2.1 Reagents and antibodies

Arctigenin (purity > 98%) was purchased from the Tianjin Shilan Technology Company. Cell culture reagents were purchased from Gibco (Life Technologies Corp.). Cell Counting Kit-8 (CCK-8) was bought from DOJINDO (Kumano, Japan). Recombinant human IL-1β protein was supplied by Novoprotein. Type II collagenase, sodium carboxymethyl cellulose (CMC-Na) and dimethyl sulphoxide (DMSO) were received from Solarbio. The primary antibody against Lamin B and GAPDH was obtained from Abcam, iNOS antibodies were obtained from Sigma-Aldrich, and goat anti-rabbit and antimouse IgG-HRP were from Bioworld (OH). The anti-COX-2, anti-p65, anti-IκBα, anti-PI3K, anti-p-PI3K, anti-Akt and anti-p-Akt antibodies were acquired from Abcam. The second antibody (Alexa Fluor® 488-labelled goat anti-rabbit IgG) was achieved from Yeasen. The 4', 6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime.

2.2 Primary human chondrocyte culture

Following the standards of the terms of the Medical Ethical Committee of the Second Affiliated Hospital, Wenzhou Medical University (ethic cord: LCKY-2017-37), and adhering to the guidelines of the Declaration of Helsinki,45 the human articular cartilage samples with OA were collected from 10 eligible patients, and only patients undergoing the total knee arthroplasty aged between 62 and 68 years were included in the study (five men and five women). All patients signed the informed consent. The diagnosis of OA matches the classification criteria of the American College of Rheumatology (ACR).46

The hyaline cartilages of collected tissue were rinsed in PBS and cut up into pieces and subsequently digested with collagenase II (2 mg/mL) in DMEM/F12 at the temperature of 37°C for 4 hours. Afterwards, chondrocytes were seeded in a 25-cm² task at a density of 2 × 105 cells/mL in complete DMEM/F12 medium (with 10% FBS and 1% antibiotic) in a 5% CO₂ atmosphere at 37°C. To avoid the phenotype loss of chondrocytes, cells at least in two passages were employed for subsequent experiments.

2.3 Cell viability

The toxicity of ATG towards human OA chondrocytes was determined via the CCK-8 kits obeying the protocols of the manufacturer. Firstly, the chondrocytes in the second passage were cultured in 96-well plates (50 000 cells/cm²) with serum-free culture medium for 24 hours. Then, the cells were incubated with respective concentrations of ATG (5, 10 and 50 μmol/L) for the duration of 24 and 48 hours. At the appointed time, followed by rinsing in PBS thrice, the cells were disposed with 100 µL 10% of CCK-8 solution (diluted in serum-free DMEM/F12) and subsequently incubated at the temperature of 37°C for 2 hours. The medium was then collected and assessed at 450 nm by a micro-plate reader. The experiment was performed five times.

2.4 Nitrous oxide measurement and ELISA

The activity of NO in culture medium was appraised via the Griess reagent as previous descriptions did.47 According to the manufacturer's introductions, the PGE2, TNF-α, IL-6, collagen II, aggrecan, MMP13 and ADAMTS-5 level in culture medium were assessed using the ELISA kit (R&D Systems). The experiment was performed five times.

2.5 Western blotting

The total protein was gained from cells using the RIPA lysis buffer with 1% phenylmethanesulphonyl fluoride (PMSF) on the ice bath for 10 minutes, and subsequent centrifugation (15 338 g) was at 4°C for 20 minutes. Later, the concentrations of protein were detected by virtue of the BCA protein assay kit. The protein (30 mg) of the respective group was separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, followed by transferring to a polyvinylidene difluoride (PVDF) membrane (Millipore). After being blocked with 5% BSA for 2 hours, the obtained membranes were then incubated with the primary antibodies: Lamin B (1:5000), AKT (1:1000), p65 (1:1000), PI3K (1:1000), IκBα (1:1000), COX-2 (1:1000), GAPDH (1:5000), P-PI3K (1:1000), P-AKT (1:1000) and iNOS (1:1000) overnight at 4°C. At the appointed time, the membranes were treated with HRP-labelled secondary antibodies for 2 hours at indoor temperature. After rinsing in TBST for three times, the blots were visualizable by virtue of the electrochemiluminescence plus reagent (Invitrogen), and the intensity of blots was quantified with Image Lab 3.0 software (Bio-Rad).

