Alpinetin Inhibits RANKL-Induced Osteoclastogenesis and Ovariectomy-Induced Bone Loss by Modulating NFATc1 Transcription and Lysosomal Function

Postmenopausal osteoporosis is a chronic metabolic bone disease caused by excessive osteoclast activation, and osteoclasts are considered to be the sole participants in the degeneration and resorption of bone matrix for controlling bone integrity and continuity. The biological functions of osteoclasts depend critically on the number and activity of fused polykaryon. Hence, targeting osteoclast differentiation and activity can modulate bone resorption and alleviate osteoporosis. Alpinetin is widely used for excellent anti-inammatory activities and little side-effect, but its role in osteoporosis remains unknown. this we investigated for the the ability of to inhibit reduced thereby inhibiting differentiation in a concentration- and time-dependent manner. Additionally, alpinetin inhibited F-actin ring formation and bone resorption, as well as reduced the activation levels of NF-κB, ERK, and AKT signaling cascades. In mature osteoclasts, alpinetin remarkably inhibited integrin-mediated migration and lysosomal biogenesis and tracking by modulating the PKCβ/TFEB and ATG5/LC3 axes. Importantly, alpinetin treatment in mice alleviated ovariectomy-induced bone volume loss.


Introduction
Postmenopausal osteoporosis is a relatively common chronic condition resulting in decreased bone mass [1][2][3]. Abnormal bone metabolism, which is associated with the imbalance between bone resorption and bone formation [4], plays a signi cant role in osteoporosis [5] and thus contributes to a series of complications, including systemic pain, muscle weakness, and increased risk of fractures [6,7].
Osteoporosis-induced bone fractures pose signi cant challenges in individuals, families, and national medical nancial systems [8], and total expenditure on osteoporosis-related health care is still rising year by year [9].
Osteoclasts are giant multinucleated cells derived from monocytes or macrophages in response to receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling [10,11]. Osteoclasts play a critical role in regulating osteolysis [12]. M-CSF stimulation of macrophage precursors induces the expression of RANK, which upon binding to RANKL, triggers the activation of osteoclast-associated genes and osteoclast differentiation by activating mitogen-activated protein kinases (MAPKs), protein kinase B (AKT), and nuclear factor (NF)-κβ signaling cascades [13].
Excessive bone resorption by osteoclasts has been implicated in various bone metabolism-associated diseases. Lysosomes are key regulators of bone metabolism, facilitating the degradation and dissolution of the bone extracellular matrix (ECM) by promoting environmental acidi cation [14]. Lysosomes are also involved in the accumulation of proteases at the ru ed border membrane of the resorption lacuna, the primary site of active bone resorption [15].
Various anti-osteoporosis drugs have been approved for clinical use by the US Food and Drug Administration (FDA). However, most of these agents suffer from limited e cacy or cause severe side effects, including hepatorenal damage and gastrointestinal complications [16][17][18]. Hence, the development of novel, safer, and more effective agents targeting pathological osteolysis are needed.
Chronic in ammation in the bone microenvironment results in osteoclast differentiation and activation, thereby promoting excessive bone resorption [29]. Although alpinetin exerts strong anti-in ammatory effects, its potential usefulness in the treatment of osteoporosis remains unknown. Herein, we report that alpinetin inhibits RANKL-induced osteoclast formation and prevents ovariectomy (OVX)-induced bone mass loss in vivo. We also show that alpinetin modulates osteoclast activity by regulating integrinmediated migration and lysosome function.

Cell isolation and cell viability assay
Long bones were obtained from 8-week-old C57BL/6 mice, and bone macrophage precursors were isolated by rinsing the bone medullary cavity. Bone marrow macrophages (BMMs) were obtained by treating macrophage precursors with 40 ng/mL M-CSF for at least 3 days. Subsequently, adherent cells were used for the cell viability assay and osteoclast differentiation.
BMMs and MC3T3-E1 cells were seeded in 96-well plates and treated with increasing concentrations of alpinetin (0-50 µM) for 4 days. Subsequently, CCK-8 solution (10 µL/well) was added, and cells were incubated for an additional 4 h to assess cell viability. The optical density was measured at 450 mm using the ELX808 absorbance microplate reader (BioTek, Winooski, VT, USA).

