The procedure for the preparation of Rhubarb E is shown in <Fig. 1A>. Briefly, dried rhubarb (Rheum palmatum) purchased from the herbal medicine market in Gwangju was finely ground using a grinder and blended with 70% alcohol at a ratio of 1:10. The resulting mixture was kept at room temperature for 3 days, and the obtained extract was filtered twice using a Whatman No. 2 filter paper (Whatman International Ltd.). The filtrate was concentrated under reduced pressure then lyophilized to obtain powdered Rhubarb E, which was further dissolved in dimethyl sulfoxide (DMSO; VWR Life Science, Solon, OH, USA) to obtain a concentration of 50 mg/mL for experimental use.

To isolate primary bone marrow-derived macrophages (BMMs), the femur and tibia of 5 week old male C57BL/6 mice were obtained from Damool Science (Daejeon, Korea) were separated. Both ends of the tibia and femur were cut, and BMMs were collected from the intramedullary cavity using a 1 mL syringe. The collected BMMs were cultured for 3 days in MEM Alpha (Gibco, Carlsbad, CA, USA) rowth medium containing 10% FBS (FBS, 10%, Gibco, Carlsbad, CA, USA), 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA), and 30 ng/mL mouse macrophage colony-stimulating factor (M-CSF, 100 ng/ml, BioLegend, USA). Subsequently, the attached BMMs were treated with various concentrations of Rhubarb E in the presence of RANKL (100 ng/mL) to induce differentiation. The culture medium was replaced every other day.

The cytotoxicity of Rhubarb E was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BMMs were seeded in 96-well plates and exposed to different concentrations of Rhubarb E for 24 and 48 hours. Subsequently, the MTT reagent was added and the plates were incubated for 4 hours. The formazan crystals formed were solubilized with DMSO and the absorbance of the samples was measured at 565 nm absorbance using a microplate reader (Multiskan SkyHigh Microplate Reader, Thermo Fisher, USA). The viability of the BMMs was determined from the absorbance values recorded.

To assess the effect of Rhubarb E on the viability of BMMs, a live/dead cell imaging assay was employed. BMMs were treated with different concentrations of Rhubarb E (20 and 50 μg/mL) and cultured for 5 days. After the incubation period, the cells were stained with a fluorescent dye to between live cells (evidenced by green fluorescence) and distingush dead or compromised cells (indicated by red fluorescence). The stained cells were visualized using a fluorescence microscope imaging system (DMIL LED/DFC450C, Leica, Germany).

BMMs seeded in 24-well plates (2×10⁴ cells/well) were treated with different concentrations of Rhubarb E (10, 20 and 50 μg/mL) and cultured for 5 days in the presence of 30 ng/mL M-CSF and 100 ng/mL RANKL. The medium was changed every 2 days. The plates were washed three times with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 20 min, and then stained with a staining solution (0.12 M sodium acetate, 50 mM sodium tartrate, LB fast red violet salt, and naphthol AS-MX phosphate, pH 5.2). Subsequently, the plates were washed with PBS and air-dried. Finally, the TRAP-positive MNCs with three or more nuclei were counted under a quantified using Image J software (Media Cybernetics, Inc., Rockville, MD, USA).

Total RNA extraction from BMMs was performed using the Cell&Tissue RNA Extraction Kit (Infusion Tech, Gyeonggi-do, Korea). The obtained total RNA was used for cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed using Power SYBR Green PCR Master Mix (Life Technologies Ltd., Woolston Warrington, UK) and a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Target gene expression was quantified by the comparative CT method using StepOne Software ver. 2.10 (Applied Biosystems, Waltham, MA, USA). GAPDH was employed as an internal control. The primer sequences used for qPCR are shown in <Table 1>.

F-actin ring formation was evaluated to determine the extent of osteoclast maturation. BMMs were plated a 12-well plate at a density of 1×10⁵ cells/well and treated with different concentrations of Rhubarb E (10, 20, and 50 μg/mL) or left untreated. They were stimulated with 100 ng/mL RANKL for 5 days. The BMMs were washed, fixed, and stained with rhodamine-phalloidin. Then, the F-actin ring Actin-Stain™ Fluorescent Phalloidins (Red, Thermo Fisher, USA) formation was visualized using a fluorescence microscope (DMIL LED/DFC450C; Leica, Germany), and the size and number of the F-actin rings formed were calculated.

Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using the GraphPad Prism 9 software (GraphPad Software Inc, San Diego, CA, USA). Results are presented as Mean±SD. Significance levels are inficated as ^*p≤0.05, ^**p≤0.01.

To directly assess the effect of Rhubarb Ｅ on the viability of osteoclasts, the cytotoxicity of Rhubarb Ｅ on BMMs was examined. As shown in <Fig. 1B>, Rhubarb Ｅ showed no apparent cytotoxicity at the concentrations used in this study (10, 20, 50, and 100 μg/mL). A live/dead assay further demonstrated that Rhubarb Ｅ did not affect the viability of BMMs at the concentrations used <Fig. 1C>. These results suggested that the inhibitory effects of Rhubarb Ｅ were not related to its cytotoxic potential. Subsequent experiments to study osteoclast differentiation were based on these viability results.

For TRAP staining, BMMs were cultured with Rhubarb Ｅ at concentrations of 10, 20, and 50 μg/mL in the presence of 30 ng/mL M-CSF and 100 ng/mL RANKL for 5 days. interestingly, Rhubarb Ｅ significantly suppressed the number of TRAP-positive MNCs in the BMM cultures in a concentration-dependent manner <Fig. 2A, 2C>. Among the studied concentrations, the most robust inhibitory effect on RANK-induced TRAP positivity was observed at 50 μg/mL of Rhubarb Ｅ.

To validate the inhibitory effect of Rhubarb Ｅ on osteoclastogenesis, mRNA expression of the treated BMMs was analyzed. The expression of osteoclast-specific genes, such as cathepsin K, NFATc1, and TRAP, showed a significantly increase in the RANKL-treated group. However, the expression of these genes was significantly suppressed during osteoclastogenesis in the presence of Rhubarb Ｅ <Fig. 3A, 3B>. This downregulation of critical genes associated with osteoclastogenesis underscores the inhibitory role of Rhubarb Ｅ on osteoclast differentiation.

To further confirm the inhibitory effect of Rhubarb Ｅ on actin ring formation (an essential feature of mature osteoclasts induced by RANKL), immunofluorescence staining of F-actin rings was performed. While BMMs cultured with RANKL and M-CSF showed the formation of actin ring structures, as indicated by the rhodamine-phalloidin staining, Rhubarb Ｅ inhibited the development of F-actin ring structures in a concentration-dependent manner, as shown in <Fig. 4A, 4B>.This result further supports the role of Rhubarb Ｅ in inhibiting the RANKL-induced formation of mature osteoclasts.