2.1 Plant materials
EMS is a combination of equal parts Phellodendri Cortex (Huangbai, specimen number 1901200062), the bark of Phellodendron chinensis Schneid (Rutaceae), and Atractylodis Rhizoma (Cangzhu, specimen number 1902120322), the rhizome of Atractylodes lancea (Thunb.) DC.(Asteraceae) in 1:1 ratio. All herbs were obtained from Anhui Puren Herbal Pieces Co., Ltd (Bozhou, China) and identified by Dr. Liu SJ (School of Pharmacy, Anhui University of Chinese Medicine). An authenticated sample for each sample was preserved (ID: EMS-19-01) in the Herbarium of herbal medicinal, School of Pharmacy, Anhui University of Chinese Medicine (Hefei, China) (20).
First, dried and crushed Phellodendri Cortex and Atractylodis Rhizoma, each weighted 105 g. Then, the samples were extracted using water at ratios of 10:1, 8:1, and 6:1 for 1.5 h, 1 h, and 0.5 h, respectively. After filtered the mixture three times, combined the filtrate and place it in a water bath at 60°C to evaporate. Concentrated the filtrate until it reaches 500 to 800 mL, then extracted it with petroleum ether and ethyl acetate in equal parts five times. Finally, the ethyl acetate part from EMS was concentrated to the dosage required for the experiment (calculated as the amount of crude drug) used a rotary evaporator. Our research team had previously conducted UPLC testing on EMS extracts(12).
2.2 Reagents and Antibodies
NEK7 antibody was purchased from Abcam (Cambridge, UK). NLRP3 antibody and Caspase-1 antibody were obtained from Servicebio (Guangzhou, China). ASC antibody, β-actin antibody and Secondary antibodies were purchased from Proteintech (Wuhan, China). The BCG vaccine was purchased from Beijing Biological Products Institute Co., Ltd (Beijing, China). Lyophilized chicken type II collagen was purchased from Chondrex (Washington DC, USA). Liquid paraffin was purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). MTX was supplied by Shanghai Xinyi Medical Technology Co., Ltd (Shanghai, China). Lymphocyte separation solution was purchased from Tianjin Haoyang Biological Muanufacture Co., Ltd (Tianjin, China). NEK7 small interfering RNA (NEK7 siRNA) and Mus NEK7 were purchased from GenePharma (Shanghai, China). Dynabeads™ Protein G was purchased from Beyotime Biotechnology (Shanghai, China).
2.3 Animals
Eight-week-old male DBA/1 mice were acquired from the SLAC Laboratory Animal Centre (SCXK (Hu) 2017-0005) and housed at the Laboratory Animal Centre, Anhui University of Traditional Chinese Medicine. The mice were housed 5 per cage (130×200×300 mm) under standard laboratory conditions (22–26°C controlled temperature, 12 hours of light and 12 hours of darkness). The Anhui University of Traditional Chinese Medicine Ethics Committee approved the research methodology [protocol number (202214)].
2.4 Establishment of CIA mouse model and treatment
Ten milligrams of freeze-dried type II collagen from chicken was dissolved overnight at 4°C under gentle stirring in 5 mL of 0.05 M acetic acid to generate a stock solution. On day 0, CII was emulsified in equal volumes with complete Freund's adjuvant (CFA) at a concentration of 4 mg/mL. Mice were then administered 150 µl of the emulsion intradermally at the back. On day 21, a boosted vaccine was administered using 100 µl emulsion containing CII emulsified with CFA(23).
The collagen-induced arthritis (CIA) model was induced on day 0. The mice were randomly divided into 6 groups: normal, model, EMS(1 g/kg), EMS(2 g/kg), EMS(4 g/kg), MTX(2 mg/kg). Day 28 after primary immunisation. The EMS (1 g/kg), EMS(2 g/kg), EMS(4 g/kg) groups were administered the treatment by gavage once per day for 28 day. The MTX (2 mg/kg) group was administered the treatment by gavage every three days for 10 times.
