Cell Source, Culture, Grouping, and Intervention
Mouse macrophage cell line RAW264.7 (STCC20020P, Servicebio, China) was cultured in DMEM (SH30022.01, Hyclone, USA) supplemented with 10% fetal bovine serum (16000044, Gibco, USA) and 1% penicillin-streptomycin (SV30010, Hyclone, USA). Cells were seeded in 6-well plates, each well containing 2.5 ml of medium, and incubated at 37°C in a humidified atmosphere of 5% CO2. Cells were divided into five groups: control (Ctrl), iron (Fe), and three concentrations of desferrioxamine (DFO) groups - DFO1, DFO2, and DFO3.
Iron solution was prepared as per literature22: 2.5 mg of ferric citrate (F5879, Sigma-Aldrich, USA) was dissolved in 1 mL ultrapure water and filtered through a 0.2 μm filter to prevent bacterial contamination. When macrophages reached a density of approximately 1×108 cells per well, iron solution was added to the Fe, DFO1, DFO2, and DFO3 groups. After 4 hours, the medium was discarded in the DFO groups, and replaced with 2.5 ml of 50μmol/L, 100μmol/L, and 200μmol/L DFO solution (138-14-7, Sigma-Aldrich, USA), dissolved in the culture medium.
Animal Source, Grouping, and Intervention
Animal experiments were approved by the Ethics Committee of Xinjiang Medical University (Approval No: 2017D01C267) were performed according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) and the Animal Care and Use Committee of Xinjiang Medical University. All methods were reported in accordance with the ARRIVE guidelines.
C57BL/6 male rats, 8 weeks old, were procured from the Animal Research Institute of Xinjiang Medical University. They were housed under SPF conditions with a 12-hour light/dark cycle at 21±2°C, with free access to food and water. The rats were divided into five groups (6 rats per group, 30 in total): animal control (A-Ctrl), animal iron (A-Fe), and three animal DFO groups (A-DFO1, A-DFO2, A-DFO3). Following an adaptation period of one week, iron overload osteoarthritis models were established in all groups except A-Ctrl, as per Jing X et al23, by intraperitoneal injection of ferric dextran (500 mg/kg). A-Ctrl received an equal volume of saline. After 16 weeks, anterior cruciate ligament transection (ACLT) surgery was performed on the left knee of all rats. A-DFO1, A-DFO2, and A-DFO3 groups received weekly intra-articular injections of DFO at concentrations of 25mg/kg, 50mg/kg, and 100mg/kg, respectively.
At 25 weeks of age, rats were anesthetized, and blood was drawn from the abdominal aorta. The rats were euthanized by cervical dislocation, and liver and knee synovium samples were processed into 0.4μm sections.
Cell Morphology Observation
Cells were observed under a high-power microscope (400x magnification) to preliminarily assess macrophage polarization.
Prussian Blue Staining
Prussian Blue staining was used to detect iron overload in macrophages, liver, and synovium. For cell staining: cells were washed after culture removal, fixed in 4% paraformaldehyde at room temperature, incubated with Prussian Blue stain for 30min, and then examined under a high-power microscope. For tissue staining: sections were deparaffinized to water, stained with Perl’s stain for 30min, and examined under a high-power microscope.
qPCR
Total RNA was extracted from macrophages using Trizal and chloroform, following the product manual, and its concentration was measured. Reverse transcription was performed using the Takara Reverse Transcription Kit (RR0470A, Takara, Japan), and the resulting cDNA was quantified by qPCR using SYBR-Green (RR820A, Takara, Japan) in a fluorescent quantitative qPCR detection system, with GAPDH as an internal reference. Primer sequences were as follows: iNOS, forward: 5'-GTTACCATGAGGCT GAAATCC-3', reverse: 5'-CCTCTTGTCTTTGACCCAGT AC-3'; Arg-1, forward: 5'-CATATCTGCCAAAGACATCGTG-3', reverse: 5'-GACAT CAAAGCTCAGGTGAA TC-3'; GAPDH, forward: 5'-GGACCTCATGGCCTACATG G-3', reverse: 5'-TAGGG CCTCTCTTGCTCAGT-3'.
