Female DBA/1J, C57BL/6 and ICR mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. (Jiangsu, China). All mice were raised at the Animal Center of Jiangsu Province Academy of Traditional Chinese Medicine. This study was performed according to the Animal Welfare Law of China and approved by the Animal Ethics Committee of Jiangsu Province Academy of Traditional Chinese Medicine (AEWC-20200819-125).

Methotrexate (MTX, Shanghai Xinyi Pharmaceutical Co., Ltd.) and processed scorpions, scolopendra, astragali radix, and black soybean seed coats were purchased from the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine (Nanjing, Jiangsu, China). Freund’s adjuvant and bovine type II collagen were obtained from Chondrex (Redmond, WA, United States). Tartrate-resistant acid phosphatase (TRAP) staining kits were obtained from Sigma‒Aldrich (St. Louis, MO, United States). M-CSF was purchased from Pepro Technology (Rocky Hill, NJ, United States). IL-6, RANKL, OPG and Cathepsin K antibodies were purchased from AiFang Biological Company (Changsha, Hunan, China). Antibodies against phosphorylated p65, p65 and MMP9 were obtained from Cell Signaling Technology (Danvers, MA, United States).

The SF formula consists of 20 g Astragali Radix (dried root of A. membranaceus (Fisch.) Bge), 0.5 g scorpion (dried body of B. martensii Karsch), 0.5 g scolopendra (dried body of Scolopendra subspinipes mutilans L. Koch), and 30 g black soybean seed coats (seed coats of G. max (L.) Merr). The SF used in both animal and cell experiments was provided by the pharmacy of Jiangsu Province Hospital on Integration of Chinese and Western Medicine (Jiangsu, China) and prepared according to our previously published methods (Hua et al., 2021). Specifically, the animal components (scorpion and scolopendra) were ground through an 80-mesh sieve and further pulverized using a cryogenic ball mill. The botanical components (astragali radix and black soybean seed coats) were decocted in water, followed by freeze-drying at −80 C to obtain powder. In in vitro and in vivo experiments, the combined powders were dissolved in sterile water for use. The SF used for clinical administration in RA patients was manufactured by Jiangyin Tianjiang Pharmaceutical Co., Ltd., batch No. 2105317. The formulation and preparation method of SF for in vitro and in vivo experiments was consistent with that of the clinically used SF manufactured by Jiangyin Tianjiang Pharmaceutical Co., Ltd. Moreover, the SF batch used in the pharmacological and mechanistic studies was the same as that used in our recent article (Fan et al., 2025), thus the chemical profile characterized by UHPLC-QE-MS in that article applies directly here.

ICR mice were randomized divided into a normal control group and an SF-treated group (n = 5). Based on clinically equivalent human doses and our previous publication (Hua et al., 2021), SF was administered orally at the dose of 183 mg/kg/day for 3 months. The mice in the normal group were given an equal volume of double distilled water. After 3 months of continuous treatment, blood samples were collected via retro-orbital bleeding prior to euthanasia by cervical dislocation. Potential toxicity of SF was assessed by detecting serum biochemical indices.

The CIA model was established according to our previously published protocol, with an approximate success rate of 80% (Hua et al., 2021; Ling et al., 2021). The female DBA/1J mice were randomly divided into five groups (n = 6): the normal control group (Normal), CIA vehicle group (CIA Vehicle), MTX group (920 mg/kg), high-dose SF group (SF-H, 550 mg/kg) and low-dose SF group (SF-L, 183 mg/kg). The dosing regimens for MTX and SF were determined according to the clinically recommended doses for humans in reference to our previous publication (Hua et al., 2021). Drug administration was initiated 28 days after the first immunization with bovine type II collagen. SF was administered orally at 183 or 550 mg/kg/day, and MTX was administered at 920 mg/kg twice a week. Both normal and model control groups received equivalent volumes of double distilled water.

Mice were monitored for arthritis progression through regular assessments of arthritis scores, paw swelling, and body weight every 3–4 days after the first immunization. Arthritis severity was scored on a 0–4 scale for each limb according to established criteria (Hua et al., 2021), with the cumulative score of all four limbs (maximum 16) representing the individual mouse’s total arthritis score (Filippin et al., 2013). The swelling was evaluated by a paw swelling meter (Woodland Hills, CA, United States).

The mice were sacrificed by neck dislocation 90 days after treatment. The hind limb tissue sections were processed and stained with hematoxylin-eosin (H&E). The degree of histopathological damage was scored according to our previously described criteria (Ling et al., 2021). The hind limbs were scanned with a compact micro-computed tomography (micro-CT) scanner (Bruck SkyScan 1176, Germany). The bone mineral density (BMD) and bone volume fraction (BV/TV), which reflect bone mass, were evaluated with the built-in software.

Osteoclasts in the ankle joint sections of the mice were identified by TRAP staining. IL-6, RANKL, OPG and cathepsin K were immunologically localized by the respective primary antibodies. The images were processed by optical microscopy and analyze dby Aipathwell (Dogan et al., 2019; Guo et al., 2019; Paschalis et al., 2019; Maclean et al., 2020).

