Introduction: Osteoarthritis (OA) is a degenerative disease common in the elderly and is characterized by joint pain, swelling, and restricted movement. In recent years, heparanase has been reported to play an important role in the development of osteoarthritic cartilage. PG545 is a heparan sulfate mimetic with heparanase inhibitory activity. In this study, the therapeutic effects and possible mechanisms of PG545 were investigated in a chondrocyte injury model induced by interleukin-1β (IL -1β). Methods: Following treatment with PG545 or the autophagy inhibitor 3-methyladenine (3-MA), chondrocyte viability was detected using Cell Counting Kit-8 and fluorescein diacetate/propidium iodide double staining. The apoptosis rate of chondrocytes was determined by flow cytometry. Expression of light chain 3 and P62 was monitored by immunofluorescence labeling. Western blot, lentivirus infection with red fluorescent protein and green fluorescent protein, and quantitative real-time polymerase chain reaction were used to determine the expression levels of chondrocyte markers, apoptosis-related factors, autophagy proteins, and key proteins of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. The expression and activity of stress-specific enzymes such as malondialdehyde, superoxide dismutase, and catalase (CAT) were investigated. Chondrocytes with ATG5 knockdown were used to investigate the relationship between the therapeutic effect of PG545 and autophagy. The therapeutic effect of PG545 was verified in vivo. Results: PG545 had a significant protective effect on chondrocytes by reducing oxidative stress, apoptosis, and degradation of chondrocytes and increasing chondrocyte proliferation. PG545 was effective in inducing autophagy in IL-1β-treated cells, while 3-MA attenuated the effect. The PI3K/Akt/mTOR pathway may be involved in the promotion of autophagy and OA treatment by PG545. Conclusion: PG545 was able to restore impaired autophagy and autophagic flux via the PI3K/Akt/mTOR pathway, thereby delaying the progression of OA, suggesting that PG545 may be a novel therapeutic approach for OA.

Osteoarthritis (OA) is a degenerative disease that often occurs in older people and is characterized by chronic intra-articular inflammation and cartilage degeneration [1]. Both obesity and an aging population could increase the incidence of OA [2]. It is estimated that more than 28% of people over 60 are affected by OA [3], which is mainly manifested by joint pain, swelling, and reduced mobility and is a severe social and economic burden [4]. The first step in OA is loss of the extracellular matrix (ECM), followed by rupture of the articular cartilage and massive fibrotic growth, culminating in complete cartilage loss [5]. Due to the complexity of the disease, there is currently no effective treatment [6]. Although there are some drugs that could alleviate OA by acting on the subchondral bone and synovium. However, the severe side effects of these drugs are a non-negligible problem that limits the quality of life of OA patients [7]. Apoptosis inhibition and cell-based clinical treatments such as autologous chondrocyte implantation have become hotspots in recent years, and OA researchers hope to use them to preserve cartilage function and maintain chondrocyte activity [8, 9]. However, most patients with severe OA have to resort to arthroscopic surgery or artificial joint replacement [10, 11]. Therefore, there is an urgent need for research into new medical treatments to delay, halt, or reverse the progression of OA.

The pathological process of OA is mainly related to apoptosis, oxidative damage, aging, and autophagy of chondrocytes [12, 13]. Autophagy is a highly conserved catabolic process that plays an important role in maintaining cellular homeostasis and recycling broken organelles [14]. The role of autophagy in regulating proliferation, differentiation, and homeostasis of healthy chondrocytes has been demonstrated [15]. Caramés et al. [16] found that the level of autophagy was significantly reduced in chondrocytes from patients with OA, which was confirmed by inhibition of the expression of the proteins ULK1, Beclin-1, and light chain 3 (LC3).

Heparan sulfate (HS) is a highly anionic glycosaminoglycan side chain on the cell surface and ECM that plays a role in regulating cellular metabolism [17]. Heparanase, the only HS-degrading enzyme in mammals, cleaves the HS side chain of the proteoglycan and, through its endo-β-glucuronidase activity, releases HS-linked growth factors, cytokines, chemokines, and other ligands stored in the ECM that are closely associated with cartilage metabolism [18, 19]. Heparanase has been shown to be involved in chondrocyte metabolism in both humans and mice, causing upregulation of catabolic genes and downregulation of anabolic genes in human chondrocytes [19, 20]. Due to the important role of heparanase in tissue remodel and signal transduction, PG545, a HS mimetic with inhibitory effects on heparanase, was introduced [21]. The aim of this study was to uncover the possible chondroprotective role of PG545 in interleukin-1β (IL-1β)-induced chondrocyte injury and to determine the possible mechanisms. We hypothesized that PG545 modulates chondrocyte autophagy via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) axis, thereby delaying the progression of OA. The autophagy inhibitor 3-methyladenine (3-MA) was used to test the effect of PG545 on autophagy-related signaling pathways.

