Reagents and antibodies

AM1241 (purity > 97%), type II collagenase were purchased from Thermo Fisher (Shanghai, China). Anti-type II collagen, Aggrecan, ADAMTSS, MMP-13, GAPDH, Histone H3 primary antibody, anti-P65, anti-Nrf2, anti-HO-1, anti-Iκb-α, anti-TGF-β1, anti-TNF-α, anti-IL-6, LPS, DMSO were purchased from Beyotime (Shanghai, China). DMEM/F12 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotics and EDTA were purchased from Thermo Fisher (Shanghai, China).

Primary mice chondrocytes culture

Mice below 10 days old were euthanized by overdosed sodium pentobarbital. Articular cartilage of mice was collected under sterile conditions using a dissecting microscope. The tissue was treated with 2 mg/mL (0.1%) type II collagen at 37 °C for 4 h. Subsequently, the digested cartilage tissue was inoculated in suspension in tissue culture flasks. The chondrocytes were grown in DMEM/F12 with 10% FBS and 1% penicillin/streptomycin antibiotics (Thermo Fisher Shanghai, China) at 37 °C. The culture medium was changed immediately after 24 h of incubation. When the fusion rate reached 80–90%, cells were collected via 0.25% trypsin EDTA (Thermo Fisher) Then, they were transferred into 10 cm culture plates at the appropriate density and cultured to P2-P3 for experiments. The chondrocytes were cultivated at 5% CO2 and 37 °C, and the complete medium was changed every other day.

Animal model

A total of 28 C57BL/6 male mice (20–25 g) aged 10 weeks were purchased from Sipeifu Biotechnology Co., Ltd. The experiment was performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Medical Ethics Committee of Nanning First People’s Hospital. The mice were kept in a controlled environment with a 12-hour light/dark cycle and free access to food and water. The mice OA model was established by a modified Hulth method that severed the anterior cruciate ligament and medial meniscus of the mice (Rogart et al. 1999). After preoperative fasting without water for 12 h and weighing, mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg), and the skin was incised about l cm long along the medial edge of the patellar ligament. After separation, the joint capsule was incised and the anterior cruciate ligament and medial meniscus were severed. Penicillin (500,000 U) was administered intramuscularly for 5 consecutive days after surgery to combat infection. Mice with arthrotomy without cutting the medial meniscus ligament on the left knee were conducted as a sham group. After surgery, the mice were randomly divided into sham-operated group, Hulth group, Hulth + 3 mg AM1241 (12 mg/ml) and Hulth + 9 mg AM1241 (36 mg/ml) group.

Experimental design

In the in vitro study, chondrocytes from mice were treated with 100 ng/ml LPS alone or pretreated with AM1241 (5, 10 µM) for 4 h, followed by stimulation with 100ng/ml LPS for 12 h. The control group only changed the culture medium without treatment. Cells were harvested after 24 h of incubation. For in vivo evaluation, mice were subjected to the Hulth method as described above. After the successful establishment of the OA model, the groups were treated as follows: Hulth + 3 mg AM1241 group (3 mg/kg/day; once a week; 12 mg/ml AM1241 dissolved in 5% DMSO saline for five weeks in the joint cavity) and Hulth + 9 mg AM1241 group (9 mg/kg/day; once a week; 36 mg/ml AM1241 dissolved in 5% DMSO saline was injected for five weeks in the joint cavity). At the same time, equal amounts of 5% DMSO saline were administered to the sham group and the control group. Postoperatively, the animals were driven for 30 min daily for 5 weeks. After that, the animals were sacrificed and cartilage tissue specimens were collected for histological and MicroCT scan analysis.The experiment was approved by the Medical Ethics Committee of Nanning First People’s Hospital (2022-172-01).