Bone marrow-derived mononuclear cells from the tibia and femur were isolated from 6-8-week-old C57BL/6 mice. The cells were cultured in α-MEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 30 ng/mL M-CSF at 37°C with 5% CO₂ for 3 days. The adherent cells left at the bottom of the culture dish were considered bone marrow-derived mononuclear cells.

SF was filter-sterilized through a 0.22 μm PVDF filter membrane for in vitro experiments. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay. Bone marrow-derived mononuclear cells were cultured in medium containing 30 ng/ml M-CSF. After adhesion, cells were treated with different concentrations of SF (0.01–20 mg/mL) for 72 h. Absorbance values were measured 2 h after the addition of CCK-8 reagent.

Bone marrow-derived mononuclear cells were pretreated with or without SF (0.1, 0.5, or 1 mg/mL) for 2 h and then stimulated with RANKL (100 ng/mL) for 5 days, after which the solution was changed every 2 days. On the fifth day of culture, osteoclasts were stained using a TRAP staining kit and observed with an optical microscope.

Bone marrow-derived mononuclear cells were pretreated with or without SF for 2 h and then stimulated with RANKL for 48 h. Total RNA was subsequently extracted and reverse-transcribed to cDNA. RT-qPCR analysis was performed using SYBR Green qPCR Master Mix with cDNA as a template. Relative gene expression was calculated using the ^2−ΔΔCT method. The primer sequences are presented in Table 1.

The expression levels of cathepsin K, p65 and phosphorylated p65 (p-p65) in bone marrow-derived mononuclear cells were detected by Western blotting. Analyses were performed using ImageJ software. In addition, bone marrow-derived mononuclear cells were treated with or without SF for 12 h and then stimulated with RANKL for 1 h. The nucleation of p65 was detected by immunofluorescence staining.

All antibodies and reagents used for Western blotting are shown in the Table 2.

The study followed the principles of the Declaration of Helsinki for humans and was approved by the ethics committee of the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine (2022-LWKY-038). Informed consent was obtained, and the study was registered at the Chinese Clinical Trials Registry (ChiCTR2200066446). All patients in the study met the American College of Rheumatology/European League Against Rheumatism-2010 (ACR/EULAR-2010) diagnostic criteria for RA. The inclusion criteria for the participants were as follows: age between 18 and 70 years; no use of slow-acting anti-rheumatic drugs or use of slow-acting anti-rheumatic drugs with a stable dosage for more than 6 weeks; no use of NSAIDS/glucocorticoids or use of NSAIDS/glucocorticoids with a stable dosage for more than 2 weeks. Exclusion criteria included: pregnancy, hepatic/renal dysfunction, hypertension (grade ≥2), significant cardiovascular/cerebrovascular diseases, malignancies, or other serious medical conditions.

Following the eligibility assessment, 16 RA patients were enrolled in this study and randomly divided into an MTX group and an SF + MTX group at a 1:1 ratio. Patients in both groups were scheduled to receive MTX tablets at an initial dosage of 10 mg/week, and the dosage was gradually reduced according to the symptom relief of joint swelling and pain. In addition to MTX, patients in the SF + MTX group were administered 6.3 g of SF orally per day. The treatment continued for 3 months.

The primary outcomes included the symptoms of RA (swollen joint count (SJC), rheumatoid factor (RF), tender joint count (TJC) and visual analog score (VAS)) and the serum levels of osteoclast-related proteins (IL-6, IL-10, OPG and TRACP). The secondary outcomes included liver and kidney function indices.

RF, liver and kidney function indices were tested in the clinical laboratory before medication (at 0 months), 1 month and 3 months after medication. The levels of IL-6, IL-10, OPG, and TRACP in patient serum were measured using ELISA kits (MULTISCIENCES) according to the manufacturer’s protocol. Due to the COVID-19 epidemic, several patients missed their scheduled hospital visits for follow-up examinations.

All data are presented as mean ± SEM. Intergroup comparisons were analyzed using one-way or two-way ANOVA. Tukey’s multiple comparison test was performed to evaluate differences between groups. Statistical significance was defined as P < 0.05.

The SF prescription contains two animal-derived medicines and two plant-derived medicines. Toxicity evaluation in ICR mice revealed no abnormalities in hepatic/renal function parameters or hematological indices following 3 months of SF administration (Figures 1A,B), indicating that SF is safe and non-toxic in ICR mice.

Drug administration was initiated 28 days after primary immunization (designated as day 0 in Figure 2A). To assess the therapeutic effect of SF, we measured the hind paw joint swelling and arthritic scores in CIA mice. Compared with the CIA vehicle group, both the low-dose SF (183 mg/kg) and high-dose SF (550 mg/kg) groups exhibited significant reductions in the joint swelling from day 10, with arthritis scores showing significant improvement from day 20 through the end of administration (Figure 2A). No significant differences were observed between the MTX and CIA vehicle groups for the joint swelling and arthritis scores. Furthermore, both low and high doses of SF significantly attenuated weight loss in CIA mice compared to CIA vehicle group, whereas MTX group showed no significant protective effect (Figure 2A). SF was significantly more effective than MTX in improving joint swelling and body weight in CIA mice.

