2.1 Cell culture and reagents

Recombinant CCN3 protein and recombinant human IL-1β were purchased from R&D systems. SW1353 human chondrosarcoma cell lines were obtained from the Institute of Life Science Cell Culture Centre.

Rat chondrocytes were obtained from the knee joints of 1-week-old Sprague Dawley rats by sequential enzymatic digestion, as previously described.20 Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10% foetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C, in a humidified atmosphere containing 5% CO2.

2.2 Patients selection and informed consent

Osteoarthritis human articular cartilage specimens were collected from patients (n = 6) with knee OA undergoing total knee arthroplasty. Normal human articular cartilage specimens were collected from patients (n = 6) without knee arthropathy undergoing lower limb amputation because of trauma. X-ray and Kellgren-Lawrence Grading Scale were used here to assess the stage of OA. Informed consent forms were signed by all patients. Cartilage tissues were cut into 5-μm sagittal sections and embedded in paraffin for histological analysis. All experimental procedures were approved by Ethical Committee of Tongji Medical College, Huazhong University of Science and Technology.

2.3 Animal model

Twelve 8-week-old male Sprague Dawley rats were purchased from the Animal Care and Use Committee of Tongji Medical College. A rat OA model was conducted by surgical destabilization of the anterior cruciate ligament transection (ACLT) and medial meniscus (DMM) of the right knee joint. Rats were divided to a sham group and an ACLT + DMM group. The joint capsule of the sham operation group was opened without transaction of the ACLT and DMM. Twelve weeks after surgery, the rats were killed and the joints were collected for histological evaluation. All the animal procedures were approved and under supervision by the Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.

2.4 Lentivirus transfection

CCN3 was overexpressed via transfection of lenti-CCN3 (GeneChem). Cells were seeded in 6-wells plate and were transfected with lenti-CCN3 or lenti-NC at a confluence of 50%. Fourteen hours after incubation, the medium was changed and cells were cultured for further experiments. The transfection efficacy of lenti-CCN3 was detected by western blotting.

2.5 Western blot analysis

Protein concentrations in the supernatants were measured using a BCA protein assay kit (Boster). Then, equal amounts of protein lysate were loaded on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% BSA at 37°C for 60 minutes, and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used in this study: MMP1 (#10371-2-AP; 1:1500), MMP3 (#17873-1-AP; 1:1000), HMGB1 (#10829-1-AP; 1:1000), TLR4 (#19811-1-AP; 1:1000) and PI3K (#20584-1-AP; 1:1000) from Proteintech Group; Collagen2 (#ab34712; 1:1000), MMP13 (#ab39012; 1:1000), iNOS (#ab3523; 1:500), Aggrecan (#ab3778; 1:1000) from Abcam (Cambridge, MA); CCN3 (#8767; 1:1000), P-AKT (#4060; 1:1000), AKT (#4685; 1:1000), P-mTOR (#5536; 1:1000), mTOR (#2983; 1:1000), P-PI3K (#4228; 1:1000), Atg5 (#12994; 1:1000), Beclin1 (#3495; 1:1000) and LC3-I/II (#12741; 1:1000) from Cell Signalling Technology; ADAMTS5 (#BA3020; 1:500) and GAPDH (#BM3876; 1:500) from Boster. Next, the blots were incubated with goat anti-rabbit or goat anti-mouse IgG secondary antibodies (Boster, 1;5000) for 1 hour at 37°C. Protein bands were then detected using a super ECL chemiluminescence solution (Boster). The protein expression was normalized to GAPDH and quantified using ImageJ software.

2.6 Histochemistry and immunohistochemistry

Human and rat articular cartilages sections were stained with hematoxylin and eosin (H&E) as well as Safranin-O-Fast Green. The paraffin-embedded tissue sections were firstly deparaffinized and blocked with 5% BSA. Next, primary antibodies against CCN3 (1:100, 55449-1-AP, Proeteintech) and HMGB1 (1:200, 10829-1-AP, Proteintech) were applied and incubated overnight at 4°C. Finally, slides were incubated with conjugated secondary antibodies for 1 hour at 37°C. Images of the histology slides were captured under a light microscope, and the number of CCN3 positive cells was quantified.

2.7 Immunofluorescence

The treated cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes and blocked with 1% BSA for 1 hour at 37°C. Primary antibodies against HMGB1 (1:100, 10829-1-AP, Proteintech) were applied and incubated overnight at 4°C. Next, cells were incubated with CY3-goat anti-rabbit IgG (1:100, BA1032, Boster) for 1 hour in dark, and diamidino-2-phenylindole (DAPI) was added for 10 minutes to stain the nuclei. Protein expression was quantified by integrated optical density (IOD) with the Image-J image analysis system.

