Animals
SD rats of SPF grade, 96 rats, male and female, 6 weeks old, body mass (200±20) g, purchased from Beijing HFK Bio-Technology Company. The experiments were reviewed and approved by the Experimental Animal Welfare Ethics Committee of the China-Japan Friendship Hospital, approval number: zryhyy21-21-03-03.
Drug
WBT tablets, specification: 0.5g/tablet, GMP Z20044066, manufactured by Liaoning Good Nurse Pharmaceutical (Group) Co Ltd; Methotrexate tablets, specification: 2.5mg/tablet, GMP H31020644, manufactured by Shanghai Xinyi Pharmaceutical Factory.
Main Reagents
Immunization Grade Bovine Type II Collagen (Item No. 20022), Freund's Incomplete Adjuvant (Item No. 7002), Chondrex; Protein Extract (Item No. MDL91201), Protease Inhibitor (Item No. MD912893), BCA Protein Concentration Assay Kit (Item No. MD913053), SDS-PAGE Prepared Gel Kit (Item: MD911919), DEPC (Item: MD911875), Interleukin 4 (IL-4) Kit (Item: MD123461), IL-1β Kit (Item: MD6758), OPG Kit (Item: MD123455), RANKL kit (item no. MD120008), MDL; medium protein molecular weight marker (item no. 26617), Thermo; β-actin (item no. ab8226), abcam; NFATc1 (item no. A1539), TRAF6 (item no. A16991) DKK1 (item no. A2562), WNT3A (Item: A0642), WNT10B (Item: A16717), β-catenin (Item: A19657), abclonal; TRIZOL (Item: 10296028), Invitrogen; anhydrous ethanol (Item: 10009218), isopropanol (Item: 40064360), abcam; NFATc1 (Item: A1539), TRAF6 (Item: A16991) DKK1 (Item. (item no. 40064360), Sinopharm; UltraPure Agarose (item no. 16500100), SuperScript III RT reverses transcription kit (item no. 11752050), Sybr qPCR mix (item no. 4472920), ABI-Invitrogen.
Main instruments
Microscope (DM3000 model), Leica; Decolorisation shaker (TS-100 model), Haimen Kylin-Bell Lab Instruments Company; Electrophoresis apparatus (BG-subMIDI model), Beijing Baygene Biotech Company; Cryogenic centrifuge (3-30K model), Sigma, Germany; SDS-PAGE electrophoresis system (Mini-PROTEAN® Tetra Cell with Mini Trans-Blot® Module And PowerPac™ Universal Power Supply), Enzyme Labeler (Model 550), BIO-Rad, USA; Gel Imaging System (GelDoc-It310), UVP, USA; Chemiluminescence Imaging System ( ChemiScope 6100), CLINX, China; benchtop high-speed frozen centrifuge (LEGEND MICRO 21R), spectrophotometer (Nanodrop lite), THERMO; electrophoresis instrument (EPS 300), gel imager (2500), biorad; fluorescence quantitative PCR instrument ( StepOne Software), Applied biosystems (USA), micro-CT (Siemens INVEON mmCT).
Animal grouping and models preparation
After 1 week of acclimatization, the rats were grouped by the random number table method with 12 animals in each group. They were divided into 8 groups: blank control group, CIA group, renal deficiency group, kidney deficiency pattern CIA group, CIA + WBT group, kidney deficiency pattern + WBT group, kidney deficiency pattern CIA + WBT group, and CIA + MTX group. The kidney deficiency pattern model was firstly prepared using the castration operation (removal of both ovaries and testes), and then the collagen-induced arthritis (CIA) model was prepared 4 weeks later using bovine type II collagen and incomplete Freund's adjuvant induction.
