Imperatorin promotes osteogenesis and suppresses osteoclast by activating AKT/GSK3 β/β‐catenin pathways

Abstract Osteoporosis is caused by disturbance in the dynamic balance of bone remodelling, a physiological process, vital for maintenance of healthy bone tissue in adult humans. In this process, a new bone is formed by osteoblasts and the pre‐existing bone matrix is resorbed by osteoclasts. Imperatorin, a widely available and inexpensive plant extract with antioxidative and apoptotic effects, is reported to treat osteoporosis. However, the underlying mechanism and specific effects on bone metabolism have not been elucidated. In this study, we used rat bone marrow‐derived mesenchymal stem cells and found that imperatorin can activate RUNX2, COL1A1 and osteocalcin by promoting the Ser9 phosphorylation of GSK3β and entry of β‐catenin into the nucleus. Imperatorin also enhanced the production of phospho‐AKT (Ser473), an upstream factor that promotes the Ser9 phosphorylation of GSK3β. We used ipatasertib, a pan‐AKT inhibitor, to inhibit the osteogenic effect of imperatorin, and found that imperatorin promotes osteogenesis via AKT/GSK3β/β‐catenin pathway. Next, we used rat bone marrow‐derived monocytes, to check whether imperatorin inhibits osteoclast differentiation via AKT/GSK3β/β‐catenin pathway. Further, we removed the bilateral ovaries of rats to establish an osteoporotic model. Intragastric administration of imperatorin promoted osteogenesis and inhibited osteoclast in vivo. Our experiments showed that imperatorin is a potential drug for osteoporosis treatment.

resorption. Although these agents increase the bone mass considerably, they have some limitations and side effects, including thromboembolism and oesophageal stimulation. [5][6][7] The ultimate goal of an anti-osteoporosis treatment is to improve the function of osteoblast and inhibit the function of osteoclast, simultaneously, without any adverse reactions. To achieve this, we studied imperatorin, a natural compound as a potential anti-osteoporosis drug.
Canonical Wnt/β-catenin pathway plays a key role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts. 8 The entry of β-catenin into the nucleus initiates the transcription of downstream osteogenesis-related genes and aids the osteogenic differentiation of the cells. [9][10][11] When the Wnt pathway is not activated, the cytoplasmic β-catenin gets destroyed by a complex of casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). This complex leads to ubiquitination of β-catenin and finally, the proteolytic degradation by phosphorylation of its N-terminal four serine/threonine residues. 12 Whereas, the GSK3β is phosphorylated by activated AKT into inactive p-GSK3β. 13 Thus, we assume that AKT/GSK3β/β-catenin pathway affects osteogenesis.
Jiake Xu, Jun Zou et al, by down-regulating the levels of Ca2 + sig-

nal transduction and ROS, inhibiting MAPK/NF-κ B and its downstream
NFATc1 and other osteoclast-related factors to find drugs to inhibit osteoclast activity in the treatment of osteoporosis. Although these drugs showed inhibitory effect on osteoclast activity, they had no effect on osteogenic activity. [14][15][16] Simply reducing osteoclast activity may interfere with bone formation, reduce bone transformation and bring side effects such as mandibular osteonecrosis. Studies have shown that the proliferation of early osteoclasts requires the activation of β-catenin; however, the activated β-catenin inhibits the differentiation of osteoclasts. 17 This view is consistent with reports by Modarresi et al, who found that overexpression of β-catenin by adenoviruses can inhibit the osteoclasts differentiation. 18 John D. Shaughnessy Jr used human osteoclast progenitor cells isolated from multiple myeloma patients and found that its differentiation was inhibited by Wnt pathway. 19 Imperatorin [9-(3-methyl-2-enoxy)-7H-furan [3,2g]chromene-7one] (molecular weight 270.28 g/mol) is a linear furocoumarin compound. It is isolated from Angelica archangelica. and Peucedanum praeruptorum., that are components of traditional Chinese medicine.
Being a plant extract, imperatorin is widely available and inexpensive and has antioxidative and anti-apoptotic effects. 20 cells. 24 However, the underlying mechanism and specific effect of imperatorin on bone metabolism have not yet been fully elucidated.