Osteoclast differentiation and TRAP staining
To determine the inhibitory effect of alpinetin on osteoclast differentiation, BMMs were seeded in 48-well plates (2 × 10 4 cells/well) and treated with 40 ng/mL M-CSF and 75 ng/mL RANKL for 4 days.
Additionally, cells were exposed to different concentrations of alpinetin (0, 5, 10, and 20 µM). After incubation, cells were carefully washed three times with phosphate-buffered saline (PBS) and xed in 4% paraformaldehyde for at least 30 min. The number and size of mature osteoclasts were determined using ImageJ (National Institutes of Health, Bethesda, MD, USA); only cells containing at least three nuclei were considered differentiated osteoclasts.

F-actin ring and immuno uorescence staining
BMMs were incubated with RANKL and different concentrations of alpinetin. After treatment, cells were xed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After a 1 h incubation with rhodamine-phalloidin, cells were carefully washed three times and stained with DAPI for 5 min. Stained cells were observed under a confocal microscope.
BMMs treated with different concentrations of alpinetin in osteoclastogenic medium were also used for immuno uorescence staining. Fixed and permeabilized cells were blocked with 2% bovine serum albumin for 20 min and incubated with uorescently labeled primary antibodies overnight. Subsequently, cells were incubated with secondary antibodies for 1 h at 37℃ and stained with DAPI for 5 min. Cells were observed under a confocal microscope, and uorescent signals were analyzed using ImageJ.

Bone resorption assay
BMMs were seeded onto bone discs (2 × 10 4 cells/well) and grown in complete α-MEM containing 40 ng/mL M-CSF overnight. Subsequently, cells were stimulated with 75 ng/mL RANKL and increasing concentrations of alpinetin (0, 5, 10, and 20 µM) for 7-8 days. Adherent cells were thoroughly removed from the bone discs, and the resorption pits were imaged using a Hitachi S-3700N scanning electron microscope (Chiyoda, Tokyo, Japan). Three random elds were used to determine the bone absorption area.

qRT-PCR
To assess the effects of alpinetin on osteoclast-associated gene expression, BMMs were seeded in 12well plates and cultured with or without different concentrations of alpinetin. Total RNA was isolated using TRIzol reagent (Takara, Dalian, China) and reverse-transcribed into cDNA, which was used for qRT-PCR. The expression levels of target genes were normalized to those of Gapdh. qRT-PCR assays were repeated at least three times. The murine primer sequences of osteoclast-speci c markers were presented in Table 1. Table 1 Primer sequences for RT-PCR

Western blotting
Cell lysates were obtained by high-speed centrifugation (14,000 rpm) for 15 min, and proteins were collected from the supernatants. Equal amounts of proteins were resolved by SDS-PAGE and transferred onto polyvinylidene di uoride (PVDF) membranes for 2 h. After blocking with 10% milk, PVDF membranes were incubated with primary antibodies overnight at 4℃ followed by secondary antibodies for 2 h at 4℃. Protein signals were detected using the Bio-Rad XRS chemiluminescence detection system (Hercules, CA, USA) and analyzed using ImageJ.

Establishment of an osteoporosis mouse model and bone histological analysis
To con rm the inhibitory effects of alpinetin on estrogen de ciency-induced bone mass loss and osteoclast activity, we established an osteoporosis mouse model. Twenty 8-week-old female C57BL/6 mice were randomly divided into four different groups: sham-surgery, OVX, low dose (LD), and high dose (HD) groups. One week after surgery, mice in the LD and HD groups were intraperitoneally injected with 5 mg/kg or 25 mg/kg alpinetin, respectively, every 2 days. Mice in the sham-surgery and OVX groups received an equal volume of PBS. After 4 weeks of treatment, mice were euthanized by anesthetic overdose. Bilateral femurs were isolated, xed in 4% paraformaldehyde solution for 3 days, and evaluated by microcomputed tomography (micro-CT). Subsequently, bones were decalci ed with 10% EDTA for half a month and cut into 4-µm sections, which were subjected to histological analysis.

Alkaline phosphatase (ALP) and alizarin red staining (ARS)
To determine the effects of alpinetin on osteogenesis in vitro, we seeded MC3T3-E1 cells in 12-well plates, and after overnight incubation with complete α-MEM, cells were treated with alpinetin (0, 20, and 50 µM) in osteogenic medium for 3 or 14 days. Cells treated for 3 days were subjected to ALP staining, and those treated for 14 days were used for ARS.