2.8 Statistical analysis

All data are displayed as means ± standard deviation (SD). Student's t test or one-way analysis of variance (ANOVA) were utilized to determine the statistical significance among different groups. P ≤ .05 was deemed statistically significant.

3.1 CCN3 expression is decreased in human and rat knee OA articular cartilage

To determine changes in CCN3 levels in OA, western blot and immunohistochemistry were applied to human and rat knee OA articular cartilage tissue and chondrocytes. In Figure 1A, Safranin-O-Fast Green staining showed significant cartilage erosion in OA cartilage. According to immunohistochemistry results, a decrease in CCN3 expression was observed in human OA cartilage. After SW1353 cells were treated with IL-1β for 24 hours, the CCN3 expression was decreased according to western blot analysis (Figure 1C). Similar results were observed in rat cartilage chondrocytes as well as in the sham and rat OA cartilage tissues (Figure 2A-C). We observed decreased expression of CCN3 in rat OA cartilage compared with that in sham group. CCN3 expression was decreased after treatment with IL-1β for 24 hours in rat chondrocytes.

3.2 CCN3 ameliorates the IL-1β-induced catabolism, cartilage matrix degradation and promotes autophagy process in vitro

The overproduction of catabolic factors such as MMPs, ADAMTS5, iNOS lead to the degradation of extracellular matrix. During OA, autophagy is also increased to regulate changes in OA-like gene expression. To determine the role of CCN3 in OA, SW1353 cell were treated with recombinant CCN3 (30, 60, 120 ng/mL) and IL-1β for 24 hours. As shown in Figure 3A,B, western blot analysis indicated that IL-1β increased MMP1, MMP3, MMP13, ADAMTS5, iNOS accumulation and decreased collagen 2 and aggrecan expression in a dose-dependent manner, and 10 ng/mL IL-1β was the optimal concentration to simulate the OA-like changes in vitro. However, recombinant CCN3 (30, 60 ng/mL) reversed induced IL-1β changes in MMPs, ADAMTS5, iNOS, collagen 2 and aggrecan expression (Figure 3C,D). We also observed that recombinant CCN3 could reverse the decreased expression of autophagy markers such as Atg5, Beclin1 and LC3-II induced by IL-1β (Figure 3E,F).

3.3 HMGB1 is negatively correlated with CCN3 in IL-1β induced osteoarthritis responses

Previous studies have demonstrated that HMGB1 proteins are secreted abundantly in OA. High-mobility group box 1 is not only an important inflammatory mediator of OA but also a potent regulator of autophagy in chondrocytes. Thus, we have been suggested that CCN3 is associated with HMGB1 levels. As shown in Figure 4A, we observed the significant increased HMGB1 expression in human OA cartilage according to immunohistochemistry results. As shown in Figure 4B-E, western blot analysis revealed that HMGB1 is negatively correlated with CCN3 in IL-1β-treated SW1353 cells in both time- and dose-dependent manners. TLR4, one of the main HMGB1 receptors, was also changed with increased HMGB1 levels.

3.4 HMGB1 is involved in the protective effect of CCN3 in osteoarthritis

To determine the relationship between HMGB1 and CCN3 in osteoarthritis, western blot and immunofluorescence assays were used. As shown in Figure 5A,B, recombinant CCN3 dose-dependent decreased HMGB1 and TLR4 expression. CCN3 (30, 60 ng/mL) reversed the increased HMGB1 and TLR4 production induced by IL-1β (Figure 5C,D). Moreover, as shown in Figure 5E,F, CCN3 reduced the amount of HMGB1.

3.5 CCN3 overexpression decreases the expression of HMGB1 and reverses the IL-1β-induced MMP expression

To further investigate the relationship of CCN3 and HMGB1, CCN3 was overexpressed via transfection of lenti-CCN3. As shown in Figure 6A,B, overexpression of CCN3 could decrease the protein levels of HMGB1. Furthermore, CCN3 overexpression attenuated the increased MMP1, MMP3, MMP13 and HMGB1 expression induced by IL-1β (Figure 6C,D). Moreover, HMGB1 release induced by IL-1β could also be inhibited by CCN3 overexpression (Figure 6E,F).

3.6 CCN3 induces cartilage protection via blocking the PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR signalling pathway is central fora plethora of cellular mechanisms. In our experiment, we examined the effects of recombinant CCN3 (30, 60 ng/mL) on the activation of the PI3K/AKT/mTOR pathway. As shown in Figure 7A,B, CCN3 attenuated the increased protein levels of P-PI3K, P-AKT and P-mTOR activated by IL-1β. Moreover, we observed that CCN3 overexpression inhibited the activation of the PI3K/AKT/mTOR pathway (Figure 7C,D).