Primary chondrocytes isolation and culture

5-day-old male C57BL/6J mice were anesthetized and humanely euthanized by cervical dislocation. Cartilage from the knee joints was cut into small pieces and subjected to a 0.25% trypsin solution for 30 min and 0.25% type II collagenase for 4–6 h. After centrifugation and resuspension, primary chondrocytes were isolated and cultured in an incubator under conditions of 5% CO2 at 37 °C.

Western blot assay

The cells were lysed in RIPA buffer containing a protease inhibitor cocktail for 15 min on ice, followed by centrifugation at 12,000 g. The total protein concentration was determined using the BCA protein assay kit (Beyotime, China). A total of 20 micrograms of protein was loaded onto a SDS-PAGE gel and separated. The separated proteins were then transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% BSA, then incubated with primary antibodies. TFR1(#ab84036) and HIF-1α(#ab179438) were purchased from Abcam. BNIP3(#3769S), IL-6 (#12,912), IL-1β(#12,242), iNOS (#sc-7271), COX2 (#4842), p65 (#8242), Phospho-p65 (#3033), SOX9 (#A00177-2) were purchased form CST. MMP3 (#17873-1-AP), COL2 (#28459-1-AP), MMP13 (#18,165- 1-AP), SLC7A11 (#26864-1-AP), GPX4 (#67763-1-Ig), cGAS(#A8335), STING(#19851-1-AP), DRP1(#12957-1-AP), MFF(#17090-1-AP), GAPDH (#10494-1-AP) were purchased from Proteintech. COL10 (#BA 2023, Boster) was purchased from Boster. Following an overnight incubation at 4 °C, the membranes were washed three times with Tris-buffered saline with Tween (TBST) and then incubated with the appropriate anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature. The signal intensity on the membranes was visualized using a Bio-Rad scanner (Bio-Rad, Hercules, CA).

Immunofluorescence staining

Chondrocytes were seeded in a 12-well plate and cultured until 80% confluence. After fixation and permeabilization, the cells were blocked with 5% Bovine Serum Albumin (BSA) for 1 h. Subsequently, the cells were respectively treated with primary antibodies against COL2 (1:500), GPX4 (1:500), STING (1:500), and TfR1 (1:200) at 4 °C overnight. Afterward, they were treated with Cy3-conjugated goat anti-rabbit secondary antibody (#A0516, Beyotime, Shanghai, China, 1:500) for 1 h at 37 °C in the dark. The cells were then subjected to a washing step and stained with DAPI (Boster, AR1177) for 5 min.

To investigate the colocalization of mitochondria with BNIP3 and Drp1, cells were incubated with a diluted Mito-Tracker Red CMXRos solution (#C1049B, Beyotime, Shanghai, China, 1:500) in the dark at 37 °C for 30 min. After fixation and permeabilization, the cells were then incubated with BNIP3 (1:200) and Drp1 (1:200) antibodies at 4 °C overnight. Subsequently, the cells were treated with FITC-conjugated goat anti-rabbit secondary antibody (A0562, Beyotime, Shanghai, China, 1:500) at 37 °C for 1.5 h in the dark. Following a wash with PBS and labeling with DAPI, fluorescence microscopy (Axio Observer 3; Carl Zeiss) was used to capture images and detect differences in the fluorescence expression of the corresponding proteins.

siRNA transfection

To induce the knockdown of STING and TfR1 in mouse chondrocytes, specific siRNA transfection procedures were conducted using the riboFECTTMCP kit (Ribo Bio, Ribobio Co. Ltd., Guangzhou, China). The chondrocytes were transiently transfected with 100 nM of each siRNA for 48 h following the manufacturer’s instructions. After transfection, the efficiency of silencing was confirmed via western blot analysis, and the most effective siSTING and siTfR1 were selected for subsequent analysis.

Assessment of intracellular ROS and mitochondrial membrane potential (MMP)

Intracellular ROS production was assessed using a Reactive Oxygen Species Assay Kit (S0033, Beyotime, Shanghai, China) following the manufacturer’s instructions. Chondrocytes were washed three times with serum-free media. Subsequently, dichloro-dihydro-fluorescein diacetate (DCFH-DA) was diluted to 10 μM in serum-free medium and added to the cells for a 30-minute incubation in the dark. After washing the cells with serum-free media, they were examined using a fluorescence microscope (Axio Observer 3; Carl Zeiss).