Cell viability assay

The impact of AM1241 on chondrocyte viability of mice was detected via a Cell Counting Kit-8 (CCK-8) kit (Beyotime). First, P2 chondrocytes were transferred to 96-well plates (2000 cells/well) for inoculation and incubated in different concentrations of AM1241 (0-250 µM) for 24/48 h. Before assay, 200 µL of DMEM/F12 medium containing 10 µL of CCK-8 reagent was added to each well and incubated for 2 h at 37 ℃ in the dark. Then the incubated medium was transferred to an enzyme-labeled plate and cultivated by measuring the absorbance at 450 nm using an enzyme marker. Five replicate wells were set up for each group, and the experiment was carried out five times.

TGF-β1, TNF-α, and IL-6 determination

The expression levels of TGF-β1, TNF-α, and IL-6 were measured by ELISA kits (E-EL-0162c, E-EL-M0049c, E-EL-M0044c, Elabscience, Wuhan, China) according to the manufacturer’s instructions. The optical density was read at 450 nm with a Microplate Reader.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA of chondrocytes treated with 100ng/ml LPS alone or in combination with different concentrations of AM1241 (0, 5, 10 µM) were extracted from 6-well culture plates using TRIzol (Beyotime). 1000 ng of total RNA was reverse transcribed to synthesize cDNA. For qPCR, 10 µl reaction volume in total was applied, including 5 µL of 2 × SYBR master mix, with 0.25 µL of each primer. RT-PCR parameters: 10 min 95 °C for 10 min followed by 15 s 95 °C for 15s and 60 °C 1 min for 40 circles with CFX96 real-time PCR system. Cycle threshold (Ct) values were collected and normalized to GAPDH levels. The relative mRNA levels of each target gene were calculated using the 2−∆∆Ct method. All the primers were designed via NCBI primer blast (Table 1).

Table 1 List of primer sequences of mice for real-time PCRWestern blot

Total proteins were extracted from chondrocytes using RIPA lysis buffer with 1 mM PMSF (phenylmethane sulfonyl fluoride). Next, they were separated on ice for 10 min and then centrifuged at 12,000 rpm and 4 °C for 15 min, followed by a measurement of protein concentration using the BCA protein analysis kit (Beyotime). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) was applied to separate 40ng protein which would be transferred to polyvinylidene difluoride membranes (Bio-Rad, USA). After being blocked by 5% skim milk for 1 h, the membranes were incubated with the antibodies of type II collagen, Aggrecan, Nrf2, MMP-13, ADAMTS-5, TGF-β1, TNF-α, IL-6, p65, Histone H3, and IκBα overnight at 4 °C, and subsequently incubated with the corresponding secondary antibodies at room temperature for 1 h. After washing twice with PBST and once with PBS (10 min/time), the blots were observed with electrochemiluminescence plus reagent (Invitrogen). Finally, the intensity of these blots was quantified using imagelab 3.0 software (Bio-Rad).

Immunofluorescence

For type II collagen, Aggrecan immunofluorescence staining was employed. Chondrocytes were treated with 100 ng/mL LPS for 12 h, or pretreated with AM1241 (5 and 10 µM) for 4 h before being stimulated with 100 ng/mL LPS for 12 h. For p65 and Nrf2 staining, the duration of LPS treatment was shortened to 2 h. After treatment, samples were washed with PBS three times and permeabilized with PBS diluted with 0.1% Triton X-100 for 15 min. Chondrocytes were then blocked with 5% bovine serum albumin for 1 h at 37 °C, washed with PBS, and incubated overnight in a humid chamber at 4 °C with PBS-diluted primary antibodies: type II collagen, Aggrecan, p65 (1:200) and Nrf2 (1:400). The next day, plates were washed and incubated with Alexa-Fluor® 488 labeling or Alexa-Fluor® 594-conjugated secondary antibody (1:400) for 1 h at room temperature and labeled with DAPI for 5 min. Finally, five areas of each slide were randomly selected for microscopic observation using a fluorescence microscope (Olympus Inc., Tokyo, Japan) and fluorescence intensity was measured using Image J software 2.1 (Bethesda, MDUSA).

