Human bone samples
Human bone samples were harvested from postmenopausal and nonmenopausal female patients with femoral neck fracture at The First Affiliated Hospital of Hainan Medical University (Haikou, China). The postmenopausal female patients were divided into the osteoporosis and severe osteoporosis groups based on bone mineral density, and the nonpostmenopausal female patients were allocated to the Control group; informed consent was obtained from all the subjects. All the procedures involving human patients were approved by the ethics committee of The First Affiliated Hospital of Hainan Medical University. Bone samples were immediately fixed in 10% neutral buffered formalin (Sigma, USA) for 24 h for microcomputed tomography (micro-CT) analysis, and some of the samples that were used for histological analysis were decalcified in 15% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 4 weeks. In addition, some bone samples were incubated in 2.5% glutaraldehyde for 24 h at 4°C and decalcified in 15% EDTA (pH 7.4) for 4 weeks for transmission electron microscopy (TEM). The clinical characteristics of the patients are summarized in Table 1.
Reagents
Necrostatin 2 racemate (Nec-1s), GSK’872 and Resatorvid (TAK-242) were obtained from Selleck (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma‒Aldrich (St. Louis, MO, USA). TNF-α was purchased from PeproTech.
Animals
The study was approved by the ethics committee of The First Affiliated Hospital of Hainan Medical University. All the procedures involving animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Female Sprague Dawley (SD) rats (Animal Laboratory Center of Chongqing Medical University, Chongqing, China) were housed in a standard laboratory environment (12 h light/dark photocycle, temperature of 22 ± 2°C, and humidity of 50%-60%) with free access to water and a standard rodent diet for a 7-day adaptation period. Only 16-week-old female rats were used in this study, and random grouping was performed.
Ovariectomy (OVX) rat model
A total of 48 SD rats were randomly assigned to the ovariectomy (OVX) group (OVX surgery) and Control group (sham operation). OVX surgery and sham operation were performed as previously described (ref. 16). Briefly, rats were anesthetized with pentobarbital sodium (30–50 mg/kg), and bilateral OVX surgery and sham operation were performed on rats in the OVX group and Control group, respectively. All the rats gradually completely recovered and exhibited good wound healing after the OVX and sham operations.
Drug administration
Drugs included necrostatin 2 racemate (Nec-1s, Selleck, Shanghai, China) and GSK’872 (Selleck, Shanghai, China). Nec-1s and GSK’872 were used to block RIPK1 and RIPK3, respectively (ref. 47,48). Four weeks after the operation, 36 rats in the OVX group were randomly divided into the Vehicle group (10% dimethyl sulfoxide, intraperitoneal injection), Nec-1s group (300 µg/kg/d, intraperitoneal injection) and GSK’872 group (5 mg/kg/d, intraperitoneal injection). The animals in the Control group were intraperitoneally injected with Vehicle solution. All the rats in this study were sacrificed by cervical dislocation at 8 weeks after surgery as previously described (ref. 12).
Sample collection
All the animals were weighed at the beginning and end of the experiments. Eight weeks after the operation, bone samples were harvested as previously described (ref. 12,16). Briefly, both left and right proximal femurs (5 mm distal and proximal to Ward’s triangle) were dissected, and some of the samples were fixed in 10% neutral buffered formalin for 24 h for micro-CT. Then, some of these samples were decalcified in 15% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 4 weeks for H&E staining, immunofluorescence staining and immunohistochemical staining. Other bone samples were fixed in 2.5% glutaraldehyde for 24 h at 4°C and decalcified in 15% EDTA (pH 7.4) for 4 weeks for transmission electron microscopy (TEM).
H&E staining
Histopathological analysis was performed as described in a previous study (ref. 16). Paraffin-embedded bone sections from patients and OVX model rats were deparaffinized with xylene, rehydrated with an ethanol gradient and stained with hematoxylin and eosin (H&E). Histomorphometric analysis of bone microstructure and quantification of osteocyte numbers were performed with a light microscope (Olympus, Tokyo, Japan) equipped with cellSens Dimension 3.1 (Olympus, Tokyo, Japan).
Micro-CT
Bone samples from patients and rats were fixed in 10% neutral buffered formalin for 24 h. All bone tissues were scanned with a Bruker Micro-CT Skyscan 1276 system (Kontich, Belgium) using the following conditions: voxel size of 8.033484 µm, medium resolution, 85 kV, 200 uA, 1 mm Al filter and integration time 384 ms. Analysis was performed using the manufacturer’s evaluation software. Reconstruction was accomplished by NRecon (version 1.7.4.2). 3D images were obtained from contoured 2D images by methods that were based on the distance transformation of the grayscale original images (CTvox; version 3.3.0). 3D and 2D analyses were performed using CT Analyser software (version 1.20.3.0). Analyses of the bone microarchitecture were carried out in a region of interest (ROI), and the analyses included measurements of bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp).
Transmission electron microscopy (TEM)
TEM was performed as previously described (ref. 16). Bone tissues from patients and rats were dissected, fixed in 2.5% glutaraldehyde for 24 h at 4°C, washed with 0.1 M PBS (pH 7.4), and subsequently demineralized in 15% EDTA for 4 weeks at room temperature. After washing with 0.1 M PBS (pH 7.4), tissue fragments were treated with 2% osmium tetroxide for 2 h and block-stained with 2% uranyl acetate. After dehydration with an ethanol gradient, the bone tissues were embedded in epoxy resin and sliced into 80 nm ultrathin sections. The tissue sections were stained with uranyl acetate and lead citrate, and the sections were subsequently observed by transmission electron microscopy (Hitachi-7500, Hitachi, Tokyo, Japan).
