Male Sprague–Dawley (SD) rats (8-week-old) were obtained from Guangdong medical laboratory animal center. Rats were randomly assigned to NC and MSU groups (three rats per group). The necessary sample sizes were biometrically estimated. All the animals were housed in an environment with a temperature of 22 ± 1 ºC, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Guangzhou University institutional animal care and conducted according to the AAALAC and the IACUC guidelines (20211201006).
The rat model of gouty arthritis was established as previously described . Briefly, adaptive feeding was applied for one week. Rats in the MSU group were injected with 0.2 mL of 25 mg/mL MSU crystals solution into the right ankle joint cavity, while the NC group received the same volume of PBS at the same site. The rats were sacrificed at 24 h, 7 d, and 14 d, after which the joint cavity was exposed, dissected, and rinsed three times with 3 ml PBS (supplement Fig. 1).
Human neutrophil isolation
This study was approved by the Institutional Medical Ethics Committee of the Fourth Clinical Medical College of Guangzhou University of Chinese Medicine (K2021-082–01). The peripheral blood analyses of normal healthy donors (NHDs) were performed in accordance with institutional guidelines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood of NHDs by routine Ficoll density gradient centrifugation using standard protocols. In three independent experiments, the experiment was performed in triplicate with the same donor.
A total of 15 ml of human peripheral blood was collected and separated with Ficoll lymphocyte separation solution. Thereafter, the blood was centrifuged at 2000 rpm for 23 min to push the neutrophils into the upper layer of red blood cells. The erythrocyte lysate was then added at 1:3 for 20 min, centrifuged again at 1800 rpm for 5 min, and the concentration of neutrophils was measured.
NETs formation and collection
The neutrophils were incubated in a 6-well plate at 1 × 106 cells ml−1 and cultured for 30 min. After stimulating with MSU crystals (200 μg/ml) or PBS for 4 h at 37 ℃, the bottom of the plate was washed gently with cold PBS, and 1 ml preheated phenol red-free 1640 medium was added to the dish. Next, the bottom of the plate was repeatedly blown with a pipette to promote the dissolution of NETs into the liquid. Then, the liquid was centrifuged at 350 g, 4℃ for 10 min, and the NETs were collected.
SV40 transfected human osteoblasts (hFOB 1.19) were kindly provided by Guangzhou saiku biotechnology Osteoblastic Co.Ltd. hFOB resuscitation. Cells were passaged several times and then divided into 5 groups: (1) NC Group: the supernatants obtained from neutrophils were treated with PBS; (2) MSU Group: the supernatants obtained from neutrophils were treated with MSU crystals (200 μg/ml); (3) MSU + DNase Group: the supernatants obtained from neutrophils were treated with MSU crystals (200 μg/ml) and with specific DNA endonuclease DNase for 30 min; (4) MSU + Sivelestat Group: MSU crystals (200 μg/ml) for 30 min followed by addition of 44 nM Sivelestat sodium; (5) Sivelestat + MSU Group: 44 nM sivelestat sodium for 30 min followed by addition of MSU crystals (200 μg/ml).
The method of OC formation induced by THP-1 cells kindly provided by Procell life science and technology Co., Ltd, was performed as previously described . THP-1 cells were incubated with 100 ng/ml PMA for 48 h to induce osteoclast progenitor cells. Then, cells were cultured at 37 °C in the presence of 5% CO2; the media were replaced every three days. In order to further determine whether NETs affect osteoclast differentiation through OB, the osteoclast progenitor cells induced by THP-1 cells were co-cultured with the supernatant obtained in the previous step (NC group, MSU group) for 24 h.
Preparation of the MSU
MSU crystal was prepared following the protocol .
Cell Counting Kit-8 Assay
CCK-8 assay was used to detect the OB cell viability and proliferation. Briefly, the cells were cultured with different supernatants for 48 h. A 3 × 104 cells/ml were then added into 96-well plates (100 μl per well) and incubated at 37℃ under water-saturated 95% air and 5% CO2 atmosphere. After 24 h, 10 μL of CCK-8 reagent was added into 100 μL medium in each well, and the optical density (OD) was measured at a wavelength of 450 nm every 30 min until the OD was 1.0–2.0. The experiment was run in triplicate.
