Bovine serum albumin (bsa)

The rats were anaesthetized using 1% pentobarbital sodium (40 mg/kg). After anesthetisation, the brains were removed from cranial cavity after perfusion by saline and then 4% paraformaldehyde according to the aforementioned protocol. The tissues were fixed using 4% polyformaldehyde at 4°C overnight, embedded in paraffin and sectioned to a thickness of 5 µm. Briefly, sectioned specimens were deparaffinized using xylene for 20 min, twice, and then rehydrated using a graded ethanol series (100, 95, 80 and 70%) for 5 min at room temperature. Antigen recovery was performed using microwave antigen retrieval set on high for 5 min, 3 times, with citric acid buffer (pH, 6.0). After inactivation of the endogenous enzymes using 3% H₂O₂ for 10 min at room temperature, sections were blocked using bovine serum albumin (cat no. AR1006, Wuhan Boster Biological Technology, Ltd.) for 30 min at room temperature. Then the sections were incubated at 4°C overnight with diluted primary antibodies against HMGB1 (1:200, cat no. 10829-1-AP, Wuhan Sanying Biotechnology) and Iba1 (1:1,000, cat no. 012-26723, FUJIFILM Wako Pure Chemical Corporation). They were then rewarmed in an incubator at 37°C for 1.5 h. According to the manufacturer's protocols for the immunohistochemical kit (cat. no. PV9001, OriGene Technologies, Inc.), the sections were incubated at 37°C for 30 min with the Bio-goat anti-rabbit IgG and streptavidin-POD solution (1:100), then washed using PBS. Sections were stained using DAB colour liquid (cat. no. B1072, ApplyGen Technologies, Inc.) according to the manufacturer's protocols, in a dark environment at room temperature and the staining reaction was terminated when moderate tan particles were observed under the microscope. The slices were differentiated with hydrochloric acid alcohol differentiation solution (0.5%) for 2 sec, dehydrated for 5 min with each specific concentration of alcohol (70, 80, 95 and 100%) and neutral gum sealed after haematoxylin staining for 40 sec at room temperature. Finally, immunohistochemical images were captured using a CX43 light microscope (Olympus Corporation). The protein expression levels of HMGB1 and activation of microglia were assessed using ImageJ 1.52a (National Institutes of Health).

CEP chondrocytes were isolated and seeded onto a 12-well plate. The cells were subsequently subjected to 5 ng/ml TNF-α treatment with or without Nrf2 siRNA when reaching 80% confluence. Following fixation at room temperature for 20 min with 4% paraformaldehyde and permeabilization with 0.1% Triton X-100 (cat. no. T8787; Sigma-Aldrich; Merck KGaA), the cells were blocked with 5% BSA (Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature and incubated with primary antibodies against COL2 (cat. no. 28459-1-AP, 1:500; Proteintech Group, Inc.), GPX4 (cat. no. 67763-1-lg; 1:200; Proteintech Group, Inc.) and NCOA4 (cat. no. ab86707; 1:500; Abcam) at 4°C overnight. Subsequently, the cells were incubated with Cy3-conjugated goat anti-rabbit secondary antibody (cat. no. A0516; Beyotime Institute of Biotechnology; 1:500) in the dark for 1 h at 37°C. The cells were then subjected to a washing step with PBS and labeled with DAPI (cat. no. AR1177; Wuhan Boster Biological Technology, Ltd.) for 5 min with a fluorescence microscope (Olympus Corporation).
To examine the colocalization of mitochondria and Parkin, as well as Drp1, cells were incubated with diluted Mito-Tracker Red CMXRos solution (cat. no. C1049B; Beyotime Institute of Biotechnology) in the dark at 37°C for 30 min. After MitoTracker incubation, cells were subjected to fixation and permeabilization as aforementioned, then the cells were incubated with Parkin (cat. no. 14060-1-AP; 1:200; Proteintech Group, Inc.) and Drp1 (cat. no. 12957-1-AP; 1:200; Proteintech Group, Inc.) antibodies at 4°C overnight, and were then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibodies (cat. no. A0562; Beyotime Institute of Biotechnology; 1:500) in the dark at 37°C for 1.5 h. After washing with PBS and labeling with DAPI, fluorescence microscopy (Axio Observer 3; Carl Zeiss AG) was employed to capture the images and detect the differences in the fluorescence expression of the corresponding proteins.