Western Blot
Total protein was extracted from cells, and equal amounts of protein (20 μg) were subjected to electrophoresis on 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked at room temperature for 1 hour, then incubated overnight at 4°C with the following antibodies: F4/80 (70076S, Cell Signaling Technology, diluted 1:1000), iNOS (68186S, Cell Signaling Technology, diluted 1:1000), Arg-1 (93668S, Cell Signaling Technology, diluted 1:1000), 4E-BP1 (9644S, Cell Signaling Technology, diluted 1:125), phosphorylated 4E-BP1 (2855S, Cell Signaling Technology, diluted 1:125), p70S6K (34475S, Cell Signaling Technology, diluted 1:125), phosphorylated p70S6K (9208S, Cell Signaling Technology, diluted 1:125), and GAPDH (2118S, Cell Signaling Technology, diluted 1:1000). After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (BA1055, Boster, China, diluted 1:10000) at room temperature for 1 hour, and protein bands were visualized using the Western ECL Substrate Kit (BL523B, Biosharp, USA). Images were captured with a Bio-Rad scanner (Hercules, CA) and analyzed for density using ImageJ software (USA). GAPDH served as an internal standard for normalization.
ELISA
TNF-α and IL-6 concentrations in the culture supernatants were measured using ELISA kits (JL20268 and JL10484, Shanghai Jianglai Industrial Limited By Share Ltd, Shanghai), following the manufacturer’s instructions. Each experiment was repeated three times.
Serum Iron Concentration Measurement in the Abdominal Aorta
The serum iron concentration was measured using the Ferrozine method kit (G1212F, Suzhou Gereisy Biotechnology Co., Ltd, China), following the manufacturer's instructions. All reagents from the kit were mixed thoroughly, standard and sample solutions were added, and after sealing and incubating the plate, washing was performed. Streptavidin-HRP was added, followed by incubation and addition of TMB. The reaction was stopped as required and read.
H&E and Safranin O-Fast Green Staining
H&E and Safranin O-Fast Green staining were performed following the Hematoxylin and Eosin Staining Kit (BL700B, Biosharp, China) and Safranin O-Fast Green Staining Kit (DB0082, LEAGENE, China). For H&E staining: synovial tissue sections were deparaffinized to water, stained with hematoxylin and eosin, and examined under a high-power microscope. For Safranin O-Fast Green staining: sections were deparaffinized to water, stained with Weigert’s iron hematoxylin and Safranin O stain, and examined under a high-power microscope. The OARSI Modified Manking Score (Fig. 4 B3) quantitatively assessed the extent of left knee joint cartilage damage24.
Immunohistochemistry
Immunohistochemistry was performed on rat synovial membrane sections, which were routinely deparaffinized to water. After washing, inactivation, and blocking, sections were incubated overnight at 4°C with antibodies: F4/80 (70076S, Cell Signaling Technology, diluted 1:125), iNOS (68186S, Cell Signaling Technology, diluted 1:125), Arg-1 (93668S, Cell Signaling Technology, diluted 1:125), 4E-BP1 (9644S, Cell Signaling Technology, diluted 1:125), phosphorylated 4E-BP1 (2855S, Cell Signaling Technology, diluted 1:125), p70S6K (34475S, Cell Signaling Technology, diluted 1:125), and phosphorylated P70S6K (9208S, Cell Signaling Technology, diluted 1:125). The next day, after returning to room temperature, sections were washed and incubated with secondary antibody (ZB-2036, Zhongshan Golden Bridge, China), followed by washing, color development, counterstaining, differentiation, dehydration, and mounting.
Statistical Analysis
Data were analyzed using GraphPad Prism 10.0 and ImageJ software. Data are presented as means±SEM. The unpaired Student’s t-test was used to establish the statistical comparison between the two groups. To compare differences between several groups, we utilized one-way analysis of variance followed by the Tukey test. At P<0.05 or less (including P<0.01 and P<0.001, etc.), differences were deemed statistically significant.