MicroCT scan

After 6 weeks, the knee joints of mice were taken and immersed in 4% paraformaldehyde. The samples from each group were fixed and then scanned by micro-CT (Skyscan 1176, Bruker, Belgium) at a voltage of 50 kV, a current of 500 µA and a resolution of 18 μm per pixel. The region of interest (ROI) was set to the subchondral bone, and the bone trabecular parameters in this region were analyzed to assess the bone healing.

Three-dimensional anatomical structures including bone volume/total tissue volume (BV/TV), bone surface area/bone volume (BS/BV), trabecular thickness (Tb.Th), and separation (Tb.Sp) were calculated with CTAn (Bruker MicroCT, Kontich, Belgium). CTVox software (Bruker MicroCT, Kontich. Belgium) was utilized to reconstruct the 3D images.

Histopathological analysis

After MicroCT, the knee was treated with a decalcification solution for 4 weeks. The tissue was dehydrated and embedded with paraffin. Sagittal sections in 6-µm thickness were cut along the knee joint, and each joint section was stained with fast green and safranin-O. IHC staining was applied to type II collagen, MMP-13. Slides were dewaxed and then stained with Fast Green (Beyotime) for 6 min, washed at 20˚C, dehydrated, and stained with Safranin O (Beyotime) for 3 min at 20˚C. For type II collagen and MMP-13 IHC staining, endogenous peroxidase in the tissue was first blocked with 3% H2O2. Then, the samples were treated with 2.5% hyaluronidase (Beyotime) for 1 h at 37˚C for epitope retrieval. The sections were blocked with fetal bovine serum for 1 h and incubated with type II collagen and MMP-13 monoclonal antibody at 37 °C for 4 h. Finally, the sections were counterstained with hematoxylin. The extent of cartilage lesions was assessed using the modified light microscopic cartilage assay evaluation criteria, the Mankin score, with a blinded evaluation. Disease course: 1–5: early stage; 6–9: intermediate stage; 10–14 late stage. Five mice per group were subjected to histomorphometric analysis.

Molecular docking and molecular dynamics simulation

The 3D structure of the AM1241 compound was downloaded from the PubChem database and then imported into Chemdraw 3D for energy minimization using the MM2 module. The target protein crystal structures of Keap1 protein as well as the NEMO/IKKβ structures were downloaded from the PDB database and subsequently visualized separately using pymol. Next, the ligands were docked to the receptors using Autodock vina 1.1.2 and the docking results were analyzed after water removal, hydrogenation, charge calculation, and merging of non-polar hydrogens. After that, protein binding was visualized using Pymol and discovery studio software to analyze the specific binding sites and interaction forces of AM1241 with NEMO/IKKβ and Keap1 proteins. Molecular dynamics simulations were performed using the Gromacs software package, the physical conditions were then set to constant temperature (310 K), constant pressure (101 kPa), and periodic boundary conditions, and the TIP3P water model was employed to simulate the human environment in a 0.145 mol/L neutral sodium chloride solution. After the state of all environments was balanced, the screened compound target complex system was utilized to carry out a 50nsMD simulation, in which the confirmation storage calculation was performed every 10 ps. Then, compounds with docking fractions below the cut-off value were validated using the Gromacs embedded program and the root mean square deviation (RMSD), root mean square fluctuation (RMSF), the radius of gyration (Rg), and intermolecular hydrogen bond (H bond) from the visualized MD simulation results. Finally, the stability of AM1241 binding to its target protein was analyzed.

Statistical analysis

The results were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS statistical software program 20.0. Data were compared using a one-way analysis of variance (ANOVA) followed by Tukey’s test. Non-parametric data (e.g., Mankin scores) were analyzed using the Kruskal-wallish test. p-values less than 0.05 were considered a significance.