N-Butanol Extract of Modified You-Gui-Yin Attenuates Osteoclastogenesis and Ameliorates Osteoporosis by Inhibiting RANKL-Mediated NF-κB Signaling

Postmenopausal Osteoporosis (PMOP) is the most prevalent primary osteoporosis, attributable to an imbalance in osteoblast and osteoclast activity. Modified You-Gui-Yin (MYGY), a traditional Chinese herbal formula, is able to effectively treat PMOP, while the critical components and pharmacological mechanisms of MYGY are still unclear. In this study, we aimed to investigate the therapeutic effects and underlying mechanisms of N-butanol extract of MYGY (MYGY-Nb) in ovariectomized (OVX)-induced osteoporosis mice. Histological staining and micro-computed tomography (μCT) analysis showed that MYGY-Nb was more effective in the suppression of OVX-induced bone loss than MYGY original formula. Subsequently, liquid chromatography and mass spectrometry analysis identified 16 critical compounds of MYGY-Nb and some of them are reported to affect osteoclast functions. Furthermore, in vivo and in vitro experiments demonstrated that MYGY-Nb significantly attenuated osteoclastogenesis by down-regulating RANKL-mediated NF-κB signaling. In conclusion, our study indicated that MYGY-Nb suppresses NF-κB signaling and osteoclast formation to mitigate bone loss in PMOP, implying that MYGY-Nb and its compounds are potential candidates for development of anti-PMOP drugs.


INTRODUCTION
Osteoporosis is a skeletal disease characterized by reduced bone mass density and strength, increased skeletal fragility and fractures (1). As the population ages, the worldwide patients caused by osteoporosis might well reach 221 million by 2050 (2)(3)(4), and the medical and socioeconomic burden of osteoporosis, particularly postmenopausal osteoporosis (PMOP), will increase further (5). PMOP, mainly contributed by menopausethe cessation of ovarian function, is the most common primary osteoporosis. However, due to the cost and side effects of available drugs, most cases of PMOP have not been effectively treated so far (6). Therefore, the development of novel effective therapeutic agents with minimal side effects for the PMOP treatment is urgently needed (7).
Traditional Chinese medicine (TCM) has been used for thousands of years to treat various chronic diseases, especially osteoporosis (8)(9)(10)(11). The Modified You-Gui-Yin (MYGY) is a well-known TCM formula, which is derived from You-Gui-Yin classic prescription recorded in Jing Yue Quan Shu written by Jingyue Zhang in the Ming Dynasty. The MYGY consists of ten herbs: Persicae semen, Cinnamomi cortex, Monkshood root, Eucommiae cortex, Lycii fructus, Dioscoreae rhizome, Carthami flos, Corni fructus, Glycyrrhizae radix and Preparata rehmanniae radix (Table 1), and has been widely applied to treat various chronic diseases with fewer side effects (12,13). Our recent study has shown that MYGY-based therapies achieve significant clinical outcomes in PMOP management (14). In addition, several herbs of MYGY have been reported to ameliorate osteoporosis by inhibiting osteoclast activity, such as Glycyrrhizae radix, Dioscoreae rhizoma, Corni fructus (12,15,16). However, the underlying mechanisms and functional compounds of MYGY in the treatment of PMOP have not been fully elucidated, which limits the further development and application of MYGY.
In the process of bone reconstruction, the cumulative imbalance in the activity of osteoblasts and osteoclasts can lead to a decrease in bone mass, which is fundamental to the pathogenesis of PMOP (17). Of note, this imbalance is mainly due to the osteoclast reabsorption of bone in excess of osteoblasts production of mineralized extracellular matrix (18). Osteoclasts are produced by the differentiation of precursors from a monocyte/macrophage lineage that is derived from bone marrow hematopoietic stem cells (19). Thus, the prevention of these stem cells from differentiation of osteoclast is one of the most important strategies in PMOP treatment.
The survival, proliferation, differentiation and activation of osteoclasts require the presence of RANKL (receptor activator of nuclear factor kappa B ligand) and M-CSF (macrophage colonystimulating factor) (20). In the process of osteoclastogenesis, RANKL interacts with RANK (receptor of RANKL) to induce the activation of NF-kB through the recruitment of adapter molecules such as TRAF6 (TNF receptor-associated factor 6), the degradation of IkB (the suppressor of NF-kB), and the phosphorylation of p65 (the subunit of NF-kB). These signaling cascades subsequently activate NFATc1 (nuclear factor of activated T cells, cytoplasmic 1), known as the primary transcription factor for osteoclastogenesis (21,22). During the postmenopausal period, the expression of various inflammation inhibitors was found to decrease, but RANKL increased significantly, suggesting a critical role for RANKL in PMOP (23). And NF-kB signaling pathway participates in regulating the proliferation and function of osteoclasts (24)(25)(26)(27). Thus, the suppression of osteoclast formation by targeting the RANKL-mediated NF-kB signaling pathway is probably a potential treatment strategy for osteoclastogenesis-related disorders, especially for PMOP.
In this study, we investigated the impact of N-butanol extract of MYGY (MYGY-Nb) on OVX mice and primary bone marrow-derived monocytes (BMMs). We found that MYGY-Nb possessed a stronger anti-osteoporosis effect with attenuating bone loss and osteoclast formation. Moreover, the mechanism by which MYGY-Nb suppressed osteoclastogenesis might be that MYGY-Nb inhibited RANKL-mediated NF-kB signaling. Taken together, our findings implicated that MYGY-Nb might be an effective medicine for treating PMOP.