Reagents

PG545 was purchased from Progen Pharmaceuticals (Brisbane, Australia) and stored at −20°C [22]. IL-1β and 3-MA were purchased from Sigma-Aldrich (St Louis, MO, USA). A lentivirus expressing red fluorescent protein (RFP), green fluorescent protein (GFP), and LC3 were purchased from Pudong Genomeditech (Shanghai, China). LY294002 and antibodies against GADPH, cleaved-caspase 3, cleaved-PARP, collagenase type II (Col II), aggrecan, P62, cyclooxygenase-2 (COX-2), matrix metalloproteinase 13 (MMP13), PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR were from Abcam (Cambridge, MA, USA). The antibody against Beclin-1 was from Novus Biologicals (Littleton, CO, USA). The antibody against LC3 and the DyLight™ 800 4X PEG-conjugated secondary antibody were from Cell Signaling Technology (Beverly, MA, USA).

Isolation and Culture of Chondrocytes

One-week-old Sprague-Dawley mice were euthanized and primary chondrocytes were isolated from knee joint cartilage as described in the literature [12]. Primary chondrocytes were digested with 2 mg/mL Col II for 4 h and cultured in DMEM/F-12 containing 1% penicillin/streptomycin (Solarbio, Beijing, China) and 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA) in an incubator with 5% CO2 at 37°C. Second-generation cells were used for all experiments. All experimental procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Kunming Medical University.

In vitro Treatment of Chondrocytes

Following previous reports [12, 23], chondrocytes were seeded in 96-well plates (5 × 103 cells/well) and divided into IL-1β, IL-1β+PG545, and IL-1β+PG545+3-MA groups. Cells in the IL-1β group and IL-1β+PG545 group were pre-treated with 50 μm LY294002 in some wells for 1 h to block the PI3K/Akt pathway. Then, 10 ng/mL IL-1β was treated for 24 h to establish the chondrocyte injury model. Then, 10 μg/mL PG545 (or the same volume of phosphate-buffered saline [PBS]) was cultured for 4 h, followed by 5 mm 3-MA (or the same volume of PBS) for 2 h. Untreated cells served as controls. Chondrocytes with ATG5 gene knocked out were generated by lentivirus infection for the next experiment.

Cell Viability Test

Cell Counting Kit-8 (CCK-8, Dojindo Co., Kumamoto, Japan) was used to evaluate the cytotoxicity of various reagents to chondrocytes. Chondrocytes were seeded in 96-well plates (100 μL DMEM/F12 medium per well, containing approximately 1 × 104 cells). 10 μL CCK-8 solution was added to each well for 2 h at 37°C. Finally, the absorbance of each well was measured using a microplate reader (Thermo, USA) at a wavelength of 450 nm to determine the proliferative potential and viability of the chondrocytes.

Cell Apoptosis

Treated chondrocytes were collected and interacted with Annexin V-binding buffer (Sigma, St. Louis, MO, USA) and stained with Annexin V-FITC and propyl iodide (PI). Cells were analyzed by flow cytometry (BD Bioscience, Fullerton, CA, USA), and apoptosis rate was expressed as percentage of (Annexin V-FITC+PI) cells.

Determination of Malondialdehyde, Superoxide Dismutase, and Catalase

First, the treated chondrocytes were disrupted with Western and IP cell lysis buffer (Beyotime, Shanghai, China) and the proteins were collected for further experiments. The values of malondialdehyde (MDA), superoxide dismutase (SOD) activity, and catalase (CAT) activity were determined by thiobarbituric acid, xanthine oxidase, and ammonium molybdate spectrophotometry method. The OD values of MDA, SOD, and CAT activities were measured at 530, 450, and 570 nm, respectively, using a 722 N spectrophotometer (Shanghai Scientific Instruments Co. Ltd., China) and then calculated.

Fluorescein Diacetate/Propidium Iodide Staining

Chondrocytes were incubated with 2 μm fluorescein diacetate (Invitrogen Life Technologies, CA, USA) and 2 μg/L propidium iodide (Invitrogen, CA, USA) for 5 min at 37°C in the dark. The ratio of live and dead cells was determined using a confocal laser scanning microscope (LSCM; Nikon America, Inc., Melville, NY, USA).