2.5 Evaluation of arthritis
The arthritis score consisted of two parts: the arthritis index score and the joint swelling score. Beginning on day 28 after immunization, arthritis index and joint swelling were evaluated every three days. The arthritis index based on the scoring criteria listed in the Supplementary table1 (24). The joint swelling score was based on the ankle and knuckle joints of the mice, with each joint swelling counting for 1 point, with a maximum score of 24 points(25).
2.6 Radiological examination of the ankle joint
After the mice were sacrificed, the left hind ankle joint of each group was placed under X-ray, and the bone and joint were analyzed to observe whether the joint was swollen, broken or deformed.
2.7 Measurement of thymus index and spleen index
On day 60, the mice were sacrificed. Than, the thymus and spleen tissues were harvested for weighing and recording. The ratios of thymus/spleen weight to the body weight of each mouse were used to calculate the thymus and spleen index (g/g).
2.8 H&E staining
The ankle joints of mice were isolated, fixed with 4% paraformaldehyde, and subsequently embedded in paraffin. The mouse ankle joints within the embedding were sliced into 5 mm segments, subsequently dyed with H&E, and thoroughly analyzed via light microscopy.
2.9 Anti-tartrate-resistant acid phosphatase (Trap) staining
The paraffin sections were deparaffinized and the substrate solution was prepared according to the instructions of the Trap staining kit (Sevicebio, China). The sections were mixed with the substrate solution, incubated for 1 h away from light, washed with tap water for 1 min and then stained with hematoxylin for 2 min. The sections were dehydrated, sealed, and observed under the microscope.
2.10 Immunohistochemistry
Immunohistochemistry experiment was performed using a one-step immunohistochemistry kit (Keygen Biotech, China). Tissue sections were treated with peroxidase blocking solution followed by incubation with fetal bovine serum protein to block non-specific binding. Sections were then incubated overnight at 4°C using primary antibody. The next day the sections were washed and then incubated with secondary antibody at 37°C for 1 h. Sections were treated with diaminobenzidine (DAB) staining solution and then sealed with neutral resin adhesive and the sections were visualized under the microscope.
2.11 Cell Preparation Extraction
After the mice were sacrificed under anesthesia with sodium pentobarbital, the mice were soaked in bromogeramine and transported to a sterile environment. The abdominal fur of each mouse was cut off, and approximately 5 mL PBS (containing 1% penicillin and streptomycin) was injected into the abdominal cavity. The abdomen of the mouse was massaged for 3–5 min to enrich the macrophages in the abdominal cavity. Finally, separation of the cells through centrifugation(26).
T/B-cell. Tissues from the spleen and thymus of mice were aseptically extracted from the ultraclean table and ground with glass syringes separated by 120 nylon cloth, and the cell suspension was collected. After centrifugation, the cells were suspended in 10% FBS supplemented RPMI 1640 and then added to 96-well plates at a density of 1×105 cells/well.
2.12 T/B-cell proliferation
T and B cells were stimulated with 5 mg/L Con A or 4 mg/L LPS for 48 h. CCK-8 (10/µl) was added to each well 2 hours before the culture ended, and the microplate reader was used to measure the absorbance at 450 nm.
2.13 Peparation of EMS-containing serum
Thirty SD rats were taken and randomly divided into blank control group and EMS(3g/kg) group.The EMS group was given the EMS by gavage for 7 days, once a day. One hour after the last administration, the rats were anaesthetised with pentobarbital sodium, blood was taken from the abdominal aorta. EMS-containing serum was collected after centrifugation and filtration, which was inactivated at 56℃ for 1h, filtered and sterilized by 0.22 µm filtration, and put into − 20℃ for spare.
