Collagenase II (Worthington Biochemical Corp., Lakewood, NJ, USA) was dissolved in DMEM-F12 at 1.5 mg/ml to digest articular cartilage. Erastin (USA, E-7781) and ferrostain-1 (USA, SML0583) were purchased from Sigma-Aldrich. BODIPY 581/591 C11 (USA, D3861) was purchased from Thermo Fisher. The LDH Cytotoxicity Assay Kit and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Biotechnology (Shanghai, China). Nrf2 antibody (16396-1-AP), collagen Ⅱ (18165-1-AP) and SLC7A11 antibodies (26864-1-AP) were purchased from Proteintech (Wuhan, China). GPX4 antibody (A11243) and FTH1 antibody (A19544) were purchased from Abcam. ASP was purchased from Shanghai Yilin Biotech. Co., Ltd. (Shanghai, China). ASP had a purity greater than 92% pure, and the average molecular weight was 85.0 kDa.
The study was approved by the ethics committee of the Second People’s Hospital of Changzhou, Jiangsu, China. Cartilage specimens were collected from the knees of patients with clinical stage III or IV (K-L score) OA when they underwent joint replacement, and tissue was obtained from non-damaged regions and obviously damaged regions of the joints (Fig. 1A). All participants signed an informed consent form before the operation. All participants signed an informed consent form before the operation. Patient information is shown in Supplementary Table 1.
Isolation and Culture of Chondrocytes
All tissues were cut into pieces as finely as possible, partially digested by collagenase II at 1 mg/mL in Dulbecco’s modified Eagle’s medium DMEM/F12 (Gibco BRL, Grand Island, NY, USA) at 37℃ for 4 hours, and filtered through a 70-µm cell strainer (BD, Durham, NC, USA). Finally, 10% fetal bovine serum, 50 µg/mL ascorbic acid (AA, Sigma), 100 U penicillin and 100 µg/ml streptomycin were added to the DMEM/F12 medium for culture in a standard cell culture chamber containing 5% CO2. Nonadherent cells were removed 2 days later. Adherent cells were split at a ratio of 1:2 until 90% confluence. Passage 2–3 chondrocytes were used in subsequent experiments. The remaining specimens were cryopreserved and then ground with liquid nitrogen and preserved.
Cell Viability Assay
The Cell Counting Kit-8 (CCK-8) was used to detect chondrocyte activity in the experiment. Chondrocytes were cultured in 96-well plates at a density of 5×103 cells/well. After 12 hours, the chondrocytes were pretreated with 30 µg/ml ASP for 24 hours. Next, the cells were treated with 5 µM erastin, 5 µM erastin and 30 µg/ml ASP, and 5 µM erastin and 10 µM Fer-1 for 24 hours and 48 hours. After incubation, the cells were washed twice with PBS, and then 100 µl of 10% CCK-8 solution was added to each well, followed by incubation at 37℃ for 1.5 hours. The absorbance at 450 nm-650 nm was detected by an absorbance microplate reader (Epoch Bio-Tek Instruments, USA).
Detection of lipid ROS
Chondrocytes were spread in a 24-well plate at a density of 3×104 cells/well. After 24 hours, the chondrocytes were treated with 5 µM erastin, 5 µM erastin and 30 µg/ml ASP, or 5 µM erastin and 10 µM Fer-1 for 24 hours. Lipid ROS levels were measured with the C11 BODIPY fluorescent probe according to the manufacturer’s instructions. Briefly, the chondrocytes were cleaned three times with PBS and treated with 5 µM C11 BODIPY for 25 min at 37℃ in the dark. After incubation, the chondrocytes were washed with PBS three times and observed under a fluorescence microscope (Nikon Eclipse Ti, Japan).
Western blot analysis
Chondrocytes were spread on 6-well plates at a density of 4×105 cells/well. After 12 hours, the chondrocytes were treated consistently with the above conditions. Then, the chondrocytes were lysed on ice for 15 min with 200 µl RIPA buffer containing 1% proteinase inhibitor cocktail and boiled for 5 min after removal of the medium. The protein concentration was determined by a BCA Protein Assay kit. Equal amounts of protein were electrophoresed on a 15% SDS–PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the membranes were blocked with 5% skimmed milk at room temperature for 1 hour and incubated overnight at 4°C with the primary antibody according to the instructions (1:1000 dilution). Then, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Protein bands were detected with SuperSignal West Pico Plus (Thermofisher Scientific, USA) ECL kit, and relative expression levels were quantified using ImageJ software.