Immunofluorescence

Chondrocytes were rinsed with PBS and fixed in 4% paraformaldehyde for 15 min. After permeabilization in 0.4% Triton X-100 for 15 min and blocking with 5% goat serum for 30 min, the chondrocytes were incubated overnight at 4°C with antibody LC3 II and antibody P62. After washing with PBS 3 times, the cells were cultured with the secondary antibody at 37°C for 1.5 h and 4′,6-diamino-2-phenylindole for 3 min. Finally, the samples were visualized with a fluorescence microscope (Olympus Inc., Tokyo, Japan), and the fluorescence intensity was evaluated with ImageJ version 2.1 software (NIH, Bethesda, MD, USA).

Measurement of Autophagic Flux

The first generation of chondrocytes was cultured overnight in 6-well plates and infected in serum-free medium with a carefully designed RFP-GFP-LC3 lentivirus with an infection multiplication (MOI) of 50. After 12 h, the medium was replaced with fresh medium. Cells were incubated for 24 h and then treated with IL-1β, PG545, and/or 3-MA. Samples were examined with LSCM (Nikon America Inc., Melville, NY, USA) and autophagy flux was measured by manually counting RFP and GFP puncta in at least 50 cells per sample.

Western Blot

Proteins were extracted from the chondrocytes of each group using RIPA lysis buffer (Thermo Fisher Scientific, MA, USA), which contains protease inhibitors. Equal amounts of protein (60 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Microwell, Billerica MA, USA), and sealed for 1 h. The primary antibodies LC3, Beclin-1, COX-2, MMP13, Col II, aggrecan, P62, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, and GADPH were then used for sample blotting at 4°C. Wash 3 times with TBST and then assay with the appropriate DyLight™ 800 4X peg-conjugated secondary antibody (1:10,000) for 1 h. The chemiluminescence signals of the protein bands were visualized using the Gel Doc 2000 imager (Bio-Rad, USA), and protein levels were quantified using ImageJ software (NIH).

Quantitative Real-Time Polymerase Chain Reaction

Chondrocyte RNA was isolated with TRIzol (Invitrogen, MA, USA), purified with the RNeasy mini kit (QIAGEN, Valencia, CA, USA), and reverse transcribed with the Transcription First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). SYBR Green, reverse transcripts, and gene primers were mixed for quantitative real-time polymerase chain reaction (qRT-PCR) on the Applied Biosystems 7500 Real-Time Cycler (Applied Biosystems, CA, USA). PCR conditions were as follows: 95°C for 10 min, 95°C for 15 s, 60°C for 1 min, with 40 cycles to establish the standard melting curve. Gene expression was determined using the 2−ΔΔCT method with GADPH as an internal reference. The primer pairs of the target genes are listed in Table 1.

Table 1.

Primer sequences for RT-PCR

GeneForward primer (5′ to 3′)Reverse primer (5′ to 3′)
Collagen Ⅱ ATT​GCC​TAC​CTG​GAC​GAA​GC CAA​CCC​TCT​GAT​GGG​TCT​CG 
Aggrecan CCT​TGG​TGC​CCA​CAT​TGA​GT CCT​CCA​CGT​GTT​CCC​ATT​CA 
MMP13 ACC​ATC​CTG​TGA​CTC​TTG​CG TTC​ACC​CAC​ATC​AGG​CAC​TC 
COX-2 GTA​CAA​GCA​GTG​GCA​AAG​GC TCA​GCA​ACC​GTT​TCT​CAC​CT 
LC3 Ⅱ GTG​TTG​TGT​GCT​GCT​GAG​TG ACA​AGA​AGC​GGC​CAG​ATA​CC 
P62 GTG​CTC​ATC​TCG​ACC​TAG​CC GAG​ATC​GCG​AGT​CCT​CTT​CG 
Beclin-1 TTC​CGT​ACA​GGT​GAG​TGT​GC ACC​ATC​AAC​GCC​ATG​TGA​CT 
GAPDH AGG​ACC​AGG​TTG​TCT​CCT​GT GAG​GGC​ACC​AAA​CCT​TCA​GT 
GeneForward primer (5′ to 3′)Reverse primer (5′ to 3′)
Collagen Ⅱ ATT​GCC​TAC​CTG​GAC​GAA​GC CAA​CCC​TCT​GAT​GGG​TCT​CG 
Aggrecan CCT​TGG​TGC​CCA​CAT​TGA​GT CCT​CCA​CGT​GTT​CCC​ATT​CA 
MMP13 ACC​ATC​CTG​TGA​CTC​TTG​CG TTC​ACC​CAC​ATC​AGG​CAC​TC 
COX-2 GTA​CAA​GCA​GTG​GCA​AAG​GC TCA​GCA​ACC​GTT​TCT​CAC​CT 
LC3 Ⅱ GTG​TTG​TGT​GCT​GCT​GAG​TG ACA​AGA​AGC​GGC​CAG​ATA​CC 
P62 GTG​CTC​ATC​TCG​ACC​TAG​CC GAG​ATC​GCG​AGT​CCT​CTT​CG 
Beclin-1 TTC​CGT​ACA​GGT​GAG​TGT​GC ACC​ATC​AAC​GCC​ATG​TGA​CT 
GAPDH AGG​ACC​AGG​TTG​TCT​CCT​GT GAG​GGC​ACC​AAA​CCT​TCA​GT 