H&E staining suggested collagen-induced synovial hyperplasia and cartilage destruction in the ankle joints of the mice, while SF reversed these effects, and the pathological scores were much lower than those of the CIA vehicle group (Figure 2B). TRAP staining was used to identify osteoclasts in the ankle joints of the mice. A large number of osteoclasts appeared in the ankle joints of CIA mice, and the number decreased significantly after treatment with SF (Figure 2B).

Micro-CT analysis was used to assess ankle joint bone erosion in CIA mice. In the normal group, the bone surface of the mice was smooth, and the bone structure was intact, whereas the CIA vehicle group exhibited severe bone erosion, joint bone destruction, and cavitation lesions (Figure 3A). Administration of both low- (183 mg/kg) and high- (550 mg/kg) dose SF significantly attenuated bone erosion in CIA mice (Figure 3A). Quantitative analysis revealed that bone mass parameters BMD and BV/TV were significantly reduced in the CIA vehicle group compared with controls. Notably, SF treatment markedly improved both BMD and BV/TV in CIA mice (Figure 3B). However, MTX treatment failed to significantly alter BMD or BV/TV compared with the vehicle, indicating that it had little effect on bone erosion.

Immunohistochemical analysis revealed the expression levels of osteoclast-related proteins, including IL-6, cathepsin K, OPG and RANKL, in mouse ankle joints. The expression levels of these proteins were significantly higher in the CIA vehicle group than in the normal group (Figure 4). Both low and high doses of SF significantly reduced the expression levels of IL-6 and cathepsin K in the ankle joints but had no significant effect on OPG or RANKL expression in CIA mice. These findings suggest that SF might modulate the downstream signaling pathway of RANKL.

In vivo experiments demonstrated that SF reduced osteoclasts numbers, however, its effects on osteoclast differentiation and mechanism remained unclear. To investigate this, we examined SF’s inhibitory effects on osteoclastogenesis in vitro. Figures 5A,B shows that TRAP-positive cells (> three nuclei) were observed after stimulation with RANKL, and the number of TRAP-positive osteoclasts decreased in a dose-dependent manner after SF (0.1, 0.5, or 1 mg/mL) treatment. We next evaluated the effect of SF on the expression of the osteoclast-related genes cathepsin K and MMP9. Quantitative PCR analysis revealed that RANKL-stimulated bone marrow-derived mononuclear cells showed significantly elevated mRNA levels of cathepsin K and MMP9, which were markedly downregulated by 48-h SF treatment for (Figures 5C,D). The viability of bone marrow-derived mononuclear cells was not affected by SF at concentrations up to 5 mg/mL (Figure 4E).

The NF-κB signaling pathway is crucial for RANKL-stimulated osteoclast differentiation (Lin et al., 2017). Therefore, the effect of SF on the RANKL-stimulated NF-κB signaling pathway in bone marrow-derived mononuclear cells was further investigated. After RANKL stimulation, the protein expression level of p-p65 increased significantly but decreased after treatment with different concentrations (0.1, 0.5, or 1 mg/mL) of SF (Figures 5F,G). p65 translocated into the nuclei of bone marrow-derived mononuclear cells after RANKL induction, and SF blocked the nuclear translocation of p65 (Figure 5H). Consistently, the expression of the cathepsin K protein was also inhibited by 48 h of SF treatment (Figures 5F,G). These results indicated that SF might suppress osteoclast differentiation by inhibiting the RANKL-stimulated NF-κB signaling pathway.

In this randomized clinical trial, RA patients were allocated to an MTX group and an SF + MTX group at a 1:1 ratio for 3 months of continuous drug administration. With the prolongation of medication time, the RF of patients in the SF + MTX group decreased significantly (Figure 6A). Notably, the addition of SF effectively reduced the TJC, SJC and VAS scores of pain in RA patients, while MTX alone had no significant effect (Figure 6A).

SF not only ameliorated clinical symptoms but also improved the levels of bone erosion-related proteins in RA patients. After t the 3-month intervention, the inflammatory state of the RA patients in the SF + MTX group improved, the levels of the proinflammatory cytokine IL-6 decreased, and the levels of the anti-inflammatory cytokine IL-10 increased in the serum (Figure 6B). OPG is a decoy receptor of RANKL and can inhibit the activation and promote the apoptosis of osteoclasts. TRACP is an important marker of bone destruction and osteoclast activation. As shown in Figure 6B, the serum level of OPG increased, and that of TRACP decreased significantly after 3 months of SF treatment (Figure 6B).

To examine the safety of SF, the hepatic and renal function parameters of each patient were measured at one and 3 months after SF treatment. Hepatic function tests revealed that ALT, AST, ALP, and γ-GT levels remained within normal limits throughout the study period before and after SF treatment (Figure 7A). Similarly, renal function parameters BUN and CRE levels were maintained within normal ranges and showed no significant difference before and after SF treatment (Figure 7B). These findings demonstrate that SF has no obvious toxicity to the liver or kidney at the clinically used dose and is a safe and effective drug for treating RA.