Measurement of reactive oxygen species (ROS)
To assess the effects of alpinetin on cellular ROS, we seeded RAW 264.7 cells in 12-well plates (5 × 10 4 cells/well) and pretreated them with alpinetin for 12 h. Subsequently, alpinetin-pretreated cells were incubated with 10 mM 2′,7′-dichlorodihydro-uorescein diacetate (H 2 DCFDA) for 30 min in the dark, and then stimulated with 75 ng/mL RANKL for another 30 min. Cellular ROS levels were measured on a multimodal microplate reader (SpectraMaxM5; Molecular Devices, Sunnyvale, CA, USA).

Statistical analysis
All results are presented as means ± standard deviation (SD). Statistical signi cance was determined by Student's t-test or one-way ANOVA. P-values < 0.05 were considered statistically signi cant.

Alpinetin suppresses RANKL-induced osteoclast differentiation but does not affect osteogenesis in vitro
Cell viability results revealed that alpinetin (0-50 µM) did not signi cantly affect the viability of BMMs or MC3T3-E1 cells (Fig. 1B), although it suppressed RANKL-induced osteoclast differentiation in a concentration-and time-dependent manner (Fig. 1C-G). Next, we assessed the effects of alpinetin on the early (0-2 days) and late stages (2-4 days) of osteoclast formation; we found that the number and size of TRAP-positive cells were reduced both at the early and late stages, although the inhibitory effects were more profound during late-stage osteoclast formation (Fig. 1H-J). These data suggest that alpinetin strongly suppresses RANKL-induced osteoclast formation. However, ARS and ALP staining results indicated that alpinetin had no effect on osteogenesis in vitro (Fig. 1L-N).

Alpinetin inhibits bone resorption and RANKL-induced ROS production
The F-actin ring is a characteristic cytoskeletal structure essential for the osteolytic function of osteoclasts [30]. To determine the effects of alpinetin on F-actin ring formation, we treated BMMs with different concentrations of alpinetin during osteoclastogenesis. Then, we stained alpinetin-treated BMMs with rhodamine-phalloidin and measured the number and area of F-actin rings; we found that alpinetin inhibited actin ring formation in a dose-dependent manner ( Fig. 2A-C). Additionally, we found that alpinetin treatment remarkably decreased the bone resorption area (Fig. 2D, E), suggesting that alpinetin suppresses bone resorption by osteoclasts.
Aberrant oxygen metabolism leads to the accumulation of ROS, thereby promoting the development of osteoporosis and other in ammatory diseases [31]. Interestingly, we found that alpinetin treatment signi cantly reduced intracellular ROS levels in RANKL-treated cells (Fig. 2F, G).

Alpinetin blocks RANKL-mediated activation of ERK/AKT/NF-κβ signaling
To elucidate the molecular mechanism by which alpinetin affects osteoclast differentiation, we pretreated RAW 264.7 cells with 20 µM alpinetin and investigated activation of the osteoclast-related signaling pathways MAPK, AKT, and NF-κβ in response to RANKL stimulation (75 ng/mL; 0-60 min). Alpinetintreated RAW 264.7 cells exhibited profoundly lower p-ERK levels than control cells. However, activated p38 and JNK levels, downstream components of the MAPK pathway, were not affected by alpinetin treatment.
AKT is a master regulator of osteoclast differentiation and survival [32]. Notably, AKT signaling has been shown to promote osteoclast fusion and subsequent osteoclast formation [33]. In this study, we found that alpinetin signi cantly decreased p-AKT levels. NF-κβ signaling also plays a pivotal role in osteoclast differentiation and function [34]. We found that alpinetin (20 µM) signi cantly reduced the phosphorylation levels of p65. Consistent with the decrease in p-p65 levels, IκBα pathway activation was inhibited by alpinetin treatment (Fig. 3A, B). To further con rm the effects of alpinetin on the transcriptional activity of NF-κB, we performed immuno uorescence analysis and found that alpinetin suppressed the nuclear translocation of p65 (Fig. 3C, D).

Alpinetin inhibits NFATc1 induction and osteoclastrelated gene expression
The transcription factors NFATc1 and c-Fos are crucial for osteoclast differentiation [35]. Interestingly, we found that alpinetin suppressed the expression of NFATc1 and c-Fos in a time-dependent manner; when used at 20 µM, the inhibitory effects of alpinetin on NFATc1 and c-Fos expression lasted for 4 days.
Additionally, the expression of CTSK, which also plays a key role in osteoclast formation and function, was also suppressed by alpinetin (Fig. 3A-E). Immuno uorescence staining con rmed that alpinetin reduced the levels of mature NFATc1 in a dose-dependent manner (Fig. 3F, G). Moreover, alpinetin signi cantly reduced the mRNA levels of the osteoclast-related genes Nfatc1, Trap, Dc-stamp, Ctsk, V-ATPase-a3, and Mmp9 in a concentration and time-dependent manner (Fig. 3H, I), further con rming the ability of alpinetin to suppress osteoclastogenesis.