Preparation of MYGY-Nb
All herbs of MYGY (Table 1) were provided by the First Affiliated Hospital of Zhejiang Chinese Medical University (Hangzhou, China). The N-butanol extract of MYGY was prepared as follows: Rou gui and Tao ren were mixed in a ratio of 1:2, soaked in 60% ethanol for 1 h, and then extracted 3 times by reflux method for 1.5 hours each. Other eight herbs including Fu zi, Du zhong, Gou qi, Shan yao, Hong hua, Shan zhu yu, Gan cao and Shu di huang were mixed in a1:3:2:2:2:1:2:1 ratio, steeped in 12 volumes of distilled water for 1 hour, and then extracted 3 times for 1.5 h each. Afterwards, the above two extracts were mixed and concentrated into MYGY extractum (3.4 g crude drug/mL). The above procedure is the preparation method of MYGY concentrate. After that, to extract its optimal anti-osteoporosis fraction, 95 ml MYGY extractum was dissolved into 300 ml distilled water, then, 300 ml N-butanol was mixed in equal proportion. After overnight emulsification and precipitation, the supernatant was collected and again extracted by the above method 3 times. Then, the supernatant was concentrated under reduced pressure until the N-butanol was completely evaporated to obtain the crude extract powder, namely MYGY-Nb. The MYGY-Nb powder was dissolved in 300ml sterile distilled water and stored at -20°C.
The MS E continuum mode of the SYNAPT G2-Si mass spectrometer was used to detect constituents of MYGY-Nb in the scan range of m/z 50~1500. In the ESI -/ESI + modes, the capillary and cone well voltages were 3.0 kV and 40 V, respectively, and the argon flow rate was 0.15 mL/min. The cone well gas flow rate was 50 L/h at 120°C, and the desolvation flow rate was 800 L/h at 400°C. The 100 ng/mL solution of leucine-enkephalin (ESI -: m/z 555.261 5, ESI + : m/z 556.2771) was used as a calibration standard (Lock Spray TM) with a volume flow rate of 15 mL/min, and the instrument was calibrated with sodium formate for accurate mass determination. The UNIFI V1.8 software was used for data analysis.

Animal Grouping and Drug Administration
10-week-old female C57BL/6J mice, supplied by Zhejiang Chinese Medical University Animal Center (Hangzhou, China), were maintained under standard feeding conditions with fresh water and food freely available. All mouse experiments were conducted in the pathogen-free laboratory of the Animal Center of Zhejiang Chinese Medical University (Hangzhou, China). Animals were randomly assigned into four groups (n = 6 in each group): (1) sham group, subjected to sham operation, (2) ovariectomy group (OVX), subjected to bilateral ovariectomy, (3) OVX+MYGY-Nb group, subjected to bilateral ovariectomy and then orally administrated MYGY-Nb extraction (20 mL/kg/day) for 12 weeks. (4) OVX+MYGY group, subjected to bilateral ovariectomy and then orally administrated MYGY extraction (20 mL/kg/day) for 12 weeks. The mice in other groups were addressed with the same dose of physiologic saline.

Micro-Computed Tomography (mCT) Analysis
After the last administration, mice were sacrificed by pentobarbital sodium overdose, and then their femurs were isolated for mCT scanning (Skyscan1176, Belgium) at a 10 µm resolution. The three-dimensional (3D) structure reconstruction of the distal femurs was performed using the NRecon software. The bone mineral density (BMD), trabecular separation (Tb.Sp, mm), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm) and bone volume fraction (BV/TV, %) were analyzed as described previously (28).

Cell Viability Assays
Cell Counting Kit-8 (CCK-8, Bioss, Beijing, China) assay was used to examine the viability of cells. The BMMs were seeded into 96-well plates at a density of 1 × 10 4 cells/well. After 24 h, BMMs were treated with 0, 10, and 100 mg/mL MYGY-Nb for 24, 48 or 72 h. Then the CCK8 solution was added to the culture medium and incubated at 37°C for 2 h. The absorbance was measured by BioTek Synergy HT spectrophotometer at absorbance was measured at 450 nm.