Chondrocytes were uniformly seeded on a 24-well plate containing slides. After the cells were treated, they were immobilized at room temperature for 15 min with 4% paraformaldehyde. After 20 minutes of 0.5% Triton X-100 permeation at room temperature, 5% BSA was used for blocking for 1 hour. Primary antibodies diluted with 5% BSA (1:200) were incubated at 4°C overnight. After treatment, the slides were washed three times with PBS and incubated with secondary antibody at room temperature for 1 hour in the dark. The slides were washed again with PBS 3 times, the nuclei were counterstained with DAPI for 10 minutes in the dark and then observed under a fluorescence microscope.
Lactate dehydrogenase (LDH) assay
To measure the release of lactate dehydrogenase, chondrocytes were evenly distributed on a 96-well plate at a density of 5×103 per well, with 3–6 wells per group. After treatment as described above, LDH levels were determined according to the manufacturer’s instructions. In brief, the supernatant of the cells was moved to a new 96-well plate and incubated for 1 hour with LDH reagent. Then, the absorbance at 490 nm was detected by an absorbance microplate reader.
Glutathione (GSH) assay in cartilage tissue
GSH reacts with 5,5’-dithiobis-2-nitrobenoic acid (DTNB) to produce 2-nitro-5-mercaptobenzoic acid (TNB) (yellow) and oxidized glutathione (GSSG). GSH levels were determined according to the manufacturer’s instructions for the Reduced Glutathione Assay Kit (BC1175, Solarbio, Beijing, China). Briefly, the specimens were washed with PBS 3 times and frozen. Then, the specimens were ground with liquid nitrogen, mixed with GSH reagent and moved to 96-well plates, and the absorbance was measured at 412 nm.
Malondialdehyde (MDA) assay in cartilage tissue.
MDA can condense with thiobarbituric acid (TBA) and show a brownish-red color under acidic and high-temperature conditions. MDA levels were determined according to the manufacturer’s instructions for the Micro Malondialdehyde (MDA) Assay Kit (BC0025, Solarbio). Briefly, the specimens were washed with PBS 3 times and frozen. Then, the specimens were ground with liquid nitrogen, mixed with MDA reagent and moved to 96-well plates, and the absorbance was measured at 532 nm.
Cartilage tissue was taken from SD rats. The cartilage was sectioned after paraffin embedding, and endogenous peroxidase activity was eliminated by 3% H2O2. Next, 6% normal goat serum containing primary antibodies was added for incubation overnight at 4°C. Color development was observed after 25 min of incubation with the appropriate secondary antibody at room temperature.
Cells were cultured in 6-well plates (for western blot analysis), 24-well plates (for immunofluorescence and lipid ROS detection) and 96-well plates (for cell viability assay). Control siRNA (no silencing) and Nrf2 siRNA were transfected into cells using a riboFECT™ CP kit (RiboBio Co. Ltd., Guangzhou, China) according to the manufacturer’s instructions. The siRNA target sequences are shown as below: si-Nrf2#1, CATTGATGTTTCTGATCTA; si-Nrf2#2, GAGGCAAGATATAGATCTT. All the analysis was performed at least 48h after transfection.
Establishment of rat models of cartilage damage
Four-week-old male SD rats were used in this study and allowed one week of routine feeding for adaptation. The rats’ OA model was induced by destabilization of the medial meniscus (DMM) as previously described (Glasson et al., 2007; Sherwood et al., 2015). Cartilage degradation was evaluated histologically according to the Osteoarthritis Research Society International (OARSI) guidelines by two independent investigators. In addition, rats that died or appeared sick during experiments were excluded.
42 rats were randomly divided into seven groups (n = 6 per group) after OA developed.
Group 1: sham surgery;
Group 2: DMM;
Group 3: DMM and intra-articular injection of DFO (1 mg/kg, twice a week);
Group 4: DMM and intra-articular injection of ASP (3 mg/kg, twice a week);
Group 5: erastin (1 mg/kg, one time) injection into the articular cavity;
Group 6: erastin (1 mg/kg, one time) and ASP (3 mg/kg, twice a week) injection into the articular cavity;
Group 7: erastin (1 mg/kg, one time) and DFO (1 mg/kg, twice a week) injection into the articular cavity.
The same treatment was performed on each rat. After 10 weeks, the rats were sacrificed, and the knees were harvested. We performed MRI for imaging examination and hematoxylin-eosin (H&E) and Masson staining for pathological examination.
The data in this study are presented as the mean ± SD of at least three independent experiments. Differences between 2 groups were compared with Student’s t test, and one-way ANOVA was used for comparisons of more than two groups. All quoted P values were 2-tailed, and values less than 0.05 were considered statistically significant.