Alpinetin inhibits osteoclast activity by modulating integrin-mediated cell migration
Cell migration is critical for osteoclast activity, and is required for osteoclast migration and resorption ability [36]. We found that alpinetin markedly impaired the migration ability of differentiated osteoclasts and BMMs (Fig. 5A, B). Integrin β 3 and c-Src are highly expressed during osteoclast fusion and are required for osteoclast migration [37]. Here, we show that although integrin β 3 and c-Src expression levels gradually increase during osteoclast fusion, alpinetin treatment remarkably suppressed their expression (Fig. 5C, D). Consistently, alpinetin treatment (20 µM for 1 or 2 days) in mature osteoclasts derived from RANKL-stimulated BMMs signi cantly reduced integrin β 3 and c-Src levels both at the mRNA (Fig. 5E, F) and protein level (Fig. 5G-J). Collectively, these results suggest that alpinetin inhibits osteoclast activity by modulating integrin-mediated osteoclast migration.

Alpinetin inhibits lysosomal biogenesis and tra cking by modulating the PKCβ-TFEB and ATG5-LC3 axes
The ability of mature osteoclasts to resorb bones is strongly dependent on lysosomes, which are enriched at the osteoclast ru ed border and mediate degradation of the extracellular bone matrix [38]. To assess the effects of alpinetin on lysosomal biogenesis, we investigated the expression levels of TFEB, a transcription factor required for lysosomal biogenesis [39]. We found that although RANKL stimulation increased TFEB levels, alpinetin suppressed the ability of RANKL to induce TFEB expression. Additionally, alpinetin inhibited the expression of PKCβ (which is upstream of TFEB activation) and the lysosomal marker LAMP1 (Fig. 6A-D). In mature osteoclasts, alpinetin treatment reduced the protein levels of PKCβ, TFEB, and LAMP1, as well as the mRNA levels of the TFEB target genes [40] Acp5, Aatp6v0d2, Clcn7, and Tcirg1 (Fig. 6E). To further con rm the effects of alpinetin on lysosomal biogenesis, we performed immuno uorescence staining for LAMP1 and found that alpinetin reduced the levels of LAMP1 in osteoclasts (Fig. 6F, G). These results suggest that alpinetin suppresses lysosomal biogenesis by modulating the PKCβ-TFEB axis.
Lysosomal tra cking plays a critical role in osteoclast-mediated osteolysis [41]. We found that although ATG7, CTSK, and LC3 protein levels were gradually increased during RANKL-induced osteoclastogenesis, CTSK is considered one of the most effective protease, of which main function is to regulate lysosome secretion, thereby promoting bone resorption ability [42]. Alpinetin (20 µM) signi cantly decreased the levels of all these proteins (Fig. 7A, B). Similarly, alpinetin treatment profoundly decreased the levels of ATG7, CTSK, and LC3 in mature osteoclasts (Fig. 7C, D). Consistently, BMMs treated with alpinetin exhibited signi cantly lower Atg5 and Ctsk mRNA levels than control BMMs (Fig. 7E). Collectively, these data indicate that alpinetin inhibits lysosomal tra cking by modulating the ATG5-LC3 axis.

Alpinetin alleviates OVX-induced bone mass loss and inhibits osteoclast formation in an osteoporosis mouse model
To assess the therapeutic potential of alpinetin in vivo, we investigated the effects of alpinetin on bone volume loss and osteoclast activity in an OVX-induced osteoporosis mouse model. Micro-CT ndings revealed profound bone loss in the distal femurs of mice in the OVX group, con rming the establishment of an osteoporosis mouse model. Compared with mice in the OVX group, the femurs of alpinetin-treated mice exhibited a signi cantly increased bone volume, indicating the protective effect of alpinetin against bone mass loss (Fig. 8A-G). Additionally, we found that the protective effects of alpinetin against OVXinduced bone mass loss were signi cantly stronger in mice treated with 25 mg/kg alpinetin (HD group) than those treated with 5 mg/kg alpinetin (LD group), suggesting that the protective effects of alpinetin are dose-dependent.
Histological analyses (hematoxylin and eosin, Masson, and TRAP staining) of decalci ed femurs indicated that alpinetin alleviated the OVX-induced bone mass loss and osteoclast formation, especially when used at 25 mg/kg (Fig. 8H-K). Taken together, these data demonstrate that alpinetin prevents OVXinduced bone mass loss and osteoclast formation in mice with osteoporosis.