Preparation of Animal Model

Sprague-Dawley mice were obtained from the Kunming Medical University Laboratory Animal Centre. All mice were kept on a 12-h light and 12-h dark cycle, had free access to food and water, and were euthanized according to AVMA guidelines for euthanasia of animals. A mouse model of OA was constructed by partial medial meniscectomy (PMM). Briefly, mice were anaesthetized by intraperitoneal injection of 2% (w/v) pentobarbital (40 mg/kg); the right knee capsule was transected within the patellar tendon, and the medial meniscus was transected with microsurgical scissors. Thirty 10-week-old male mice were randomly divided into a control group, a PMM group, and a PMM+PG545 group (10 mice in each group). In the control group, joint incision was performed without removing the medial meniscal ligament. The mice in the control group and the PMM group were injected intraperitoneally with normal saline daily, and the mice in the PMM+PG545 group were injected intraperitoneally with PG545 in normal saline at a dose of 20 mg/kg/d daily. At 8 weeks after surgery, the mice were sacrificed and the knee joints were harvested for histological evaluation.

Histopathological Analysis

Mouse joint preparations were sliced and stained with Safranin O‐fast green staining and hematoxylin-eosin (HE). Experienced histological investigators assessed the extent of cartilage degeneration under the microscope in a blinded manner.

Immunohistochemical Analysis

Mouse preparations were embedded in paraffin, cut into slices, and treated with 3% hydrogen peroxide for 15 min. The sections were incubated with 0.4% pepsin (Biotech, Shanghai, China) in 5 mm HCl for 20 min, followed by 5% bovine serum albumin for 30 min. The sections were then incubated with the primary antibody (MMP13 [1:100], LC3 [1:100]) at 4°C overnight and finally with the enzyme-linked secondary antibody for 1 h. The positive cell rate was evaluated quantitatively.

Statistical Analysis

Data are presented as mean ± standard deviation. Differences were assessed using Student’s test, one-way ANOVA, and multi-factor analysis of variance with SPSS 22.0 (SPSS, Inc., USA). p < 0.05 was the threshold of significance. All experiments were repeated at least 3 times with similar results.

The Effects of PG545 on the Viability of Chondrocytes with IL-1β

We used the CCK-8 assay to evaluate the effects of PG545 and IL-1β on chondrocyte viability. As shown in Figure 1a, IL-1β significantly inhibited cell viability (p < 0.001), whereas treatment with PG545 for 4 h reversed inhibition (p < 0.001). In addition, 3-MA attenuated the protective effect of PG545 on chondrocytes (p < 0.05).

Fig. 1.

Protective effects of PG545 on chondrocytes induced by interleukin (IL)-1β. a The effects of PG545, IL-1β, and 3-MA on chondrocyte viability were assessed using the Cell Counting Kit-8 (CCK-8) assay. b The effects of PG545, IL-1β, and 3-MA on the apoptosis rate of chondrocytes were determined by flow cytometry. c The effects of PG545, IL-1β, and 3-MA on the expression levels of cleaved-caspase 3 and cleaved-PARP were determined by Western blot. d The effects of PG545, IL-1β, and 3-MA on chondrocyte survival/death ratio were determined by fluorescein diacetate/propidium iodide (FDA/PI) staining. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n = 3.

Fig. 1.

Protective effects of PG545 on chondrocytes induced by interleukin (IL)-1β. a The effects of PG545, IL-1β, and 3-MA on chondrocyte viability were assessed using the Cell Counting Kit-8 (CCK-8) assay. b The effects of PG545, IL-1β, and 3-MA on the apoptosis rate of chondrocytes were determined by flow cytometry. c The effects of PG545, IL-1β, and 3-MA on the expression levels of cleaved-caspase 3 and cleaved-PARP were determined by Western blot. d The effects of PG545, IL-1β, and 3-MA on chondrocyte survival/death ratio were determined by fluorescein diacetate/propidium iodide (FDA/PI) staining. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n = 3.

