ABSTRACT

   Bone homeostasis, the equilibrium between bone resorption and
   formation, is essential for maintaining healthy bone tissue in adult
   humans. Disruptions of this process can lead to pathological conditions
   such as osteoporosis. Dual‐targeted agents, capable of inhibiting
   excessive bone resorption and stimulating bone formation, are being
   explored as a promising strategy for developing new treatments to
   address osteoporosis. In this study, we investigated the effects of
   P7C3 on bone remodeling and its potential therapeutic role in
   osteoporosis treatment in mice. Specifically, P7C3 can remarkably
   suppress receptor activator of nuclear factor‐κB (NF‐κB) ligand
   (RANKL)‐induced osteoclast differentiation in bone marrow macrophages
   via the Akt‐NF‐κB‐NFATc1 signaling pathway. Additionally, RNA
   sequencing (RNAseq) analysis revealed that P7C3 promoted osteoblast
   differentiation and function through the Wnt/β‐catenin signaling
   pathway, thereby enhancing bone formation. Furthermore, μCT analysis
   and histological examination of bone tissues from P7C3‐treated mice
   showed attenuation of both Ti‐induced bone erosion and ovariectomy
   (OVX)‐induced bone loss. These findings suggest that P7C3 may have a
   novel function in bone remodeling and may be a promising therapeutic
   agent for the treatment of osteoporosis. © 2023 The Authors. JBMR Plus
   published by Wiley Periodicals LLC on behalf of American Society for
   Bone and Mineral Research.

   Keywords: BONE HOMEOSTASIS, OSTEOBLAST, OSTEOCLAST, OSTEOPOROSIS, P7C3
     __________________________________________________________________

   Maintaining bone homeostasis is crucial for healthy bone tissue in
   adults, and disruptions can cause osteoporosis. Dual‐targeted agents
   that inhibit excessive resorption and promote formation are being
   explored as new osteoporosis treatments. P7C3 was investigated on bone
   remodeling in mice and its potential therapeutic role in osteoporosis.
   P7C3 remarkably suppressed osteoclast differentiation through the
   Akt‐NF‐κB‐NFATc1 signaling pathway and promoted osteoblast
   differentiation through the Wnt/β‐catenin pathway, enhancing bone
   formation. P7C3 may have a promising therapeutic role in treating
   osteoporosis.

   graphic file with name JBM4-7-e10811-g002.jpg

Introduction

   The skeleton is a dynamic tissue that serves as the body scaffold to
   protect vital organs, facilitating body movement and supporting
   hematopoiesis.^( [48]^1 ^) Throughout the lifespan of organisms, the
   bones undergo constant remodeling through coordinated activities of
   osteoclasts and osteoblasts, which mediate bone resorption and
   formation, respectively.^( [49]^2 ^) Maintaining a balance between
   resorption and formation is critical for preserving skeletal mass.
   However, with aging or certain diseases, bone homeostasis becomes
   disrupted, leading to an imbalance of the resorption over formation.
   This imbalance further contributes to bone loss and increased
   susceptibility to fragility fracture,^( [50]^2 , [51]^3 ^) which
   affects hundreds of millions of people worldwide, especially
   postmenopausal women.^( [52]^4 ^)