2.6 Real-time PCR

The total RNA from the human chondrocytes was gained through TRIzol reagent (Invitrogen), and the procedures obey the manufacturer's guidelines. The first-strand complementary DNA (cDNA) was synthesized by dint of 1000 ng of total RNA and the QuantiTect Reverse Transcription Kit. As for the quantitative real-time PCR (qPCR), the reactions were acted in a 10 μL system (diluted cDNA (4.5 μL), reverse primer (0.25 μL), forward primer (0.25 μL) and SYBR (5 μL) Green Master Mix) through the CFX96 Real-Time PCR System (Bio-Rad Laboratories) and the parameters were set up as follows: 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C and 1 minutes at 60°C. The collected cycle threshold (Ct) values were normalized to the expression of GAPDH. The level of relating mRNA of related genes was measured by the 2^−△△Ct method.48 The NCBI Primer-Blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was applied to design and form the necessary primers, and the sequences are shown in Table 1.

2.7 Immunofluorescence

The cells were planted in a 25-mm glass in 6-well plate and incubated with serum-starved medium overnight. Subsequently, the cells were solely treated with IL-1β (10 ng/mL) or co-treated with ATG (50 μmol/L) and IL-1β (10 ng/mL) for 24 hours. However, the incubation time of the immunofluorescence staining of p65 with IL-1β was declined to 2 hours. The glass coverslips cultured with chondrocyte monolayers were firstly rinsed thrice in PBS. Next, cells were treated as follows: fixation in 4% paraformaldehyde at 4℃ for 15 minutes; permeabilization in PBS including 0.5% Triton X-100 for another 15 minutes at indoor temperature; treatment with 5% goat serum for 1 hour at 37°C; and incubation with primary antibodies against p65 (1:200), collagen Ⅱ (1:200) and MMP-13 (1:200) for the whole night at the temperature of 4°C. At the adjacent day, cells were exposed to Alexa Fluor® 488-labelled conjugated secondary antibodies (1:400) for 1 hour and DAPI for 1 minute successively. At last, cell samples were observed under fluorescence microscopy (Olympus Inc). The ImageJ software 2.1 (Bethesda) was applied to assess the fluorescence intensity by observers who were blinded to the experimental groups.

2.8 Molecular modelling

The structure of ATG and PI3K, downloaded from Chemicalbook (https://www.chemicalbook.com/) and RCSB PDB (https://www.rcsb.org/; PDB ID: 3LJ3), respectively, was applied for molecular docking study and reconstructed in PyMoL software (version 1.7.6). For the protein-ligand docking analysis, the AutoDockTools (version 1.5.6) was applied. The whole of the 3D interactions was generated in PyMoL software, and the 2D image was manufactured by Ligplus (version 1.4).

2.9 Mice OA models

Forty-five eight-week-old C57BL/6 male wild-type mice were acquired from SLAC Laboratory Animal Co., Ltd. The protocol for animal care and use was not only complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, but also ratified by the Animal Care and Use Committee of Wenzhou Medical University (ethic code: wydw3014-0127). The destabilization of the medial meniscus (DMM) as previously described 49 was used to establish the mouse OA model. Briefly, the mice were anaesthetized with 2% (w/v) pentobarbital (40 mg/kg, ip); under the direct visualization, the joint capsule of the right knee was exposed followed by transacting the medial to the patella tendon and the medial meniscotibial ligament with an ophthalmic micro-surgical knife. The sham mice were only received an arthrotomy without ligament cut down. After the operation, the mice were randomly divided into three groups (n = 15): sham control group, an OA group (DMM) and an OA treated with the ATG group (DMM + ATG). Mice in the DMM group received a gavage of vehicle (0.5% CMC-Na, a dietary cosolvent), while mice in the DMM + ATG group received oral administration of ATG (30 mg/kg, dissolved in CMC-Na) daily for 8 weeks. This dose of ATG was prepared by adapting the procedure used in the previous study39, 43, 50-53 and our preliminary experiments. The mice were maintained in a temperature-controlled room (20 ± 2°C) on a 12:12-h light-dark cycle and given free access to water and food. The animals were killed by cervical dislocation at 8 weeks after surgery. The whole knee joints were collected for further evaluation.