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The Attenuating Effect of PG545 on Chondrocyte Apoptosis

Flow cytometry analysis showed that PG545 treatment attenuated IL-1β-induced chondrocyte apoptosis. However, the therapeutic effect of PG545 was attenuated by 3-MA (Fig. 1b). The expression levels of cleaved-caspase 3 and cleaved-PARP in the IL-1β group were significantly higher than that in the control group and significantly lower than that in the IL-1β+PG545 group (Fig. 1c).

The Protective Effect of PG545 on Chondrocytes

Consistent with the chondrocyte proliferation results, live/dead cell assays based on fluorescein diacetate/propidium iodide staining showed that PG545 treatment decreased the number of dead cells (red) compared with cells treated with IL-1β alone, whereas the percentage of viable cells (green) increased (Fig. 1d). The results of the IL-1β+PG545+3-MA group indicated that the protective effect of PG545 on chondrocytes was related to the promotion of autophagy.

The Effects of PG545 on the Expression of Cartilage-Specific Markers

Using WB and qRT-PCR, we examined the effects of PG545 on ECM metabolism in chondrocytes. As shown in Figure 2a and b, IL-1β treatment significantly decreased the synthesis of cartilage-specific markers such as Col II and aggrecan protein but increased the expression of inflammatory markers MMP13 and COX-2. However, the deleterious effects induced by IL-1β were reversed by PG545 treatment. Interestingly, we observed that the chondroprotective effects of PG545 were partially abrogated by autophagy inhibitor 3-MA (Fig. 2a, b), suggesting that the chondroprotective effects of PG545 are partially mediated by autophagy. In addition, PG545 also plays a cartilage-protective role by promoting ECM synthesis and anti-inflammatory effects.

Fig. 2.

PG545 alleviated interleukin (IL)-1β-induced extracellular matrix (ECM) degradation and inflammation in chondrocytes. Western Blot (a) and quantitative real-time polymerase chain reaction (qRT-PCR) (b) were used to detect collagenase type 2 (Col II), aggrecan, COX-2, and matrix metalloproteinase 13 (MMP13). Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3.

Fig. 2.

PG545 alleviated interleukin (IL)-1β-induced extracellular matrix (ECM) degradation and inflammation in chondrocytes. Western Blot (a) and quantitative real-time polymerase chain reaction (qRT-PCR) (b) were used to detect collagenase type 2 (Col II), aggrecan, COX-2, and matrix metalloproteinase 13 (MMP13). Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3.

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The Antioxidant Effect of PG545 on the Expression of MDA, SOD, and CAT

As shown in Figure 3a–c, IL-1β stimulation significantly increased CAT activity (p < 0.001) and decreased SOD activity (p < 0.001). PG545 treatment could counteract the above effects (p < 0.001) and significantly inhibit IL-1β-induced elevation of MDA levels (p < 0.001).

Fig. 3.

The antioxidant effects of PG545 on malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) expression. Values of CAT activity (a), SOD activity (b), and MDA (c) were calculated according to kit instructions. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; **p < 0.01, ***p < 0.001; n = 3.

Fig. 3.

The antioxidant effects of PG545 on malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) expression. Values of CAT activity (a), SOD activity (b), and MDA (c) were calculated according to kit instructions. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; **p < 0.01, ***p < 0.001; n = 3.

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PG545 Induced Activation of Autophagic Flux in Chondrocytes

To investigate the possible involvement of autophagy in the protective effects of PG545 on IL-1β-induced chondrocyte damage, cells were subjected to WB and qRT-PCR to detect autophagy markers. As shown in Figure 4a and b, the expression levels of LC3 II and Beclin-1 were significantly decreased after 24 h of IL-1β alone, whereas the expression level of P62 was increased. As we suspected, all these effects were reversed after PG545 treatment, which was attenuated by 3-MA. A fluorescently labeled anti-LC3 II antibody, a marker for autophagosomes, showed that PG545 treatment increased the fluorescence intensity of chondrocytes compared with IL-1β alone. However, immunofluorescence analysis of P62 expression showed the opposite trend (Fig. 4c, d, e).

Fig. 4.

The effect of PG545 on autophagy flux was determined. Western Blot (a) and qRT-PCR (b) were used to detect LC3 II, P62, and Beclin-1. Immunofluorescence detection was used to detect the effects of PG545, IL-1β, 3-MA, and LY294002 on the expression levels of LC3 II (c) and P62 (d) in chondrocytes (green signal represents LC3 II, red signal represents P62). e The average fluorescence intensity (AU) of LC3 II and P62 was analyzed. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3.

