Complete Freund’s adjuvant (CFA) was obtained from Sigma Chemical Co. (St.Louis, MO, United States), and dexamethasone was purchased from Hubei Tianyao Pharmaceutical Co. (Wuhan, China). Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-15 (IL-15), and vascular endothelial growth factor (VEGF) enzyme-linked immunosorbent assay (ELISA) kits were purchased from ZhongShan Golden Bridge Biotechnology Co. (Beijing, China). TNF-α (bs-10802R), IL-1β (bs-6319R), IL-6 (bs-6309R), IL-10 (bs-0698R), IL-15 (bs-1829R), VEGF (bs-1313R), nuclear factor kappa-Bp65 (NF-κBp65) (bs-0465R), transient receptor potential channel-6 (TRPC-6) (bs-2393R), transient receptor potential melastatin-5 (TRPM-5) (bs-9047R) of rabbit polyclonal antibody, and goat anti-rabbit IgG H&L/HRP antibody (bs-40295G-HRP) were purchased from Beijing Biosen Biotechnology Co. (Beijing, China). High-performance liquid chromatography (HPLC)-grade methanol was purchased from American BCR company (Columbus, Ohio, United States). Watsons drinking water (Guangzhou Watsons Food & Beverage Co., Guangzhou, China) was used in this experiment. Other reagents were analytically pure (Tianjin Fuyu Fine Chemical Co., Tianjin, China.).

The plant was collected from Ceheng County, Guizhou, China, in August 2018 and was identified as Sabia parviflora Wall. by Professor Qingwen Sun. A voucher specimen (number 20180820) was deposited at the Traditional Chinese Medicine and Ethnic Medicine Laboratory of Guizhou University of Traditional Chinese Medicine.

The air-dried and crude powdered leaves of S. parviflora Wall (12.3 kg) were extracted with 70% ethanol (98 L × three times), each time for 2 hours. The combined filtrate was concentrated under reduced pressure to obtain the black extract (2,361.6 g) with a yield of 19.2%. The ethanol extract was extracted five times with petroleum ether, ethyl acetate, and water-saturated n-butanol, respectively. After vacuum drying under reduced pressure, the dried products of the petroleum ether layer (350.5 g), ethyl acetate layer (301.3 g), and water-saturated n-butanol layer (1,295.0 g) were obtained.

SPF Sprague–Dawley (SD) rats (180–200 g) were obtained from Changsha Tianqin Biotechnology Co., Ltd (Changsha, China). The standard rat chow was obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The rats were maintained under standard laboratory conditions (temperature 22 ± 1°C, humidity 60 ± 5%, and with 12 h light-dark cycle.) and given standard rodent chow and tap water before initiation of the experiment. All animal experiments were carried out in accordance with the Animal Ethics Committee of Guizhou University of Traditional Chinese Medicine on 25 June 2021 with the Approval No. 20210040.

The rats were adaptively fed for 1 week and their body weight and foot diameter were measured every week thereafter. A total of 96 SD rats were randomly divided into 12 groups of eight animals: 1) normal group, 2) model group, 3) positive control group (dexamethasone), 4) petroleum ether low-dose group, 5) petroleum ether middle-dose group, 6) petroleum ether high-dose group, 7) ethyl acetate low-dose group, 8) ethyl acetate middle-dose group, 9) ethyl acetate high-dose group 10) n-butanol low-dose group, 11) n-butanol middle-dose group, and 12) n-butanol high-dose group. The model was carried out for 2 weeks for all groups, excluding the normal group (each animal was given an intradermal injection of 0.2 ml CFA into the left hind footpad) according to a previously described method (Pan et al., 2017; Bao et al., 2019). The extracts were administered via gavage to all groups once a day for 3 weeks, with ordinary feeding after 2 weeks. The dosages of petroleum ether, ethyl acetate, n-butanol (high-, middle-, and low-dose groups), and the dexamethasone positive control groups were 0.64 g/kg, 0.32 g/kg, 0.16 g/kg, and 7.5 mg/kg, respectively.

The body weight of rats in each experimental group and the normal group were measured and recorded every 7 days from day zero to day 35. The changes in the ankle circumference were also evaluated using a Vernier caliper every 7 days from day zero to day 35.

