | 0.546 | 2.340 |
| Try4 | 0.565 | 2.317 |
| Try10 | 0.618 | 2.250 |
| Ell3 | 0.627 | 1.537 |
| Znf282 | 0.635 | 1.972 |
| Larp5 | 0.688 | 1.739 |
| Atl2 | 0.729 | 1.529 |
| LOC682968 | 0.749 | 1.508 |
ACLT, anterior cruciate ligament transection; GlcNAc, N-acetylglucosamine; sham, sham-operated.
RT-qPCR analysis
In order to confirm the results of DNA microarray, 41 genes associated with the extracellular matrix components in the articular cartilage were selected and subjected to RT-qPCR. As shown in Table III, 14 genes were upregulated by ACLT (>1.3-fold) and downregulated by GlcNAc (<0.9-fold): Periostin, osteoblast specific factor (Postn), fibrillin 2 (Fbn2), collagen type I, α1 (Col1a1), tenascin N (Tnn), sortilin-related VPS10 domain containing receptor 2 (Sorcs2), WNT1 inducible signaling pathway protein 2 (Wisp2), matrix metalloproteinase 2 (Mmp2), vascular endothelial growth factor A (Vegfa), collagen type V, α2 (Col5a2), Mmp13, A disintegrin and metalloproteinase with thrombospondin motifs 4 (Adamts4), Sushi repeat-containing protein, X-linked 2 (Srpx2) and tissue inhibitor of metalloproteinase-1 (Timp1). Furthermore, 6 genes were downregulated by ACLT (<0.7-fold) and upregulated by GlcNAc (>1.3-fold): Lipocalin-2 (Lcn2), uncharacterized anionic trypsin II like (Prss21), mesotrypsin (Try4), trypsin 10 (Try10), elongation factor RNA polymerase II-like 3 (EII3) and atlastin GTPase 2 (Atl2).
Table III.
List of genes confirmed by reverse transcription-quantitative polymerase chain reaction.
| Fold-change | |||
|---|---|---|---|
| Gene symbol | Description | ACLT/sham | GlcNAc/ACLT |
| Tnn | Tenascin N | 4.500 | 0.809 |
| Postn | Periostin, osteoblast specific factor | 2.550 | 0.675 |
| Adamts4 | A disintegrin and metalloproteinase with thrombospondin motifs 4 | 2.210 | 0.787 |
| Crabp2 | Cellular retinoic acid binding protein 2 | 1.980 | 0.934 |
| Fbn2 | Fibrillin 2 | 1.860 | 0.618 |
| Timp1 | Tissue inhibitor of metalloproteinase 1 | 1.680 | 0.815 |
| Wisp2 | WNT1 inducible signaling pathway protein 2 | 1.670 | 0.796 |
| Mmp13 | Matrix metalloproteinase 13 | 1.560 | 0.891 |
| Mmp2 | Matrix metalloproteinase 2 | 1.500 | 0.860 |
| Vwa1 | Von Willebrand factor A domain containing 1 | 1.430 | 0.930 |
| Vegfa | Vascular endothelial growth | 1.420 | 0.880 |
| Dnajc22 | DnaJ (Hsp40) homolog, subfamily C, member 22 | 1.390 | 0.791 |
| Sorcs2 | Sortilin-related VPS10 domain containing receptor 2 | 1.390 | 0.842 |
| Col5a2 | Collagen type V, α2 | 1.380 | 0.848 |
| Col1a1 | Collagen type I, α1 | 1.370 | 0.635 |
| Srpx2 | Sushi-repeat-containing protein, X-linked 2 | 1.340 | 0.776 |
| Mmp14 | Matrix metalloproteinase 14 | 1.290 | 0.853 |
| Pvalb | Parvalbumin | 1.210 | 0.190 |
| Thbs4 | Thrombospondin-4 | 1.190 | 0.870 |
| Nip30 | NEFA-interacting nuclear protein NIP30 | 1.140 | 0.886 |
| Smoc2 | SPARC related modular calcium binding 2 | 1.110 | 0.802 |
| Wtip | Wilms tumor 1 interacting protein | 1.100 | 1.060 |
| Inpp5f | Inositol polyphosphate-5-phosphatase F | 1.080 | 0.852 |
| Pdpn | Podoplanin | 1.050 | 0.930 |
| Col2a1 | Collagen type II, α1 | 0.990 | 1.071 |
| Usp25 | Ubiquitin specific peptidase 25 | 0.950 | 0.705 |
| Sertad4 | SERTA domain containing 4 | 0.940 | 0.680 |
| Lrrc28 | Leucine rich repeat containing 28 | 0.920 | 1.120 |
| Ctsk | Cathepsin K precursor | 0.920 | 1.000 |
| Slc6a8 | Solute carrier family 6, member 8 | 0.920 | 0.946 |
| Atad2 | ATPase family, AAA domain containing 2 | 0.840 | 1.024 |
| Igsf10 | Immunoglobulin superfamily, member 10 | 0.840 | 0.880 |
| Zfp282 | Zinc finger protein 282 | 0.820 | 0.902 |
| Shfm1 | Split hand/foot malformation type 1 | 0.780 | 0.950 |
| Larp4b | La ribonucleoprotein domain family, member 5 | 0.700 | 0.971 |
| Ell3 | Elongation factor RNA polymerase II-like 3 | 0.650 | 1.400 |
| Atl2 | Atlastin GTPase 2 | 0.630 | 1.349 |
| Prss2l | Prss2-like, anionic trypsin-2-like | 0.490 | 2.327 |
| Try4 | Mesotrypsin | 0.400 | 2.150 |
| Try10 | Trypsin 10 | 0.330 | 1.909 |
| Lcn2 | Lipocalin 2 | 0.260 | 4.308 |
ACLT, anterior cruciate ligament transection; sham, sham-operated; GlcNAc, N-acetylglucosamine.