Fig. 4.

The effect of PG545 on autophagy flux was determined. Western Blot (a) and qRT-PCR (b) were used to detect LC3 II, P62, and Beclin-1. Immunofluorescence detection was used to detect the effects of PG545, IL-1β, 3-MA, and LY294002 on the expression levels of LC3 II (c) and P62 (d) in chondrocytes (green signal represents LC3 II, red signal represents P62). e The average fluorescence intensity (AU) of LC3 II and P62 was analyzed. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3.

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The immunofluorescence results confirmed the role of autophagy in PG545 treatment of chondrocytes. In addition to the statically high expression of autophagy markers and autophagosomes, there is further evidence to support this conclusion. RFP-GFP-LC3 was transduced into chondrocytes and incubated for 24 h. Under the fluorescence microscope, we found that the fluorescence of RFP-LC3 became increasingly clear (p < 0.001) with the increase of PG545 concentration. Treatment with 10 µg/mlPG545 increased RFP-LC3 in the cytoplasm of IL-1β-induced chondrocytes by 618%. 3-MA reduced the RFP-LC3 increase to 338% (Fig. 5). Taken together, these data confirm that PG545 effectively increases autophagy and autophagic flux and can be partially inhibited by 3-MA.

Fig. 5.

Autophagy flux was detected after transfection of cells with adenovirus tandem red fluorescent protein-green fluorescent protein-light chain 3 (RFP-GFP-LC3). The green and red dots represent the GFP and mRFP, respectively. The yellow dots (fusion of red and green dots) represent autophagosomes and the red dots represent auto-phagolysosomes. ***p < 0.001, ****p < 0.0001; n = 3.

Fig. 5.

Autophagy flux was detected after transfection of cells with adenovirus tandem red fluorescent protein-green fluorescent protein-light chain 3 (RFP-GFP-LC3). The green and red dots represent the GFP and mRFP, respectively. The yellow dots (fusion of red and green dots) represent autophagosomes and the red dots represent auto-phagolysosomes. ***p < 0.001, ****p < 0.0001; n = 3.

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However, chondrocytes in which ATG5 was knocked down provided a different result. Although the expression levels of cleaved-caspase 3 and cleaved-PARP in ATG5 knockdown chondrocytes were similarly to those in normal chondrocytes, the results of flow cytometry were not significantly affected by PG545 and 3-MA (Fig. 6a, b). The expression levels of autophagy-related factors such as LC3 II, P62, and Beclin-1 showed no statistical difference between the IL-1β group, the IL-1β+PG545 group, and the IL-1β+PG545+3-MA group (Fig. 6c).

Fig. 6.

The effects of PG545 on ATG5 knockdown chondrocytes induced by interleukin (IL)-1β. a The effects of PG545, IL-1β, and 3-MA on the apoptosis rate of ATG5 knockdown chondrocytes were determined by flow cytometry. b The effects of PG545, IL-1β, and 3-MA on the expression levels of cleaved-caspase 3 and cleaved-PARP in ATG5 knockdown chondrocytes were determined by Western blot. c The effects of PG545, IL-1β, and 3-MA on the expression levels of LC3 II, P62, and Beclin-1 in ATG5 knockdown chondrocytes were determined by Western blot. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance; n = 3.

Fig. 6.

The effects of PG545 on ATG5 knockdown chondrocytes induced by interleukin (IL)-1β. a The effects of PG545, IL-1β, and 3-MA on the apoptosis rate of ATG5 knockdown chondrocytes were determined by flow cytometry. b The effects of PG545, IL-1β, and 3-MA on the expression levels of cleaved-caspase 3 and cleaved-PARP in ATG5 knockdown chondrocytes were determined by Western blot. c The effects of PG545, IL-1β, and 3-MA on the expression levels of LC3 II, P62, and Beclin-1 in ATG5 knockdown chondrocytes were determined by Western blot. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance; n = 3.

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Activation of Autophagy by PG545 Is Associated with the PI3K/Akt/mTOR Pathway

To further explore the molecular mechanism of PG545 in preventing IL-1β-induced chondrocyte damage and impairing autophagy, we investigated the involvement of the PI3K/Akt/mTOR pathway through WB. As shown in Figure 7a and b, PG545 reduced the phosphorylation levels of PI3K, Akt, and mTOR induced by IL-1β, whereas 3-MA attenuated the effect of PG545. These results suggest a possible mechanism by which PG545 increases autophagy levels and autophagy flux in OA by inhibiting the PI3K/Akt/mTOR pathway. Immunofluorescence assay of LC3 II and P62 was used to verify the relationship between the autophagy-promoting effect of PG545 and the PI3K/Akt/mTOR pathway. After pretreatment with the PI3K inhibitor LY294002, the autophagy-promoting effect of PG545 was significantly attenuated (p < 0.001) (Fig. 4c–e).