2.10 Histological assessment

The harvested joint samples were treated as follows: fixation in 4% paraformaldehyde for 1 day and subsequent decalcification in 10% EDTA solution at 4°C for 2 months. Afterwards, the samples were dried using graded ethanol, vitrification by dimethylbenzene and implanting into paraffin slabs. To assess the cartilage tissue degradation, sagittal sequential segments (7 μm thick) of full joints were attained with per joint, which owns 10 slides (70 μm thick) to select. Afterwards, the selected sections went through staining via Safranin O/Fast Green. The stained section images were captured with a microscope. The Osteoarthritis Research Society International (OARSI) (0-12) of both the medial tibial plateau and femoral condyle was summed to estimate the damage extent to the articular cartilage.54 The Likert scale consists of the following scoring grades: 0 signifies typical cartilage, 0.5 = undamaged surface and losing PG, 1 = shallow fibrillation with no cartilage loss, 2 = perpendicular fissures and surface lamina loss, 3 = perpendicular fissures/corrosion for 1/4 of the quadrant thickness of the calcified layer, 4 = lesion spreads to 1/2 of the quadrant thickness of the calcified cartilage, 5 = lesion spreads to 3/4 of the quadrant thickness of the calcified cartilage and 6 = lesion spreads to >3/4 of the calcified cartilage quadrant thickness. Using separate grades allows identification of the severe lesions and the universal extent of damage.

2.11 Statistical analysis

All tests were achieved independently fivefold. Outcomes were expressed as means ± SD The variances of two or more groups were analysed using (ANOVA) statistical test. Thereafter, Tukey's test analysed the disparities of the treatment and control groups using SPSS version 20.0. Statistical significance was present for values of P < .05.

3.1 Arctigenin did not show toxicity on chondrocyte at the dose of 0-50 μmol/L

The chemical structure of ATG is presented in Figure 1A. The cytotoxic effects of ATG (0, 5, 10, 50 and 100 μmol/L) on chondrocytes were determined by CCK-8 assay. No cytotoxic effect of ATG was found at the dose of 5-50 μmol/L (P < .01). Whereas the concentration increased to 100 μmol/L, the cell viability was decreased at both 24 hours (P = .0261) and 48 hours (P = .0062). Therefore, related lower concentration of ATG (5, 10 and 50 μmol/L) was utilized for the following experiments.

3.2 Arctigenin suppressed the IL-1β and increased PGE2, IL-6, NO, iNOS, TNF-α and COX-2 levels in chondrocytes

The efficacy of ATG on the expression of iNOS and COX-2 induced by IL-1β in human chondrocytes was detected through qRT-PCR and Western blotting. IL-1β greatly up-regulated both the mRNA and protein levels of iNOS (13.35-fold in mRNA and 3.86-fold in protein) and COX-2 (10.46-fold in mRNA and 3.15-fold in protein). However, although the ATG-treated group still showed higher levels of iNOS and COX-2 compared with untreated control (P < .01), it could intensively decrease this inflammatory reaction at least a half to the IL-1β group (Figure 2A,C,D). At the same time making a contrast with the control group, the IL-1β–induced group displayed up-regulation of IL-6 and TNF-α at both mRNA and protein levels (IL-6:13.32-fold in mRNA and 3.34-fold in protein; TNF-α: 11.41-fold in mRNA and 3.45-fold in protein). Not surprisingly, however, the adverse effects were reversed by the pre-treatment with ATG in a dose-dependent manner, and the 50 μmol/L of ATG group decreases these levels to 1.5-5.1-fold of control (Figure 2B,F). Moreover, application of ATG dramatically exhibited an inhibitory effect on the endogenous NO and PGE2 increases in a dose-dependent manner (decrease to around twofold of control), which were up-regulated by around sevenfold of control after IL-1β stimulation (Figure 2E). The results in this chapter indicate that ATG prominently inhibited inflammatory factor levels in human OA chondrocytes with IL-1β stimulation.

