Bacterial strain and culture
Staphylococcus aureus (S.aureus) strain ATCC 25923 provided by the Infectious Diseases Department of Nanfang Hospital, Southern Medical University was verified by PCR amplification. For preparation of S. aureus for in vivo and in vitro experiments, S. aureus were cultured in tryptic soy broth in tubes overnight at 37℃ under shaking conditions. Cultures were then harvested by centrifugation at 2500g for 10 min. The pellets were washed three times and resuspended with PBS. Next, the bacteria concentration was adjusted to an optical density of 0.5 at 600 nm, approximately equal to 1 × 108 CFU/ml. Culture and quantification methods for S. aureus and its virulence effects have been tested in our previous studies24.
Cell culture infection and treatments
MC3T3-E1 (GNM15, Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Sciences, China) cells were cultured in medium containing 10% FBS in α-MEM and Penicillin streptomycin antibiotic mix at 37℃ in the presence of 5% CO2. Caspase inhibitor Z-VAD (#161401-82-7, MedChemExpress, USA) and necroptosis inhibitor Nec-1 (#4311-88-0, MedChemExpress, USA) were used to pretreat cells for 1 h before infection. Antioxidant NAC (#616-91-1, Sigma-Aldrich, USA) were used to treat cells for 24h after infection. Small interfering RNA targeting PGAM5 and AIFM1 (chemically synthesized by Ribobio, Guanzhou, China) were transfected into cells according to the manufacturer’s recommendations. MC3T3-E1 Cells were infected with S. aureus at an MOI of 10. At 60 min after infection, cells were treated with 15µg/ml of lysostaphin and 20µg/ml of gentamicin for 30min to kill extracellular bacteria.
CCK8 assay
CCK8 assay was used to detect cell viability. MC3T3-E1 cells were seeded into 96-well plates at a density of 8000 per well. After corresponding treatments, cells were incubated with 100µl fresh medium with 10µl CCK8 solution (#C0037, Beyotime Biotechnology, China) for 2h at 37°C, and then the optical density (OD) was measured at 450nm by Model SpectraMax i3x Microplate Reader (Molecular Devices, USA).
Lactate dehydrogenase (LDH) release assay
LDH release assay was used to detect the activity of LDH released during cell injury. MC3T3-E1 cells were plated into 96-well plates at the density of 8000 cells per well. After corresponding treatments, we used the LDH Cytotoxicity Assay kit (#C0016, Beyotime Biotechnology, China) to detected the LDH levels in the supernatant according to the manufacturer’s recommendations. The OD was measured at 490nm by Model SpectraMax i3x Microplate Reader (Molecular Devices, USA).This testing process has been proven effective in the same type of study25.
Fluorescence staining of live and dead cells
Calcein/PI Live/Dead Assay Kit was used to detect the viability of cells. MC3T3-E1 cells were plated into 24-well plates at the density of 20000 cells per well and treated with corresponding treatments. According to the manufacturer's recommendation, use a Calcein/PI Live/Dead Assay Kit(#C2015S, Beyond Biotechnology, China) to perform fluorescence staining on cells and observe the staining under a fluorescent inverted microscope(IX73, OLYMPUS, USA)to determine the cell status.
DNA damage detection
DNA Damage Assay Kit by γ- H2AX Immunofluorescence can detect DNA damage markers through immunofluorescence staining γ- The content of H2AX (phosphorylated H2AX) is used to confirm whether DNA is damaged. MC3T3-E1 cells were plated into 24-well plates at the density of 20000 cells per well and treated with corresponding treatments. According to the manufacturer's recommendation, use a Calcein/PI Live/Dead Assay Kit(#C2037S, Beyond Biotechnology, China) to perform fluorescence staining on cells and observe the staining under a fluorescent inverted microscope(IX73, OLYMPUS, USA)to determine the cell status.
Western Blotting
After appropriate stimulation, equal amounts of protein (30µg) extracted from MC3T3-E1 cell were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in Trisbuffered saline Tween-20 containing 5% BSA for 1h and then incubated overnight at 4°C with primary antibodies against KEAP1 (1:2000, #28445-1-AP, Proteintech, USA), PGAM5 (1:2000, #10503-2-AP, Proteintech, USA), AIFM1(1:1000, #17984-1-AP, Proteintech, USA), phospho-AIFM1 (Ser-116)(1:1000, #AP5501, ECM Biosciences, USA) and Vinculin(1:1000, #ET1705-94, HUABIO, China). The effects of the above antibodies have been shown to be effective in studies26–30. After incubation with a secondary antibody for 1 h at room temperature, the membranes were visualized using the chemiluminescence method.