Fig. 7.

Exploration of the signaling pathway in vitro. a Western blot was used to detect PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR. b Expression of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR was analyzed. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3.

Fig. 7.

Exploration of the signaling pathway in vitro. a Western blot was used to detect PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR. b Expression of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR was analyzed. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3.

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Therapeutic Effects of PG545 on OA in vivo

The OA model in mice was established by PMM. After 8 weeks, PMM mice were injected intraperitoneally with PG545 (PG545 group) or normal saline (PMM group) once daily, and joint morphology was analyzed by Safranin O‐fast green staining, HE staining, and immunohistochemistry. Both Safranin O‐fast green staining and HE staining showed that there were significantly fewer chondrocytes in the PMM group than in the control group, and PG545 therapy partially restored these conditions (Fig. 8a, b). Immunohistochemical results showed that PG545 treatment was effective in improving the expression of LC3 II. The expression of MMP13 was slightly decreased in the PG545 treatment group, but the difference was not statistically significant. These results suggest that PG545 improves OA and reduces ECM degradation by promoting chondrocyte autophagy in OA mice (Fig. 8c, d).

Fig. 8.

Therapeutic effects of PG545 on OA in vivo. a Safranin O‐fast green staining was used to analyze the effects of PMM and PG545 on mouse articular cartilage. b HE staining was used to analyze the effects of PMM and PG545 on mouse articular cartilage. The effects of PMM and PG545 on the expression levels of LC3 II (c) and MMP13 (d) in mouse articular cartilage were determined by immunohistochemical analysis. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance; n = 5.

Fig. 8.

Therapeutic effects of PG545 on OA in vivo. a Safranin O‐fast green staining was used to analyze the effects of PMM and PG545 on mouse articular cartilage. b HE staining was used to analyze the effects of PMM and PG545 on mouse articular cartilage. The effects of PMM and PG545 on the expression levels of LC3 II (c) and MMP13 (d) in mouse articular cartilage were determined by immunohistochemical analysis. Data are presented as mean ± SD. Significant differences between treatment and control groups were determined by Student’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance; n = 5.

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In human OA cartilage, heparanase is both expressed and active, and treatment with ex vivo heparanase induces catabolic responses in human articular chondrocytes [19]. Animal studies have shown that suppression of HS expression in articular cartilage reduces the rate of OA development after joint instability, but the underlying processes remained unclear [24, 25]. In this study, we investigated the mechanism underlying the alleviation of OA by the heparanase inhibitor (PG545).

The inflammatory cytokine IL-1β promotes the synthesis and release of numerous inflammatory mediators, including COX-2, all of which play a role in chondrocyte dysfunction in progressive OA [26‒28]. Evidence suggests that blocking the ability of IL-1β to stimulate the production of inflammatory mediators may be beneficial for OA patients [29, 30]. In our study, treatment of chondrocytes with IL-1β resulted in a decrease in cell viability and an increase in the expression of COX-2, PG545 was able to attenuate these effects, whereas 3-MA was able to counteract them. In view of the above results, it appears that the anti-inflammatory effect of PG545 associated with autophagy is mediated by inhibition of COX-2.

Proteoglycans and collagens keep the articular cartilage matrix stable, and collagen II is primarily responsible for the regeneration of injured cartilage [31‒34]. MMPs play a role in the process of cartilage development, postnatal growth, and degradation [35]. The breakdown of collagen II in articular cartilage is highly dependent on MMP-13 [36, 37]. We discovered that treatment with PG545 enhanced collagen II and aggrecan and reduced IL1β-induced MMP13 overexpression in chondrocytes, and we found the same thing in an OA mice model. Treatment with PG545 seems to improve proteoglycan production and decrease cartilage injury by decreasing MMP13, according to these findings. In addition, oxidative stress plays an important role in the physiology and pathophysiology of OA, and the enzymatic cellular antioxidant defense system has a variety of enzymes, including SOD, CAT, and MDA [38]. Our experiment showed a high level of MDA and a low level of SOD after treatment with IL-1β alone in chondrocytes, in agreement with the results in OA chondrocytes [39‒41]. Serum levels CAT are elevated in OA patients due to the compensatory protective effect of CAT, although the enzyme has an oxidative effect [42, 43]. Our experiments confirm this and show that IL-1β can increase the level of chondrocytes CAT. Our results further suggest that PG545 has the potential to counteract IL-β-induced oxidative stress dysfunction.