Discussion
Bone metabolism homeostasis disruption due to imbalances in bone formation and bone resorption has been associated with various bone metabolism disorders, including rheumatoid arthritis [43], Paget's disease [44], and osteoporosis [45], imposing a serious public health challenge. Osteoporosis is a prevalent disease, affecting millions of individuals around the world. Given the importance of osteoclasts in osteolysis, targeting osteoclast activity is considered a promising approach for the management of osteolysis-related bone diseases. Although numerous drugs have gained regulatory approval for use in patients with osteoporosis, they suffer from poor e cacy and high toxicity. In this study, we con rmed that alpinetin reduced osteoclast formation in vitro and inhibited estrogen de ciency-induced bone loss by suppressing osteoclast activity, without affecting osteoblast formation in vivo.
The RANKL/RANK axis plays a critical role in osteoclast formation and function [46]. Binding of RANKL to RANK promotes the recruitment of TNF receptor-associated factor 6 (TRAF6), which activates the TGFβ-activated kinase 1 (TAK1) [47], IKK/Ikβα, AKT, and MAPKs. IKKα/β phosphorylation enhances the degradation of IκB, thereby activating NF-κβ and AP-1 [48]. Subsequently, AP-1 and NF-κB induce the expression of NFATc1, which, in turn, activates the expression of osteoclast-related genes and promotes osteoclast differentiation. In this study, we found that alpinetin inhibited RANKL-mediated activation of NF-κβ, ERK, and AKT signaling, without affecting activation of p38 and JNK.
Excessive amounts of ROS disrupt reduction-oxidation homeostasis and physiological cell metabolism [31]. Activation of RANKL-RANK signaling promotes ROS production during osteoclast formation [49], which further enhances osteoclast differentiation and bone resorption. All results demonstrated that alpinetin remarkably attenuated ROS production in RAW 264.7 cells.
Cell adhesion and migration are essential for osteoclast-mediated osteolysis, with integrin regulating both of these processes [50]. Integrin is highly expressed in differentiated osteoclasts, promoting transport of acidifying vesicles to the bone matrix surface. In this study, we found that alpinetin inhibited osteoclastogenesis and the migration of osteoclasts and osteoclast precursors by inhibiting integrin β3 and c-Src.
In addition to cell migration, lysosomal biogenesis and tra cking are also required for bone degradation and resorption. TFEB, a newly identi ed transcription factor involved in lysosomal biogenesis, has been shown to regulate the expression of various genes promoting ECM acidi cation and degradation [39], including Acp5, Atp6v0d2, Clcn7, and Tcirg1. Upon RANKL-RANK interaction and subsequent PKCβ activation, TFEB translocates into the cell nucleus, activating expression of its target genes [51]. PKCβ inhibition has been demonstrated to suppress TFEB stabilization, impairing lysosome biogenesis and increasing bone mass [52]. In line with these ndings, we found that alpinetin destabilized TFEB via inhibition of PKCβ expression, thereby suppressing lysosomal biogenesis. Autophagy and autophagyrelated proteins, including ATG5 and LC3, have been shown to regulate lysosomal tra cking and secretion. In the absence of these proteins, lysosomal tra cking in osteoclasts and subsequent bone degradation is signi cantly impaired [53]. Importantly, alpinetin inhibited lysosomal tra cking and secretion of lysosomal enzymes by modulating the ATG5/LC3 axis. However, the underlying mechanisms remain to be elucidated.
In conclusion, our ndings suggest that alpinetin inhibits NFATc1 expression and subsequent osteoclast differentiation by modulating the NF-κB, AKT, and ERK signaling cascades. Our results also indicate that alpinetin inhibits lysosomal biogenesis and tra cking by modulating the PKCβ-TFEB and ATG5-LC3 axes, respectively. Importantly, alpinetin alleviated OVX-induced bone mass loss in an osteoporosis mouse model, suggesting that alpinetin may represent a promising anti-osteoporosis agent.

Declarations Ethical Approval and Consent to participate
This work has been approved for animal ethics by the Second A liated Hospital, School of Medicine, Zhejiang University.

Consent for publication
Not applicable.

Data availability statement
All the data that support the ndings of this study are included in the manuscript.

Competing Interest
The authors declare that they have no competing interests