Western Blotting Analysis
The BMMs were cultured with 30 ng/mL M-CSF, 50 ng/mL RANKL and MYGY-Nb (0 or 10 mg/mL) for 4 days, and then mixed with RIPA buffer to extract total protein, the concentration of which was determined by bicinchoninic acid (BCA). Proteins were separated on 10% SDS-PAGE gels (20 mg/lane) and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 1 h, followed by incubating with primary antibodies (IKBa, 1:1000 dilution, Cell Signaling Technology), (p65, 1:1000 dilution, Cell Signaling Technology), (p-p65, 1:1000 dilution, Cell Signaling Technology), (NFATc1, 1:1000 dilution, Novus Biologicals) and (bactin, 1:10,000 dilution, Sigma-Aldrich) overnight at 4°C. After incubation with the corresponding secondary antibodies, proteins on the membrane were detected by the ultra ECL reagent kit (4A Biotech, Beijing, China) on an Image Quant LAS 4000 (EG, United States). The grey value was calculated by the software of Image J.

Statistical Analysis
All data were analyzed using GraphPad Prism and presented in mean ± S.D. One-way ANOVA and Student's t-test were performed to analyze the difference between groups. A value of P<0.05 was considered statistically significant.

MYGY-Nb Significantly Reduces Bone Loss in OVX Mice
To determine the effect of MYGY-Nb on PMOP, C57BL/6J mice were subjected to OVX surgery and then treated with MYGY original formula and MYGY-Nb (20 mL/kg/day) respectively for 12 weeks without any adverse effects. The mCT results showed a significant reduction in bone mass and changes of bone microarchitecture in OVX mice compared to controls ( Figure 1A). After administration with MYGY original formula and MYGY-Nb, these OVX-induced abnormal  symptoms were effectively alleviated, as illustrated by the increases in BMD, Tb.N, Tb.Th and BV/TV, as well as the decline in Tb.Sp (Figures 1B-F). Of note, MYGY-Nb was more effective in inhibiting bone loss than MYGY original formula ( Figures 1A-F).
Additionally, ABH staining revealed that compared with sham mice, OVX mice displayed sparse trabeculae and massive adiposity accumulation (Figures 2A-C). As expected, in both MYGY original formula and MYGY-Nb administration groups, these OVX-induced effects were greatly inhibited, and MYGY-Nb showed a significantly better performance than MYGY original formula (Figures 2A-C).
Therefore, we sought to identify the compounds of MYGY-Nb based on UHPLC-Q-TOF/MS analysis. Figure 3 showed the total ion chromatograms of MYGY-Nb in negative and positive ion modes. By comparison with the reference standard, retention time and published literatures, 16 main chemical constituents in MYGY-Nb were identified preliminarily ( Table 2), among which Kynnipin glycosides, Rutin (Rutinoside)_1, Glycyrrhizin and Glycyrrhizic acid have shown the inhibiting functions in osteoclast formation (30)(31)(32)(33).

MYGY-Nb Inhibits the OVX-Induced Increase in Osteoclast and Decrease in Osteoblast
Studies have shown that the main cause of PMOP is the disorder of bone metabolism contributed by an imbalance between osteoblastic bone formation and osteoclastic bone resorption (34). To determine whether MYGY-Nb regulates the formation of osteoblasts and osteoclasts, we performed TRAP histochemical staining and immunohistochemical staining of ALP. The histological results displayed that MYGY-Nb significantly suppressed OVX-induced increase in TRAP activities and decrease in ALP activities ( Figures 4A, B), implying that MYGY-Nb prevents OVX-induced bone loss by regulating the formation of osteoclast and osteoblast. Surprisingly, these results are consistent with the effects of some compounds identified by UHPLC-Q-TOF/MS analysis.

MYGY-Nb Inhibits the Activation of RANKL-Mediated NF-kB Pathway in OVX Mice
Given that RANKL-induced NF-kB activation is required for osteoclast differentiation and function, we further investigated whether MYGY-Nb inhibits osteoclast formation through the RANKL-mediated NF-kB pathway in OVX-induced mice. By using IHC assay, we found that OVX mice showed a noticeable increase in expression of RANKL, p-p65 and NFATc1, and decrease in expression of IKBa. Nevertheless, after the administration of MYGY-Nb for 12 weeks, the changes of expression of these signaling molecules was inhibited ( Figures 5A-D). Altogether, these data imply that MYGY-Nb might exert antiosteoporosis effects through the inhibition of RANKL-mediated NF-kB pathway.    Here, we used 10 mg/mL MYGY-Nb to treat BMMs in following osteoclastogenesis assays. After BMMs stimulated by RANKL with or without MYGY-Nb for 6 days, TRAP staining showed that MYGY-Nb led to an inhibition of RANKL-induced osteoclastogenesis ( Figure 6B). Additionally, we detected the activity of NF-kB signaling pathway. The results indicated that MYGY-Nb significantly suppressed the RANKL-induced degradation of IkBa as well as upregulation of p-p65 and NFATc1 ( Figures 6C-G). Together, these results indicate that MYGY-Nb inhibits RANKL-induced osteoclast formation by regulating NF-kB pathway.