Cell death of chondrocytes in OA has been associated with problems in tissue repair and maintenance. It’s important to note that apoptosis and autophagy are both forms of programmed cell death. Significantly increased expression of apoptosis markers such as caspase 3 and PARP has been found in OA cartilage [44]. The results of our study showed that IL-1β-stimulated chondrocytes and the knee joints of Sprague-Dawley mice with OA had increased expression of cleaved-caspase 3 and cleaved-PARP. Exposure to PG545 resulted in a substantial decrease in the above apoptotic markers and cells, suggesting that PG545 supports the regeneration of damaged cartilage by suppressing chondrocyte apoptosis. Autophagy is a self-degrading, non-apoptotic cell death process that is important for cartilage homeostasis [45]. There is emerging evidence suggesting that early activation of OA may be protective and represents an adaptive response to cellular stress, with the goal of enhancing cellular survival [46‒48]. In the present work, we discovered that PG545 had a counteracting effect on the increased expression of apoptotic cell death markers and the decreased activation of autophagy indicators (LC3 II, P62, and Beclin-1). The PG545 induced decrease in cleaved-caspase 3 and cleaved-PARP was abrogated by the autophagy inhibitor 3-MA. Moreover, knocking down of ATG5 in chondrocytes rendered the effects of IL-1β on chondrocyte death and autophagy indicators (LC3 II, P62, and Beclin-1) insensitive to PG545. Our results indicate that the antiapoptotic effect of PG545 prevents further cartilage injury and degradation in OA, and that this effect is closely related to autophagy.

It is well known that the PI3K/AKT/mTOR pathway controls many different cellular functions such as apoptosis, transcription, translation, metabolism, angiogenesis, and the cell cycle [49]. Many studies found that the expression of p-PI3K, p-AKT, and p-mTOR was significantly increased in IL-1β-induced chondrocytes [50, 51]. Similar results were observed in our studies; however, the ability of PG545 to reverse the elevated levels of p-PI3K, p-AKT, and p-mTOR was lower after administration of 3-MA. Studies found that curcumin has been shown to reduce cardiomyocyte pyroptosis and autophagy in response to doxorubicin, and this effect is dependent on Akt/mTOR [52]. Ablation of Akt2 in renal and skeletal muscle was accompanied by marked apoptosis and overt autophagy, the effects of which were greatly ameliorated or reversed by treatment with the autophagy inducer trehalos [53]. These data suggest a close relationship between Akt and autophagy. LY294002 is the most commonly used PI3K inhibitor. Immunofluorescence analysis showed that LY294002 administration reduced autophagy activity and that LY294002 could attenuate the ability of PG545 to activate autophagy. These results suggest that PI3K/AKT/mTOR signaling is a possible regulator of the chondroprotective effect of PG545, and that the chondroprotective effect of PG545 is also closely associated with the regulation of autophagy.

Histological analysis and staining of cartilage tissue with Safranin O‐fast green show that PG545 halts the degeneration of cartilage and reduces the progression of OA. Both cartilage surface area and cartilage thickness were repaired more effectively in PG545-treated mice. These results suggest that PG545 was effective in reducing OA damage. Together, our findings provide novel insights and treatment methods for OA by suggesting that PG545 has positive effects on osteoarthritic chondrocytes and may relieve OA by activating autophagy and modulating PI3K/AKT/mTOR signaling in cartilage.

PG545’s chondroprotective impact in an in vitro model of IL-1β-induced cartilage damage is linked to its ability to suppress the PI3K/AKT/mTOR signaling pathway and activate autophagy, indicating that it may provide a potential treatment strategy for OA.

The Laboratory Animal Welfare and Ethics Committee of Kunming Medical University approved and supervised this study (Animal Ethical Statement Number: KMMUAWEC2020011701).

The authors declare that they have no conflict of interest.

Science and Technology Project of Science and Technology Department of Yunnan Province, (202001AY070001-207).

Peiyu Guo and Hua Li conducted the experiments, analyzed the data, and wrote the manuscript; Xuming Wang and Xingguo Li conducted the research, collected, and analyzed the data; Xi Li designed the research and reviewed the manuscript. Both authors read and agreed with the published version of the manuscript.

Additional Information

Joint first authors: Peiyu Guo and Hua Li contributed equally to the work.

All data generated or analyzed as part of this study are included in this article. Further inquiries can be directed to the corresponding author.

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