Medications
The usual clinical dose of WBT is 2g tid, i.e. 6g/d. According to the "Human and Animal Body Surface Area Conversion Equivalent Dose Ratio Table", if the clinical dose for humans is Xmg/kg, the dose for rats = 6.3Xmg/kg, e.g. the human body weight is 60kg, the rat dose is 0.63g/kg QD; once a day for 8 weeks, dissolved in physiological saline to make a solution with a concentration of 0.25g/ml, ready to use. The usual clinical dose of methotrexate tablets is 10mg qw, and the calculated dose for rats is 1.05mg/kg qw; once a week for 8 weeks, dissolved in saline to make a solution of 0.25mg/ml, ready to use.
After the model was successfully replicated, the animals were performed intragastric administration. Apart from the methotrexate once a week in the CIA+MTX group, also saline the rest of the week. Equal amounts of saline every day in the blank control group, the CIA group, the kidney deficiency pattern group and the kidney deficiency pattern CIA group. The specific animal groups and intervention methods are shown in Table 1.
(Table1)
Sampling Method
In each group, blood was obtained by execution at the end of the intragastrical cycle. Whole blood was collected from the abdominal aorta and centrifuged at 3000r/min for 15 min (10 cm radius) to obtain the supernatant serum, which was stored at -80°C for subsequent enzyme-linked immunosorbent assay (Elisa). After execution, the hind limb ankle joints were quickly exposed and cut off bilaterally to remove excess skin and soft tissue around the joints. The left hind limb was fixed in 4% paraformaldehyde for 48h, then the residual fluid was washed off the joint with running water and the joint was placed in EDTA decalcifying solution, which was changed periodically until a 1ml syringe needle could easily penetrate into the joint bone as the criterion for complete decalcification, which took approximately 2 months to prepare for paraffin sectioning. The right hind limb was placed in a labeled specimen bag and immediately frozen at -80℃ for Real-Time quantitative fluorescence PCR (Real-Time PCR) and protein immunoblotting (Western Blot, WB).
Observations and methods
Elisa test for IL-1β, IL-4, OPG and RANKL in rat serum
The concentrations of IL-6, IL-1, TNF-α, IL-4, IL-10 and OPG in serum were measured according to the operation method of Elisa kit instructions. The absorbance (OD) was measured at 450nm. The regression equation of the standard curve was calculated according to the concentration and OD value, and the logistic curve (four parameters) was used to fit the model.
Real-Time PCR detection of mir-335-5p, RANKL and OPG expression in rat ankle bone tissue
Total RNA was extracted from the samples by Trizol method, and the concentration and purity of RNA were determined using a nucleic acid concentration meter. cDNA was synthesized by reverse transcription using an invitrogen reverse transcription kit, superscript III. A Real-time PCR reaction system was established and reacted on a fluorescent quantitative PCR instrument. β-actin was used as an internal reference. The relative expression of the genes was calculated by the △△CT attenuation. The primer design of this experiment is shown in Table 2.
(Table 2)
Western blot detection of DKK-1, wnt3a, wnt10b, β-catenin and RANK expression in rat ankle bone tissues
Rat ankle tissues were homogenized with liquid nitrogen, lysed in an ice bath, centrifuged at 4℃ and 12000rpm for 15min, and the supernatant was collected. The protein concentration was determined by BCA protein quantification method. The protein bands separated on the gel were transferred to PVDF membrane by SDS-PAGE gel electrophoresis. This membrane was sealed and preserved in the blocking solution for 1h. The primary antibody was diluted in the blocking solution and reacted overnight at 4°C. The secondary antibody was diluted 300 times with 1×TBST and applied for 60 min. The bands were analysed for grey scale values and relative protein expression.
Detection of bone erosion in rat ankle joint by micro-CT
The ankles of rats were fixed in 4% paraformaldehyde for 48h and the residual fluid was washed off the joint with running water. A micro-CT scan was performed and the bone was reconstructed in three dimensions to assess the bone erosion.
Statistical methods
SPSS 25.0 statistical software was used for data analysis. The data results were presented in graphical form. Measurement data were described as mean ± standard deviation. As to the comparison among the groups, one-way ANOVA was used if the data were normally distributed and met the variance, and non-parametric tests were used if they did not follow a normal distribution or did not meet the variance. All statistical tests were two-sided, and differences were considered statistically significant at p<0.05.