After collecting blood from the abdominal aorta of rats, the anesthetized rats were killed, and the thymus and spleen were immediately removed and weighed. The index of the thymus and spleen is calculated as follows: thymus index = thymus mass/body mass × 1,000 and spleen index = spleen mass/body mass × 1,000 (Jing et al., 2019).

The rats were killed on day 35, and the ankle joints of rats were cut, fixed in 4% (w/v) paraformaldehyde, and decalcified in 10% ethylene-diamine-tetraacetic acid (EDTA) at 4°C for 1 month. The decalcified ankle tissues were dehydrated, embedded in wax, sectioned, and stained with hematoxylin-eosin. The stained tissues were sealed and then observed and photographed under a microscope using a Ba200 digital micro camera system (magnification, ×100, Motic China Group Co., Ltd., Xiamen, China).

The expressions of inflammatory proteins in synovial tissue were measured according to the immunohistochemical staining method (He et al., 2018). The treated synovial tissues were incubated with primary antibodies of TNF-α (1:200), IL-1β (1:400), IL-6 (1:400), IL-10 (1:400), IL-15 (1:400), NF-κBp65(1:500), VEGF (1:500), TRPC-6 (1:400), and TRPM-5 (1:400) overnight in a refrigerator at 4°C. The following day, the tissues were washed with phosphate-buffered solution (PBS) three times for 3 min. The tissues were incubated with the anti-goat antibody for 30 min at 37°C and washed with PBS three times for 3 min. The chromogenic agent, 3,3′-diaminobenzidine (DAB), was added for 10 min. The color was observed at room temperature, and the dyeing state was observed under a microscope. Then, the tissues were stained with hematoxylin for 5 min and examined under a microscope using a Ba200 digital micro camera system (magnification, ×100, Motic China Group Co., Ltd., Xiamen, China). The protein expression in synovial tissue was quantitatively analyzed by the average optical density (AOD) of protein according to a previously described method. (Wang, et al., 2021).

The rats were anesthetized by the intraperitoneal injection of 20% urethane, and 4 ml of blood was collected from the abdominal aorta in a labeled red glass. The serum was obtained from blood by cryogenic centrifugation (4°C, 3,000 rpm, and 15 min) and the serum levels of TNF-α, IL-1β, IL-6, IL-10, IL-15, and VEGF were quantified by enzyme-linked immunosorbent assay kits (ELISAs) following the manufacturer instructions.

Chromatographic analysis was performed on an Agilent-1260 HPLC system (Agilent Technologies Inc, Santa Clara, California, United States) coupled with a rapid separation diode array detector (DAD). Chromatographic separation was performed on a YMC-Pack ODS-A column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol (A) and water (B), and the flow rate was 1.0 ml/min. A linear gradient elution was employed for elution from 35% (A) (v/v) to 100% (A) (v/v) (0–50 min). The DAD detection wavelength was 210 nm, and the column temperature was 25°C. The injection volume was 10 μL.

Statistical analysis of the experimental data was conducted using GraphPad PRISM version 5.0 software and SPSS 17 software. The results are presented as mean ± standard deviation (SD). The differences between groups were analyzed by one-way analysis of variance (ANOVA) with the post hoc test. A p-value less than 0.05 was considered statistically significant, and a p-value less than 0.01 was considered for significant differences.

The body weight of rats in the model group showed a gradual increase, compared to the normal control from day 14 to day 35 (Figure 1A). The administration of S. parviflora Wall. leaf extracts, only a fraction of n-butanol (0.64 g/kg, 0.32 g/kg, and 0.16 g/kg), and fraction of ethyl acetate (0.64 g/kg) increased body weight from day 21 to day 35, compared to that of the model group (see Supplementary Table S1).

The swelling of the ankle joint of rats from day 7 to day 35 is shown in Figure 1B. The right foot of rats in other groups began to swell on day 7 (p < 0.01) compared to the normal rats. Compared with the RA model group, the swelling of rats’ paw of fraction of n-butanol (0.64 g/kg, 0.32 g/kg, and 0.16 g/kg) and fraction of ethyl acetate (0.64 g/kg) of 70% alcohol-soluble fraction of S. parviflora Wall. leaf-treated group was alleviated from day 21 (Figure 1B, Figure 1C) (p < 0.05 and p < 0.01) (see Supplementary Table S2).