2.14 Cell culture and grouping
RAW264.7 cells were inoculated in the culture flask and cultured with DMEM (SparkJade, China) containing 10% FBS (Servicebio, China), 1%100U/mL, and 100µg/mL streptomycin at 37◦C and 5%CO2. In vitro study, the cells were divided into 6 groups: Control, model (LPS + ATP), 10% EMS-containing serum (LPS + ATP + 10%EMS), NEK7 siRNA (NEK7 siRNA + LPS + ATP), Mus NEK7 (Mus NEK7 + LPS + ATP), and Mus NEK7 + 10%EMS. The NEK7 siRNA and Mus NEK7 groups were first transfected and then RAW264.7 cells were stimulated using LPS (Sigma, USA) 100ng/ml for 4h. 5mM ATP (Solarbio, China) was added during the last 30 min according to the classical activation method of the NLRP3 inflammasome. After successful modeling, the EMS-containing serum was added to each administration group and incubated for 24 h.
2.15 Transfection of RAW264.7 cells
Reagent 1 was prepared by adding 100 pmol of NEK7 siRNA or 4µg Mus NEK7 to 250 µL of OPTI-MEM medium at room temperature for 5 min. 5 µL of Lipo 2000 was added to 250 µL of OPTI-MEM medium at room temperature for 5 min to prepare reagent 2. Reagent 2 was then slowly added to reagent 1, mixed well and allowed to stand at room temperature for 20 min. Cells were added to 500 µL of the mixture, and the medium was changed to complete medium after 6 h to continue the culture.
2.16 Immunoblotting analyses
The protein levels of NEK7 were determined by immunoblotting. Cells/Tissues were lysed by RIPA buffer (Beyotime Biotechnology) with PMSF (Beyotime Biotechnology). The protein lysates were transferred to a 1.5 mL tube and stored at a temperature of − 20°C until use. Subsequently, the cellular proteins were separated using SDS‒PAGE. Than the proteins had been transferred to PVDF membranes. Blots were probed using antibodies overnight followed by incubation with HRP conjugated secondary antibodies. Signals were visualized using ECL substrates (Sparklade). The protein expression was normalized to that of endogenous β-actin.
2.17 Co-immunoprecipitation (Co-IP)
Briefly, whole cell lysis products were prepared with RIPA lysis buffer containing protease inhibitors. The concentration of samples was determined by BCA protein assay. 200µL of protein supernatants diluted to a uniform concentration were shaking with 5µL of indicator antibody (anti-NEK7) or 5µL IgG at 4°C overnight. Next, the samples were incubated with 20µL of Dynabeads™ Protein G shaking at 4°C for 6h. Finally, the levels of proteins were analysed by western blot.
2.18 Immunofluorescence
Cells were fixed for 25 min in 4% paraformaldehyde solution, followed by treatment with Triton-100 for 15 min. The cells are then shaken 3 times with PBS and sealed for for 2 hours with goat serum. The ASC primary antibodies, diluted at a ratio of 1:1000, were added and left at 4°C to incubate overnight. The secondary antibody (1:500 dilution) was added on the second day. After cleaning, DAPI was added to the cells, followed by anti-fluorescence quencher. Finally, the staining was observed by fluorescence microscopy.
2.19 Enzyme-linked immunosorbent assay
Using tubes containing 1 mg/mL disodium ethylenediaminetetraacetate dihydrate (Na2-EDTA) concentration to collect blood samples after overnight fasting. And the IL-1β and IL-18 levels in serum were determined utilizing the IL-1β (Multi Sciences) and IL-18 (Elabscience) respectively. Performed the ELISA assays following the protocol provided by the manufacturer.
2.20 Data and statistical Analysis
The experimental results were organized using Excel, and the data were integrated and imported into SPSS 23.0 (SPSS, Chicago) for statistical analysis and GraphPad Prism 8.0 (GraphPad, USA) for graphing. Continuous variables obeying normal distribution were expressed using mean ± standard deviation (x̅±s), and normally distributed data that have homogeneous variance were compared using T-test (two groups) and one-way ANOVA. Comparisons of non-normally distributed or multiple groups of normally distributed data that did not have homogeneous variance were performed using the Kruskal-Wallis H test. The test level was two-sided 0.05, and for multiple comparisons, the Bonferroni method was used for correction.