Foetal bovine serum (FBS), Minimum Essential Medium-Alpha
Modification (α-MEM) and penicillin/streptomycin were purchased from Gibco BRL. All other chemicals were of analytical grade complying with the cell culture standards.

| Experimental animals and treatment
In this study, we explored whether the in vitro effect of imperatorin on bone remodelling could be confirmed in vivo. Thirty 3-month-old SD male rats were procured from Shanghai Laboratory Animal Center (Shanghai, China). A group of five rats were kept in a cage at 12 hours/d light duration, 25°C, ventilated and dry. Access to tap water was ad libitum and standard laboratory diet was provided by Provimi Kliba AG (Kaiseraugst, Switzerland) containing 2.5% casein, 0.8% phosphorus, 1% calcium, 70%-80% carbohydrates and 5% fat. All surgical interventions, treatments and post-operative animal care procedures were performed as per the Animal Care and Use Committee, Wenzhou Medical University. After 1 week of adaptation, ten rats were subject to sham operation and the remaining rats underwent bilateral ovariectomy (OVX) as per protocol previously reported. 25 After 12 weeks, imperatorin (Solarbio) or normal saline (NS) was administered. Further, the rats were divided into three groups (N = 10/group): SHAM group which underwent sham operation received NS (50 mL/kg/d) by gavage; OVX group which underwent bilateral ovariectomy also received (50 mL/kg/d) NS by gavage; whereas, the other ovariectomy group, IMP received imperatorin (20 mg/kg/d) by gavage. Gavage feeding was done once/day for 12 weeks.

| Osteoblastic cell culture and assays
To evaluate the effect of imperatorin on osteogenic differentiation, BMSCs were chosen. BMSC is a kind of multifunctional stem cells widely used in bone tissue engineering. Some studies have found that it has the application value in craniomaxillofacial bone regeneration. 26 Briefly, BMSCs were collected by flushing bone marrow of the 2-week-old SD rats' femurs and tibiae with α-MEM (v/v), 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were cultured at 37°C, 5% CO 2 for 48 hours to allow initial cell adherence to culture flasks. Upon 80%-90% confluence, cells were passaged, purified, seeded into 6-well plates (5 × 10 5 cells/well) and grown in complete α-MEM until confluent. Osteoblast differentiation was induced by treating cells with medium containing 50 mmol/L ascorbic acid, 10 mmol/L β-glycerophosphate, 10 nmol/L dexamethasone and 20 μmol/L ascorbic acid (Sigma), and the medium was replenished every 2 days. Alkaline Phosphatase (ALP) activity was measured using assay Kit (Beyotime) on the 7th day of differentiation. On the 21st day of differentiation, Alizarin Red S (ARS; Solarbio) staining was done to measure the degree of calcium deposition in the cells.
To obtain bone marrow-derived monocytes (BMMS), BMCs were inoculated in α-MEM supplemented with 10% FBS and 30 ng/mL macrophage colony-stimulating factor (M-CSF). After 4 days, unattached cells were removed, and adherent cells acted as the osteoclast-precursor BMMS that were subsequently seeded in 96-well plates (6000 cells/well) and cultured for 5 days with/without imperatorin in complete medium containing M-CSF (30 ng/mL) and RANKL (50 ng/mL). The culture medium was changed twice a day and the cells were fixed with 4% paraformaldehyde for 10 minutes, infiltrated with 0.1% Triton X-100 and then stained with tartrateresistant acid phosphatase (TRAP; Sigma). The TRAP-positive multinucleated cells were enumerated, and cells with ≥3 nuclei were categories as osteoclasts.

| Imperatorin cytotoxicity assay
The effect of imperatorin on the activity of BMMS or BMSC in bone marrow stromal cells was determined by cell counting kit  The amplification parameters included an initial denaturation at 95°C for 5 minutes, then denaturation at 95°C for 1 minute, annealing at 60°C for 30 seconds and extension at 72°C for 1 minute, repeating 40 cycles. Melting point analysis was used to confirm the specificity of the SYBR green assay. Data were converted to mRNA expression fold using ddCt comparative threshold cycle method.