For immunofluorescence (IF) assay
The joint cavity was repeatedly rinsed with 1 ml HANK's containing 0.1%EDTA and cytospin to collect fluid. Samples were then dried at room temperature for 6–12 h, fixed with 4% paraformaldehyde, and then incubated with 0.2% Triton X-100 for permeabilization at room temperature. Then, samples were incubated with an antibody cocktail (final concentration: anti-MPO 1:50, anti-Histone H3 1:200) overnight at 4 °C, washed with PBST 3 times before being subjected to secondary antibodies for 1 h at room temperature. Finally, samples were washed with PBST 3 times, stained with DAPI, and analyzed.
For Hematoxylin–eosin (HE) assay
The tibia was fixed in formalin, decalcified in 10% ethylenediamine tetraacetic acid solution (EDTA, pH 7.4), embedded in paraffin, and sliced at 3 μm for hematoxylin–eosin (HE) staining. The stained tibia was imaged with a light microscope.
For immunohistochemistry (IHC) assay
The appropriate antibody diluent and antigen repair treatment methods were selected and performed following the manufacturer’s instructions. The paraffin-embedded tissue was dewaxed in xylene, dehydrated in ethanol, and blocked in 3% H2O2 for 5–10 min. The antigen was extracted in 95 °C EDTA buffer (0.01 M, pH 6.0) for 10–15 min. Sections were then incubated with anti-neutrophil elastase (ab68672, Abcam) overnight at 4 °C. After washing three times with PBS, samples were incubated with the secondary antibody coupled to HRP at 37 °C for 50 min at room temperature. Finally, DAB chromogen was added, and slices were washed with water and stained with hematoxylin.
For TRAP (Tartrate-Resistant Acid Phosphatase) assay
Tibia samples were embedded in paraffin and stained using the TRAP staining kit (Servicebio, China) according to the manufacturer’s instructions. OC was defined as TRAP-positive multi-nucleated cells. The cytoplasm was colored in wine red, and the nucleus in light blue.
Alkaline phosphatase (ALP) staining and activity assay
The tissue was fixed in 4% formalin for 10 min and washed thrice with PBS. According to the manufacturer's instructions, ALP staining and activity were performed with a staining kit (Solarbio, G1480).
Total proteins in tissues or cells were extracted with RIPA lysate buffer after adding a protease inhibitor cocktail. SDS-PAGE was applied to isolate equal amounts of protein, after which samples were transferred to the PVDF membrane. Consequently, the membrane was incubated with the primary antibody GAPDH (1:8000), ALP (1:5000), OPG (1:500), RANKL (1:500), TRAP (1:5000), Ctsk (1:500), RANK (1:1000), overnight at 4 °C and with HRP coupled secondary antibody for 1 h at room temperature. Anti-GAPDH antibody was utilized as an internal control. Protein expression levels were measured using an ECL substrate.
Quantitative real-time PCR
Total RNA from each sample was extracted with Trizol (MRC, TR118-500) and then reverse-transcribed using the M-MLV Reverse Transcriptase (Promega, M1705). Amplification was performed using the GoTaq® qPCR Master Mix (Promega, A6002) with 40 cycles at 95 °C for 15 s and 60 °C for 1 min on a StepOnePlus Real-Time PCR System. Table S1 lists the primers used in this study.
The levels of IL-1β(Dakewe Biotech, 1310122), TNF-α(Dakewe Biotech, 1317202), NE (Cusabio, CSB-E08847r), and MPO (Cusabio, CSB-E08722R) were detected using ELISA kit according to the manufacturer's protocol. Absorbance was measured at 450 nm.
Statistical analysis was performed with Graphpad Prism 9.0 Software. Results are presented as means ± SD of at least three independent experiments. Statistical analysis was performed using a t-test and one-way ANOVA. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 were considered statistically significant.