Compared with the normal group, the thymus index (Figure 2B) and spleen index (Figure 2A) of the RA model group were significantly elevated (p < 0.01). In addition, the thymus index and spleen index of the n-butanol fractions (0.64 g/kg, 0.32 g/kg, and 0.16 g/kg) (p < 0.05 and p < 0.01) and ethyl acetate fraction (0.64 g/kg) (p < 0.05) of 70% alcohol-soluble fraction of S. parviflora Wall. leaves were lower than those of the model group. Also, the thymus index and spleen index of other treatment groups were higher than those of dexamethasone (p < 0.05 and p < 0.01) (see Supplementary Table S3).

The cells in the synovial tissue of the normal group were arranged orderly, without inflammatory cell infiltration and vascular proliferation, and the articular cartilage was smooth without damage (Figure 3A). The synovial tissue of the RA model group rats showed obvious synovial hyperplasia, disordered arrangement, and local joint fibrosis. The inflammatory cell infiltration appeared in the synovial stroma and small blood vessels increased in the lower synovial layer, resulting in the formation of a large number of pannus (Figure 3B). Compared with the model group, the n-butanol fractions (0.64 g/kg, 0.32 g/kg, and 0.16 g/kg) and ethyl acetate fraction (0.64 g/kg) of the 70% alcohol-soluble fraction of S. parviflora Wall. leaves reduced the pannus in the synovium and local inflammatory cells after treatment for 3 weeks (Figures 3I–L).

As demonstrated in Figure 4, the inflammatory cytokine levels of IL-1β, IL-6, IL-10, IL-15, TNF-α, and VEGF in the serum of the model group rats were significantly higher than those in the normal group rats. The IL-1β, IL-6, IL-10, IL-15, TNF-α, and VEGF levels in RA rats treated with the n-butanol fractions (0.64 g/kg, 0.32 g/kg, and 0.16 g/kg) (p < 0.05, p < 0.01) and ethyl acetate fraction (0.64 g/kg) (p < 0.05) of the 70% alcohol-soluble fraction of S. parviflora Wall. leaves decreased. In addition, the positive control dexamethasone (7.5 mg/kg/d) also reduced the release of these inflammatory cytokines with significant efficacy (p < 0.01). In addition, the inflammatory cytokine levels of other treatment groups were higher than those of dexamethasone (p < 0.05 and p < 0.01) (see Supplementary Table S4).

A large number of inflammatory proteins were expressed in RA rat synovial tissue as determined by immunohistochemical examination. Compared with the normal group, the expression of proteins IL-1β, IL-6, IL-10, IL-15, NF-κBp65, TNF-α, TRPC-6, TRPM-5, and VEGF (Figures 5–13) in synovial tissue of RA rats increased significantly (p < 0.01). Compared with the RA model group, the expression of these inflammatory proteins in synovial tissue decreased upon treatment with the n-butanol fractions (0.64 g/kg, 0.32 g/kg, and 0.16 g/kg) (p < 0.05, p < 0.01) of the 70% alcohol-soluble fraction of S. parviflora Wall. leaves. Treatment with dexamethasone (7.5 mg/kg/d) also relieved the inflammatory protein expression in synovial tissue of RA rats, with significant efficacy (p < 0.01). In addition, the expressions of proteins IL-1β, IL-6, IL-10, IL-15, and TNF-α of other treatment groups were higher than those of dexamethasone (p < 0.05) (see Supplementary Table S5 and Supplementary Table S6).

Six flavonoids: rutin (1), isorhamnetin-3-O-rutinoside (2), kaempferol-3-O-rutinoside (3), quercetin-3-O-gentiobioside (4), luteolin (5), and kaempferol 6) and three triterpenoid saponins: saikosaponin a (7), saikosaponin b₂ (8), and saikosaponin d 9) were identified from the n-butanol fraction of the 70% ethanol extract by HPLC (Figure 14A,B).