| Western blot assay
Total cell protein was obtained using radioimmunoprecipitation assay buffer (RIPA) lysis buffer (Beyotime). Nuclear proteins were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo) according to the manufacturer's instructions.
Protein concentration was determined using the BCA Protein Assay Kit (Beyotime). Protein (20 mg) was subjected to SDS-PAGE using a 10%-15% concentration gradient gel and transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was blocked with 5% skim milk diluted with 0.1% Tween-20 in tris-buffered saline (TBST) for 2 hours. The primary antibody was incubated for 12 hours at 4°C and washed three times with TBST for 5 minutes each. Secondary antibody was incubated for 4 hours at room temperature. The final step was to quantify the protein strength on the membrane using Image Lab 3.0 software (Bio-Rad).

| Specimen collection
After 12 weeks, all rats were euthanized after anaesthesia, then the bilateral femurs were removed and muscles were stripped. Soft tissue was fixed with 4% paraformaldehyde.

| Immunostaining
To detect the levels of RUNX2 and TRAP, femurs were fixed in 10% neutral-buffered formalin solution. After decalcified in 10% EDTA, tissues were dehydrated through a graded alcohol series,

| Imperatorin stimulates osteoblast differentiation and mRNA expression of osteoblastic gene markers
As shown in Figure 1A, IMP with a concentration below 400 μmol/L has no cytotoxicity to BMSCs. To evaluate the effect of imperatorin on osteogenic differentiation of BMSCs, staining was done and activity of the early osteogenic differentiation marker, ALP was examined on 7th of culture. As shown in Figure 1B To investigate the role of imperatorin in inducing mineralization, osteoblasts were stained with ARS after 21 days of culture. In differentiated osteoblasts without imperatorin, ECM calcium deposition was less; whereas, substantial ARS staining was observed in differentiated cells treated with imperatorin. The mineralization increased with increase in the concentration of imperatorin ( Figure 1C). The results indicated that imperatorin enhanced the initial differentiation of osteoblasts by increasing ALP activity and late-stage differentiation by increasing mineralization. Next, we examined the expression of genes involved in osteoblast differentiation. The expression levels of RUNX 2, OCN, COL1A1 in the imperatorin treatment group were significantly higher than that in the control group ( Figure 1D). However, no difference was observed in the expression levels of BMP2, indicating that the enhancing effect of imperatorin on osteogenesis was not through the BMP2/SMAD pathway. Further, our results of Western blot analysis were in agreement with these results.

| Imperatorin promotes osteogenesis via AKT/ GSK3β/ β-catenin axis
The results of Western blot and immunofluorescence suggested that imperatorin increases the amount of β-catenin in the nucleus (Figure 2A,B). The Wnt/β-catenin is a classical pathway that influences the osteogenic differentiation. The β-catenin enters the nucleus to promote transcription of downstream osteogenic-related genes, of which RUNX2 is the major target. 27 The rt-PCR results indicated that imperatorin did not directly increase the Wnt3a expression in BMSCs during osteogenic differentiation ( Figure 2C).