Mitochondrial membrane potential (MMP) was evaluated using a mitochondrial membrane potential kit (#C2006, Beyotime, Shanghai, China). Briefly, after incubation with the JC-1 staining working solution for 20 min at 37 °C, CEP chondrocytes were rinsed with ice-cold JC-1 washing buffer three times. Multimeric JC-1 with high red fluorescence transitions to monomeric JC-1 with high green fluorescence, indicating the loss of MMP. The changes in MMP were captured using an inverted fluorescence microscope (Axio Observer A1; Carl Zeiss, Germany).

Ferrous iron detection

After three washes with Hank’s Balanced Salt Solution (HBSS), the cells were stained with 1 μM FerroOrange (Dojindo, F374) in HBSS for 40 min at 37 °C. Subsequently, the cells underwent three additional washes with HBSS and were then subjected to imaging using a fluorescence microscope (Axio Observer 3; Carl Zeiss).

PicoGreen and Mito-tracker red co-staining

For mtDNA staining, the CEP chondrocytes were stained with MitoTracker™ Red CMXRos probes (#M7512, ThermoFisher, USA, diluted at 1:5000) for 20 min, followed by 20 min of incubation in PicoGreen dsDNA Quantitation Reagent (#12641ES, Yeasen, Shanghai, China, diluted at 1:500) at 37 °C after being washed with PBS. Then, the microscope cover glasses were observed and imaged using a confocal fluorescence microscope (TCS SP8; Leica Microsystems, Biberach, Germany).

Animal grouping and immunohistochemical assay

A total of 24 male C57BL/6 mice (8 weeks old) were randomly assigned to 3 groups (n = 8 per group): control group, OA group and OA + Ferristain II group(10 mg/kg/2d). The dosage and intervention conditions were performed according to a previous study [15]. The surgically-induced DMM model was established as detailed in prior protocols [7]. Cartilage tissues were subsequently decalcified, embedded, sectioned and stained with hematoxylin-eosin (H&E), performed to observe the morphology of cartilage and subchondral bone. The severity of OA was evaluated by OARSI assessment scoring system. All animal procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Shandong Provincial Hospital, affiliated with Shandong First Medical University (Approval No. 2022 − 812).

For immunohistochemistry (IHC) staining, as previously described, after deparaffinization, rehydration, blocking, and antigen retrieval, the sections (4 μm, coronal plane) were then incubated with primary antibodies against TfR1, STING, COL2, MMP3, overnight at 4℃. Next, the sections were stained with the HRP-conjugated secondary antibody (#GB23303, Servicebio, dilution 1:200) for 30 min at room temperature followed colored with DAB and counterstained with hematoxylin. Immune positive staining of 5 fields randomly selected was quantitatively analyzed using Image Pro Plus software. All animal experiments were approved by the Animal Care Committee of Shandong Provincial Hospital affiliated to Shandong First Medical University.

Micro-CT analysis

The samples were fixed in 4% paraformaldehyde (PFA) for 24 h. To assess alterations in the microarchitecture of the surgically modeled segment, micro-CT (Scanco Viva-CT80, Scanco Medical AG, Basserdorf, Switzerland) was then used to determine morphometric indices, such as intervertebral disc height and percent bone volume (BV/TV), from the volume of interest (VOI), with the resolution of 11.6 μm, 70kVp, and 114μA. And the three-dimensional (3D) images of the CEP were collected by built-in software.

Statistical analysis

All analyses were performed with GraphPad Prism software (version 9.0; Dotmatics). Comparisons between multiple groups were analyzed using one-way ANOVA followed by Tukey’s test. For WB data and immunohistochemistry results expressed as relative fold change in Fig. 1, a Student’s t-test or a one-way ANOVA with Dunnett’s test were used for pairwise comparisons. Results are represented as mean ± SD and p < 0.05 was considered to be significance. All analyses were performed with GraphPad Prism software (Version 9.0).

Fig. 1

The expression of TfR1 in cartilage of OA and primary chondrocytes. (A) Representative images of knee articular cartilage from control group and DMM group, showing TfR1-positive chondrocytes. Scale bars = 100 μm. The ratio of TfR1 positive cells and total chondrocytes in knee articular cartilage of control group and DMM group. (n = 8 for each group). (B) The expression of TfR1 in chondrocytes treated with 0-1-5-10ng/ml IL-1β for 12 h detected by western blotting. The density of the TfR1 immune-reactive bands was analyzed by using GAPDH expression as a loading control. (C) Representative images of immunofluorescence staining for TfR1 expression in chondrocytes treated with IL-1β for 12 h. Scale bars = 50 μm. (D) Representative images for ferrous ions in the indicated group and statistical analysis of fluorescence intensity (ferrous ions). Scale bars = 200 μm. Data are presented as the mean ± SD, * P < 0.05, **** P < 0.0001