DISCUSSION
In present study, we confirmed that MYGY-Nb is the optimal compound combination against PMOP. In OVX mice, MYGY-Nb effectively inhibits bone loss and osteoclastogenesis, as well as decreases the expression of RNAKL and NF-kB signaling genes. Subsequently, in vitro experiments further demonstrated that MYGY-Nb suppresses RANKL-induced osteoclast formation through the down-regulation of NF-kB signaling pathway. Altogether, these findings suggest that MYGY-Nb could target RANKL-mediated NF-kB pathway to attenuate osteoclast formation and bone loss. PMOP is a most common primary osteoporosis (35). However, drugs commonly prescribed for treatment of PMOP have certain side effects, which limit their long-term use. TCM usually consists of several herbs that interact with each other to produce synergistic effects, which not only improve the efficacy but also may reduce the toxic side effects produced by single drugs (36,37). It is well known that proper extraction of TCM formulas can optimize the synergy between effective compounds to avoid the overdose of ineffective compounds (38)(39)(40). MYGY is refined from the classic formula for kidney tonic, You-Gui-Yin, and has great anti-osteoporosis efficacy. In this study, we used N-butanol to extract MYGY and then explored its efficacy in OVX-induced osteoporosis mice. Surprisingly, ABH staining and mCT analysis both showed that MYGY-Nb was more effective in the inhibition of bone loss than the original MYGY formula, implying that the combination of compounds in MYGY-Nb has better therapeutic effects on PMOP.
In the pathogenesis of PMOP, over-activated osteoclast formation and excessive bone resorption are significant factors (23). Thus, the inhibition of osteoclast formation is an important strategy for PMOP treatment (41). In the present study, our data showed that MYGY-Nb significantly decreased the number of osteoclasts in OVX mice, which indicated that MYGY-Nb might regulate the process of osteoclastogenesis. The in vitro experiments further confirmed that MYGY-Nb significantly prevented RANKL-induced BMMs differentiation and suppressed the osteoclast formation. Then, we sought to clarify the combination of compounds in MYGY-Nb. UPLC/MS identified 16 major compounds of MYGY-Nb, in which kynylpinoside, rutinoside (Rutinos)_1, glycyrrhizin and glycyrrhetinic acid have been reported to have inhibitory effects on osteoclast formation (25)(26)(27)(28). Our future studies are warranted to investigate the anti-osteoporosis efficacy of these compounds in more detail.
RANKL signaling is thought to be the major target of antiresorptive agents that inhibit bone loss and osteoclast activation. RANKL-induced osteoclast differentiation activates NF-kB and NFATc1 (42)(43)(44). Under normal conditions, NF-kB protein is stored as an inactive form in the cytoplasm (the p50/p65 heterodimer associated with repressive IkBa) (45). Upon stimulation by RANKL, IkBa undergoes phosphorylation resulting in the masking of the nuclear localization signal on p65, which allows NF-kB to translocate to the nucleus. Previous studies have shown that the NF-kB inhibitor (-)-dehydroxymethylepoxyquinomycin blocks RANKL-induced osteoclast formation by down-regulating NFATc1 expression (46)(47)(48)(49). In the present study, we found that MYGY-Nb suppressed OVX-induced expression of RANKL, p-p65 and NFATc1. In addition, MYGY-Nb also decreased the expression of IkBa, p-p65 and NFATc1 in RANKL-treated BMMs. Collectively, these data indicated that MYGY-Nb might play a key role in regulation of RANKL-mediated NF-kB signaling pathway. Thus, more precise genes of the NF-kB signaling pathway targeted by MYGY-Nb need to be explored in future studies.

CONCLUSIONS
In conclusion, our data reveal that MYGY-Nb exerts antiosteoporotic efficacy by inhibiting osteoclast formation and reducing bone loss. Furthermore, we validated that the RANKLmediated NF-kB signaling pathway plays an important role in the anti-osteoporosis efficacy of MYGY-Nb. Our findings provide evidence that MYGY-Nb is a promising therapeutic drug for PMOP.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Ethics Committee of Zhejiang Chinese Medical University.