However, during osteogenic induction, the phosphorylation of
GSK3β was promoted and β-catenin was inhibited ( Figure 2D).
Studies have shown that AKT acts as an upstream signal to regulate the AKT/GSK3β/β-catenin signal axis. [28][29][30] Our results suggest that imperatorin promotes phosphorylation of AKT ( Figure 2E). To verify that AKT plays an essential role in imperatorin promoting osteogenesis, we selected ipatasertib (IPATA; Selleck), a novel, highly selective, pan-AKT inhibitor. [31][32][33][34] After pre-treatment with IPATA (1 µmol/L) for 24 hours, compared with the control group, the IMP treatment group showed more significant osteogenic P < .05 differentiation. However, the osteogenic differentiation was not as apparent as in seen the group without IPATA intervention ( Figure 3). This result indicates that the effect of imperatorin on BMSCs promoting osteogenic differentiation was inhibited by IPATA, suggesting that imperatorin acts by stimulating AKT/ GSK3β/β-catenin pathways.
F I G U R E 1 Imperatorin promotes osteogenic differentiation of BMSCs in vitro. A, Effects of different concentrations of imperatorin on the growth of BMSCs, during osteogenesis on the 7th day, as measured by CCK8 method. Osteogenic induction of BMSCs in vitro, with different concentrations of imperatorin intervention from the first day of induction; B, ALP staining on the 7th day; C, and mineralization degree by the ARS staining on the 21st day; D, the mRNA expression levels of COL1A1, RUNX2, OCN and BMP2 were measured by realtime RT-PCR; E, the protein expression levels of the above indicators were analysed by Western blot; F, representative immunofluorescence images of the above indicators, counterstained with DAPI. Data were expressed as mean ± SD, n = 5; *P < .05, **P < .01 vs IMP (50 umol/L) group | 2335 YAN et Al.

| Imperatorin inhibits RANKL-induced osteoclast differentiation
Next, we studied the effect of imperatorin on osteoclast differentiation. We pre-stimulated BMMS with imperatorin, and then induced osteoclast differentiation with RANKL. In the presence of M-CSF, RANKL can induce BMMS to differentiate into osteoclasts, and the mature osteoclasts were TRAP-positive multinucleated cells. The TRAP staining showed that imperatorin promoted osteoclast differentiation at low concentration (25 µmol/L), but inhibited at high concentration (75 µmol/L; Figure 4B). The results of Western blot showed that the expression of NFATC1, c-Fos and TRAP increased at low concentration of imperatorin and decreased at high concentration of imperatorin ( Figure 4C).
To confirm the role of imperatorin in promoting bone formation, we performed in vivo experiments in rats as aforementioned. One rat died each group that is OVX and imperatorin; whereas, all the rats from the SHAM group survived to collect femoral specimens. The bone volume of OVX rats was significantly lower than that of the SHAM group; however, imperatorin reversed this condition in OVX rats. According to the results of quantitative analysis, BV/TV, Conn.D, Tb.N, Tb.Th in IMP group were closer to SHAM group, than OVX group ( Figure 5A).
The H&E and Masson's staining revealed that imperatorin increased the number of trabecular bone, reduced the trabecular space and improved the structural arrangement of the bone in OVX rats ( Figure 5B).
Imperatorin enhanced the activity of osteoblasts and decreased the activity of osteoclasts in the metaphyseal tissue of OVX rats.
To further verify the effects of imperatorin on osteoblasts and osteoclasts in vivo, we used the aforementioned rat metaphyseal tissue sections for RUNX2 immunohistochemical, immunofluorescence and TRAP staining. As shown in the Figure 5D,E, the osteogenic activity of rat femoral metaphysis decreased after ovariectomy, but partially recovered after imperatorin intervention. The results of TRAP staining suggested that in oestrogen F I G U R E 2 Imperatorin promotes osteogenesis via AKT/GSK3β/β-catenin axis. A, Nuclear and cytoplasmic proteins were isolated, and the amount of β-catenin was detected by Western blot; B, representative immunocytochemistry images showing imperatorin increased the β-catenin in the nucleus; C, on the 1st, 7th and 21st day during the osteogenic differentiation of BMSCs, RNA was extracted to detect the transcription level of Wnt3a; D, E, Western blot examination showed that the expression of phospho-AKT (Ser473), AKT, phospho-β-catenin (Ser552) and β-catenin, p-GSK3β (Ser9) and GSK3β changed after the intervention of imperatorin on osteogenic differentiation of BMSCs deficiency, the osteoclast activity of the metaphysis of the rat was enhanced, and imperatorin could partially prevent this phenomenon ( Figure 5F).