CEP chondrocytes were treated with 0, 0.1, 1, 5 and 10 ng/ml of TNF-α (R&D Systems, Inc.) with or without Nrf2 small interfering RNA (siRNA) or 100 μM desferoxamine (DFO; cat. no. D9533; MilliporeSigma) for 24 h at 37°C. Then, cells were lysed with RIPA buffer (cat. no. CW2333; CoWin Biosciences) supplemented with a protease inhibitor cocktail for 15 min on ice; subsequently, total protein was collected following centrifugation at 2,500 × g at 4°C for 20 min. The concentration of total protein was measured using the bicinchoninic acid assay protein assay kit (Beyotime Institute of Biotechnology). A total of 25 μg total protein was added and subsequently subjected to separation by SDS-PAGE on 10% gels, followed by the transfer of the separated proteins onto polyvinylidene difluoride membranes (MilliporeSigma). After blocking with 5% BSA (Wuhan Boster Biological Technology, Ltd.) for 30 min at room temperature, the membranes were incubated with primary antibodies against Nrf2 (cat. no. 16396-1-AP; 1:1,000; Proteintech Group, Inc.), heme oxygenase (HO)-1 (cat. no. BM4010; 1:1,000; Wuhan Boster Biological Technology, Ltd.), nuclear receptor coactivator 4 (NCOA4; cat. no. ab86707; 1:1,000; Abcam), LC3B (cat. no. 4108; 1:1,000; Cell Signaling Technology, Inc.), dynamin-related protein 1 (Drp1; cat. no. 12957-1-AP; 1:1,000; Proteintech Group, Inc.), mitochondrial fission factor (MFF; 1:1,000; cat. no. 84580; Cell Signaling Technology, Inc.), ferritin heavy chain 1 (FTH1; cat. no. ab75973; 1:1,000; Abcam), type II collagen (COL2; cat. no. 28459-1-AP; 1:1,000; Proteintech Group, Inc.), SOX9 (cat. no. A00177-2; 1:500; Wuhan Boster Biological Technology, Ltd), Parkin (cat. no. 14060-1-AP; 1:1,000; Proteintech Group, Inc.), matrix metalloproteinase (MMP)3 (cat. no. 17873-1-AP; 1:1,000; Proteintech Group, Inc.), MMP13 (cat. no. 18165-1-AP; 1:1,000; Proteintech Group, Inc.), solute carrier family 7 member 11 (SLC7A11; cat. no. 26864-1-AP; 1:1,000; Proteintech Group, Inc.), COL10 (cat. no. BA 2023; 1:500; Wuhan Boster Biological Technology, Ltd.), mitochondrial fission 1 (FIS1; cat. no. 10956-1-AP; 1:1,000; Proteintech Group, Inc.), RUNX2 (cat. no. PB0171; 1:500; Wuhan Boster Biological Technology, Ltd.), glutathione (GSH) peroxidase 4 (GPX4; cat. no. 67763-1-Ig; 1:1,000; Proteintech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; 1:2,000; Proteintech Group, Inc.). Following an overnight incubation at 4°C, the membranes underwent three washes with TBS-0.1% Tween 20. The membranes were subsequently incubated with the corresponding horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:2,000; cat. nos. BA1061 and BA1062; Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature. The signal intensity of the membranes was visualized with enhanced chemiluminescence reagent (Wuhan Boster Biological Technology, Ltd.) and images were captured using a Bio-Radscanner (Bio-Rad Laboratories, Inc.).