3.3 Arctigenin enhanced ECM synthesis and reduced ECM degradation induced by IL-1β in chondrocytes

Next, to determine the ATG on ECM synthesis and degradation under the stimulation of IL-1β, we investigate the expression of CoI II and aggrecan, with or without ATG, and the production of MMP-13 and ADAMTS-5. The up-regulation of MMP-13 (2.9-fold of control) and ADAMTS-5 (2.7-fold of control) attributed to IL-1β activation was highly inverted by ATG pre-treatment (about 1.5-fold of control in 50 μmol/L of the ATG group). On the contrary, according to Figure 3A, type II collagen and aggrecan synthesis were down-modulated by the stimulation of IL-1β (decreased to 65% and 58% of control, respectively) but improved by ATG to 89% and 80% of control, respectively. All of these protective effects of ATG were in a dosage-dependent manner (5, 10, 50 μmol/L). In addition, the results of immunofluorescence imaging of collagen II and MMP-13 were in no conflict with the ELISA results (Figure 3B,C). Taken together, these results indicate that ATG alleviates ECM degradation and promotes ECM synthesis in IL-1β–induced chondrocytes.

3.4 Arctigenin attenuated IL-1β–induced NF-κB activation in chondrocytes

To assess the role ATG played in the NF-κB signalling pathway, we firstly separated the nuclear and cytosol proteins and then utilized Western blotting to examine the protein levels of IκBα in cytoplasm and p65 in nuclei, respectively. As is shown in Figure 4A,B, IL-1β markedly promoted the degradation of IκBα (decrease to 36% of control) in the cytoplasm and more dissociated p65 was phosphorylated (up-regulated to 2.58-fold of control) and be freed to translocate from cytoplasm to nucleus. However, ATG pre-treatment highly inhibited these changes. The IκBα level was recovered to 89% of control, and the p65 level in nucleus was down to 1.13-fold of control in 50 μmol/L of the ATG group. Furthermore, we carried out experiments to measure the nuclear translocation of NF-κB p65 via the immunofluorescence staining of p65 (Figure 4C). This experiment contained three groups: (a) control group, (b) IL-1β group and (c) IL-1β + ATG (50 μmol/L) group. Making a comparison between experimental groups and control group, the result shows that the p65-active proteins were transferred from the cytoplasm to the nucleus followed by IL-1β stimulation. However, ATG pre-treatment sharply decreases this motion. In brief, the findings from this study provide a new understanding that the pre-treatment of ATG highly inhibits IL-1β–induced NF-κB activation.

3.5 Arctigenin inhibited the PI3K/Akt signalling pathway induced by IL-1β in human OA chondrocytes

The association of upstream axis of NF-κB with ATG was firstly examined by performing docking analysis of ATG with the antagonist binding site of PI3K.55 According to all returned molecular models, ATG was found to exert wonderful interaction with PI3K (Figure 5A). A space-filling model illustrated that ATG was perfectly embedded in the inhibitory pocket of the crystal structure of PI3K. Similarly, the local interaction between ATG and related protein residues was revealed by the image of ribbon model and local 2D binding model. After simulated in AutoDock tools, a high affinity of −7.2 kcal/mol was calculated and a hydrogen bond formed between ATG and amino acid residues (VAL882) of PI3K was uncovered. Depending on the Ligplus, we can easily see the hydrophobic interaction was formed around ATG with the residue of Try867, Ile881, Tyr812, Met953, Ile879, Met804, Lys833, Ile963, Thr887 and Glu880 of PI3K. On the other hand, according to Western blotting, ATG pre-treatment intensively inhibit the IL-1β–stimulated PI3K and Akt phosphorylation in a dose-dependent manner. The phosphorylation levels of PI3K and Akt were increased to 1.92- and 2.28-fold of control by IL-1β, but decreased to 1.13- and 1.16-fold of control in 50 μmol/L of the ATG group (Figure 5B,C). To sum up, these results demonstrated that the pre-treatment with ATG inhibits activation of PI3K/Akt axis.

3.6 Arctigenin ameliorated OA development in a mouse DMM model

The therapeutic action of ATG in vivo was also confirmed in DMM model. As illustrated in the image of Safranin O/Fast Green staining, results indicated that the cartilage erosion, hypocellularity and a high proteoglycan loss were clearly presented in the DMM group compared with the sham one (Figure 6A). Nevertheless, oral administration of ATG (30 mg/kg) could mitigate these detrimental effects. The OARSI scores were also applied for quantitative analysis (Figure 6B). Altogether, these results indicate that ATG ameliorates OA development in vivo.