| D ISCUSS I ON
At present, bisphosphonates, raloxifene and oestrogens are the drugs used for the treatment of bone diseases. Although they all have certain curative effects, they also have different limitations including thromboembolism and oesophageal irritation. [5][6][7] The primary goal of our treatment was to restore the normal metabolism of the bones, and balance the osteogenesis and osteolysis, that is, enhance the osteogenic function and reduce the bone-weakening due to pathological condition of osteoporosis. Therefore, we intended to find a potential drug for its treatment from natural compounds. In this study, we found that imperatorin increases the bone mineral density by promoting osteogenesis and inhibiting osteoclast formation both, in vivo and in vitro. showed osteogenesis on Saos-2 cells and has the potential to treat osteoporosis. 20 But the specific mechanism is not understood. Thus, we carried out the experiment to observe the influence of imperatorin on the osteoblast and osteoclast using osteoporotic rats. Our results sug- The BMMS was induced to differentiate into osteoclasts by RANKL with 50 ng/mL concentration in vitro; B, TRAP staining was performed on the 5th day after intervention with different concentrations of imperatorin; C, detection of the expression of NFATC1, c-Fos and TRAP by Western blot; D, after BMMS was stimulated with imperatorin for 30 min, the protein was extracted and the expression of p-GSK3β(Ser9), GSK3β and p-β-catenin(Ser552) and β-catenin was analysed; E, the expression of phospho-AKT(Ser473) and AKT was analysed. Data were expressed as mean ± SD, n = 5; *P < .05, **P < .01 vs IMP (300 μmol/L) F I G U R E 5 Effect of imperatorin on osteoporosis caused by oestrogen deficiency. A, After euthanasia, the femurs of the rats were removed and the X-ray images of the longitudinal and transverse sections of the distal femurs were taken with a miniature CT device. Quantitative analysis results of BV/TV, Tb.N, Tb.Sp and Tb.Th were divided into three groups; B, H&E staining; C, Masson's staining of metaphyseal tissue sections of thigh (D) fluorescence and quantitative analysis, (E) histochemical and quantitative analysis, (F) quantitative TRAP staining and analysis of RUNX2 tissue in metaphyseal tissue section of femoral shaft. Data were expressed as mean ± SD, **P < .01 vs SHAM, ##P < .01 vs OVX Fzd receptor complex, leading to the recruitment, phosphorylation and inactivation of GSK3β. This leads to the activation of β-catenin and the accumulation of β-catenin in the nucleus [37][38][39][40] However, our results suggest that although imperatorin can inhibit the differentiation of osteoclasts and the expression of NFATC1 and TRAP at high concentration (75 µmol/L), it can promote the osteoclast differentiation and expression of these genes at low concentration (25 µmol/L). Osteoclast differentiation is a process involving cell proliferation, commitment, fusion and activation. 41 The activation of β-catenin plays an important role in the proliferation of osteoclast progenitor cells; however, it plays an inhibitory role in its differentiation to osteoclasts. 42 This study found that the overexpression of β-catenin can inhibit the differentiation of osteoclasts in vitro, supporting the earlier claims. 43  Additionally, the microscopic CT scans showed the 3D results and detailed parameters. Compared with OVX group, BV/TV and Tb. N in imperatorin group were closer to those in SHAM group. Gradually, the Tb.Sp decreased and the bone structure improved.
In conclusion, imperatorin modulates bone remodelling by stimulating osteoblast function and inhibiting osteoclast differentiation. This suggests that imperatorin could be used as a safe and an effective dual-action therapeutic agent against osteoporosis that could promote bone growth and inhibit resorption.
Although IMP has been shown to be effective in the treatment of osteoporosis, systemic administration often requires the delivery of hyperphysiological factors to achieve efficacy, leading to the risk of harmful side effects. 51 The specific peptides, aptamers and phosphate-rich compounds which can be formed in the bone microenvironment can be used as targeting carriers to enhance the bone regeneration of non-targeted drugs and reduce the effect of extra-target tissue. 52

CO N FLI C T O F I NTE R E S T
All authors have no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
All the listed authors made substantial contributions to the study. Lei

E TH I C A L A PPROVA L
The study was approved by the Animal Experimentation Ethics

Committee of Second Affiliated Hospital, Wenzhou Medical
University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.