Discussion
Several clinical trials have demonstrated that oral administration of GlcNAc has significant symptom-modifying effects and improves type II collagen biomarkers in patients with OA (12,13,27,28). However, to the best of our knowledge, no previous research exists in which the molecular mechanisms underlying the action of GlcNAc on articular cartilage metabolism are elucidated. Therefore, in the present study an ACLT surgical model was used to investigate the changes in biochemical markers of type II collagen and the expression of cartilage metabolism-related genes in addition to histopathological criteria.
The results of the present study demonstrated that ACLT-induced histopathological changes, such as erosion and degeneration, in articular cartilage, and GlcNAc administration significantly suppressed these changes. Furthermore, GlcNAc administration significantly decreased levels of C2C, a type II collagen degradation marker, in rats with ACLT, although there were no significant changes in serum PIICP levels. Notably, administration of GlcNAc significantly decreased the ratio of C2C and PIICP from the baseline. These observations suggest that GlcNAc exerts a chondroprotective action by suppressing ACLT-associated histopathological changes, possibly via inhibiting type II collagen degradation rather than increasing type II collagen synthesis in the arthritic cartilage.
To further clarify the action of GlcNAc on arthritic cartilage, genetic analyses were performed via DNA microarray and RT-qPCR. The results revealed that GlcNAc upregulated or downregulated the expression of several genes associated with the extracellular matrix in articular cartilage. In particular, Postn was substantially upregulated by ACLT, and this upregulation was suppressed by GlcNAc administration. The Postn gene encodes periostin, a protein that functions as a ligand for integrins to promote cell adhesion and migration (29). Moreover, periostin induces inflammatory cytokines and MMPs via nuclear factor κB signaling, and promotes cartilage degeneration in patients with OA (30,31). Previous studies have demonstrated that the Postn expression is upregulated in the damaged cartilage of patients with knee OA (32–34). These observations suggest that GlcNAc has the potential to inhibit the degradation of type II collagen, as evidenced by the suppression of the level of C2C, a type II collagen degradation marker, by downregulating the expression of periostin.
GlcNAc has been reported to stimulate hyaluronan synthesis via the upregulation of hyaluronan synthase-2 (HAS2) in human articular chondrocytes (11). Furthermore, hyaluronan exhibits an anti-inflammatory action by inhibiting the interleukin (IL)-1β-induced MMP-13 expression and IL-1α-induced aggrecanase expression in arthritic chondrocytes (35,36). However, in the present study HAS2 expression was not found to be affected by GlcNAc (data not shown). Therefore, it is unlikely that HAS2 expression and synthesis of hyaluronan are associated with the chondroprotective action of GlcNAc observed in the present results.
GlcNAc administration markedly upregulated the expression of Lcn2, which was downregulated by ACLT. Lipocalin 2 has a role in the regulation of chondrocyte proliferation and differentiation, and its expression decreases with age in humans (37). It may therefore be speculated that GlcNAc exhibits a chondroprotective action by maintaining chondrocyte proliferation and differentiation via the upregulation of Lcn2.
In summary, the results of the present study suggest that GlcNAc has a chondroprotective action in OA, which it achieves by suppressing histopathological changes, possibly via the inhibition of type II collagen degradation in the articular cartilage. Furthermore, it is likely that GlcNAc modulates the expression of inflammatory and chondroprotective molecules, such as periostin and lipocalin 2, which are involved in the degradation and maintenance of cartilage, thereby having a protective effect on the cartilage. However, the detailed mechanism for the protective action of GlcNAc on OA remains to be elucidated in the future.
Acknowledgements
The authors of the present study would like to thank Mr. Kei Yasui, Dr Etsushi Yamamoto, Ms. Kaori Yoshimura, Dr Tetsuro Yamamoto (Total Technological Consultant Co., Ltd., Tokyo, Japan) and Dr Shin Nakamura (Intelligence and Technology Lab, Inc., Gifu, Japan) for their helpful discussions and statistical expertise in the preparation of the manuscript.
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