Osteogenic Differentiation of Cells and ALP Staining
Alkaline phosphatase (ALP) staining assays were used to assess the effect of inhibition of cellular oxeiptosis pathway under infection conditions on osteogenic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were plated into 12-well plates at the density of 50000 cells per well and treated aforementioned infection treatments. 24 hours after cell infection, the medium was replaced with induction medium supplemented with 50µg/ml ascorbic acid (Sigma, A4544-25G), 10nmol/l dexamethasone (MCE, HY-14648) and 10mmol/l β-glycerophosphate (Sigma, G9422-10G). Cells after medium change require daily sterilization of extracellular bacteria using bacteriostatic medium. Seven days after osteogenic induction, cells were stained for ALP using the ALP staining kit for the plates (Beyotime, C3206) according to the kit guidelines. The induction medium formulation and the induction process have been shown to be effective in our previous studies31.
Animals
The experimental protocol was approved by the Southern Hospital Animal Care and Use Committee and was conducted in accordance with the Southern Hospital Southern Medical University Guide for Laboratory Animals. The animal experimental protocol in this experiment has been reviewed and approved by the Experimental Animal Ethics Committee of Southern Hospital of Southern Medical University. All the authors complied with the ARRIVE guidelines. The mice are supplied with plenty of purified water and animal feed. The number of mice per cage was controlled at 5–6, and the environment was maintained in a stable and clean environment, with the room temperature controlled at 25℃, relative humidity at 50% and simulated day-night cycle, and the bedding of the cages was changed every three days. The bedding of the cages was changed every three days to minimize errors in the experimental results caused by environmental factors affecting the state of the mice. After two weeks of acclimatization, all mice were divided into infected and control groups.
Surgical Procedures
Mice (6 weeks old) were anesthetized using intraperitoneal injection of tribromoethanol (1.25%). Alcohol cotton balls were used to sterilize the skin around the knee joint after removing the hair around the right knee joint of mice. A disposable insulin syringe (KRUUSE, China) with a 29-gauge needle was inserted percutaneously into the femoral marrow cavity at the femoral condyle position, and 2 µl of bacterial fluid containing 1×102 CFUs of S. aureus was injected. Subsequently, a stainless steel acupuncture needle (femoral prosthesis, length 10 mm, diameter 0.2 mm; Zhongyan Taihe Medical Instrument, Beijing, China) was placed in the femoral marrow cavity, and the end of the needle was cut off by withdrawing 2 mm after complete entry. The knee joints of mice were kept in passive motion to ensure that the needles completely entered the intramedullary canal. Animal samples were collected two weeks after surgery. This method of animal surgery has been tried with success in our previous studies24.
Histochemistry Staining
Bone tissue was immersed in a 4% paraformaldehyde solution for 24 hours of fixation and then transferred to 0.5 M ethylenediaminetetraacetic acid (EDTA, pH 8.0) for decalcification for 8–10 days, with the decalcification solution changed daily. The decalcified tissues were subjected to paraffin embedding and tissue sectioning. Hematoxylin and eosin(H&E) staining was performed on paraffin sections. For immunohistochemistry, primary antibodies PGAM5 (#10503-2-AP, Proteintech, USA) and AIFM1 (#17984-1-AP, Proteintech, USA) were used to incubate with tissue sections at 4℃overnight. Primary antibodies were subsequently washed off and incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature, followed by incubation for color development with a DAB kit (ZLI-9019, ZSGB-BIO, Guangzhou, China) according to the manufacturer's protocol. The histological experiments were modeled after our our previous study29; 31.
Statistics
All data were shown as mean ± SD. For cell culture experiments, all results were obtained from independent replicates of the experiments, which were repeated independently at least three times. For comparisons between two groups, independent Student’s t-tests were used. One-way analysis of variance (ANOVA) and Bonferroni post hoc tests were used for multiple comparisons. Statistical analysis software was used for the data using GraphPad, version 9.0 software (GraphPad Software, USA). P values < 0.05 considered to be a statistically significant difference. The statistical scheme of the data follows that of our previous study31.