Immunohistochemistry
Immunohistochemistry was performed as previously described (ref. 49). After deparaffinization with xylene and rehydration with an ethanol gradient, antigen recovery of the sections (4 µm thick) was performed with an Antigen Repair Kit (SBT10013, showbio, Shanghai, China) according to the manufacturer’s instructions. Then, the sections were subjected to immunohistochemical staining with the VECTASTAIN® Elite® ABC mini-PLUS Kit (Vector, Germany) according to the instruction manual as previously described (ref. 50). Briefly, bone sections from patients and rats were treated with BLOXALL (enzyme quench) solution and animal protein block, and then these sections were incubated with the following primary antibodies overnight at 4°C: anti-RIPK3 polyclonal antibody (ab152130, Abcam, Inc., Cambridge, MA, United States, 1:100 dilution), anti-RIPK3 polyclonal antibody (ab286151, Abcam, Inc., Cambridge, MA, United States, 1:100 dilution), anti-MLKL polyclonal antibody (GTX66191, GeneTex, USA, 1:100 dilution), anti-TNF-α polyclonal antibody (NBP1-19532, Novus, USA, 1:200 dilution), and anti-TLR4 polyclonal antibody (GTX31675, GeneTex, USA, 1:100 dilution). Then, sections were incubated with a biotinylated secondary antibody for 1 h. Finally, the sections were visualized by staining with DAB solution and counterstained with hematoxylin. These sections were observed under a light microscope (Olympus, Tokyo, Japan).
Immunofluorescence staining
Immunofluorescence was performed as previously described (ref. 16). After deparaffinizing and rehydrating bone sections (4 µm), antigen retrieval was performed. Then, the sections were treated with 0.3% Triton X-100 and blocked with 10% goat serum for 1 h at 37°C. Next, the sections were incubated with the following primary antibodies overnight at 4°C: anti-RIPK3 polyclonal antibody (GTX131188, GeneTex, USA, 1:100 dilution), anti-MLKL polyclonal antibody (GTX637574, GeneTex, USA, 1:100 dilution), anti-TNF-α polyclonal antibody (NBP1-19532, Novus, USA, 1:200 dilution), anti-TLR4 polyclonal antibody (GTX31675, GeneTex, USA, 1:100 dilution) and anti-TLR4 monoclonal antibody (NB100-56566, Novus, USA, 1:100 dilution). Subsequently, these sections were incubated with corresponding secondary antibodies conjugated with Alexa Fluor (Abcam, Inc., Cambridge, MA, United States) for 1 h and further stained with DAPI (P0131, Beyotime, Shanghai, China) for 10 min at room temperature in the dark. The images were captured by confocal laser scanning microscopy (FV3000, Olympus, Tokyo, Japan).
Cell culture and treatment
MLO-Y4 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). MLO-Y4 cells were cultured in DMEM (Gibco Life Technologies, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA, United States) and 1% penicillin‒streptomycin (Gibco Life Technologies, Carlsbad, CA, United States) in a humidified incubator maintained at 37°C and 5% CO2. All the cells were authenticated according to STR profiling and tested for mycoplasma contamination.
MLO-Y4 cells were pretreated with 1% DMSO, Nec-1s (100 µM), GSK’872 (0.25 µM) or TAK-242 (0.5 µM) for 30 min at 37°C and then treated with TNF-α (100 ng/ml) for 24 h when the cells had reached a proper density for further experimentation (ref. 16). After treatment, MLO-Y4 cells (5×106) were harvested and prefixed in 2.5% glutaraldehyde for 2 h. Then, these cells were observed by TEM as previously described. Cell slides were harvested, air dried, and fixed in 10% neutral buffered formalin solution for 10 min at room temperature. After treatment with 0.3% Triton X-100 for permeabilization, cells were subjected to immunofluorescence staining to detect RIPK3, MLKL and TLR4.
Western blotting analysis
Western blotting analysis was performed as previously described (ref. 16). MLO-Y4 cells (5 ×108) were homogenized and sonicated in ice-cold RIPA lysis buffer (Beyotime, Jiangsu, China) supplemented with a protease inhibitor cocktail. The total protein concentration was determined with a bicinchoninic acid protein assay kit (Beyotime, Nantong, Jiangsu, China) according to the manufacturer’s instructions. Target proteins were separated by SDS‒PAGE and then transferred to 0.22 µm polyvinylidene difluoride (PVDF, EMD Millipore, United States) membranes. After blocking with western blocking buffer (Beyotime, Shanghai, China) for 1 h at room temperature, the PVDF membranes were incubated overnight at 4°C with the following primary antibodies: anti-RIPK3 monoclonal antibody (#95702, Cell Signaling Technologies, Danvers, MA, USA; 1:1000 dilution), anti-MLKL monoclonal antibody (#37705, Cell Signaling Technologies, Danvers, MA, USA; 1:1000 dilution), anti-TLR4 monoclonal antibody (GTX57153, GeneTex, USA, 1:1000 dilution) and anti-GAPDH monoclonal antibody (AF1186, Beyotime, Shanghai, China, 1:1000 dilution). Then, the membranes were incubated with secondary antibodies. The protein bands of interest were visualized using an ECL detection kit (Beyotime, Nantong, Jiangsu, China). The intensity of the bands was analyzed using ImageJ version 1.54d. GAPDH served as the internal Control.
Statistical analysis
All the data are expressed as the mean ± standard deviation. The results were analyzed using Prism 9.0 software (GraphPad, San Diego, CA). Significance of the differences between groups was assessed by two-tailed Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s test for post hoc analysis. Correlations were analyzed with the linear regression F test. A p value of < 0.05 was considered statistically significant.