   Osteoclasts originate from the fusion of mononuclear macrophages
   lineage of hematopoietic stem cells, while osteoblasts derive from
   mesenchymal stem cells of bone marrow (BMSCs).^( [53]^5 ^) However,
   these two types of cells are intertwined with each other. During the
   remodeling process, bone resorption occurs first, followed by bone
   formation in the resorption cavity. Osteoclasts not only play a
   bone‐resorbing role, but also secrete certain bone
   osteoblast‐stimulating factors, thereby regulating osteoblast
   activity.^( [54]^2 ^) These certain factors promote the activation of
   the essential transcription factors runt‐related transcription factor 2
   (Runx2), osterix (Osx), and β‐catenin,^( [55]^6 , [56]^7 , [57]^8 ,
   [58]^9 , [59]^10 ^) which govern the expression of osteoblast‐specific
   genes, including alkaline phosphatase (ALP), osteopontin (OPN), and
   osteocalcin (OCN),^( [60]^11 , [61]^12 ^) thus exhibiting osteogenic
   function. Meanwhile, macrophage‐colony stimulating factor (M‐CSF) and
   the receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL),
   produced by osteoblast‐lineage cells, play an essential role in
   osteoclast differentiation and function.^( [62]^13 , [63]^14 ^) Binding
   between RANKL and its cell‐surface receptor RANK activates various
   downstream signaling pathways, including the mitogen‐activated protein
   kinase (MAPK: ERK, JNK, and p38), protein kinase B (Akt), and NF‐κB
   pathways.^( [64]^15 ^) Subsequently, the activation of the key
   transcription factors c‐Fos and nuclear factor of activated T cells,
   cytoplasmic 1 (NFATc1), triggers the expression of specific genes
   associated with osteoclasts such as cathepsin K (CTSK), calcitonin
   receptor (CTR), tartrate‐resistant acid phosphatase (TRAP), matrix
   metalloprotein‐9 (MMP‐9), dendritic cell‐specific transmembrane protein
   (DC‐STAMP), and V‐ATPase,^( [65]^16 , [66]^17 ^) thereby promoting the
   fusion of osteoclast precursors and exerting osteoclast activity. At
   the same time, osteoblasts also release osteoprotegerin (OPG), which
   acts as a decoy receptor of RANKL and negatively regulates osteoclast
   differentiation, affecting bone resorption function.

   Pharmacological treatments for osteoporosis that target bone remodeling
   can be classified into two main categories: antiresorptive drugs and
   anabolic drugs.^( [67]^18 ^) These interventions or treatments can
   indeed affect both osteoclasts and osteoblasts simultaneously. However,
   anti‐resorptive therapies, such as denosumab (DMAb) and
   bisphosphonates, are often associated with a decrease in osteoblast
   activity, which leads to reduced bone formation and limits their
   effectiveness.^( [68]^2 , [69]^19 ^) The gastrointestinal side effects
   and atypical femoral fractures associated with bisphosphonates have
   also raised concerns among patients and physicians.^( [70]^4 , [71]^20
   ^) The other class is parathyroid hormone (PTH) and its analog.
   However, at higher doses or for prolonged periods, PTH can also
   stimulate osteoclastic activity, leading to bone resorption. Moreover,
   PTH treatment can trigger the release of calcium from bone, increasing
   the risk of hypercalcemia.^( [72]^21 ^) In addition to the above
   mentioned mainstream clinical anti‐osteoporosis drugs, romosozumab
   provides a promising new direction for the dual regulation of bone
   remodeling by promoting bone formation and inhibiting bone resorption
   simultaneously. Regrettably, its cardiovascular risks have led to the
   US Food and Drug Administration's (FDA's) rejection of its initial
   application.^( [73]^4 ^) Thus, developing new dual target–directed
   drugs that modulate bone remodeling with limited side effects remains a
   promising strategy for the treatment of osteoporosis.

   P7C3, an aminopropyl carbazole compound that was discovered through a
   target‐agnostic screening approach, exerts remarkable neuroprotective
   efficacy with few adverse effects^( [74]^22 , [75]^23 ^) in several
   animal models of neurodegenerative diseases^( [76]^23 , [77]^24 ,
   [78]^25 , [79]^26 , [80]^27 ^) or nerve cell injury.^( [81]^28 ,
   [82]^29 , [83]^30 , [84]^31 , [85]^32 ^) Additionally, research has
   shown that P7C3 may have a protective effect against liver and spinal
   cord injuries.^( [86]^33 , [87]^34 , [88]^35 ^) However, the effect of
   P7C3 on bone remodeling has not been reported. In this study, we
   revealed that P7C3 exhibited a property to serve as a bone remodeling
   dual regulator and attenuate bone loss in various osteoporosis mouse
   models. The results showed that P7C3 inhibits osteoclast
   differentiation and affects bone resorption by targeting specific
   molecular pathways. Moreover, P7C3 promotes osteoblast differentiation
   and bone formation. These findings establish the mechanisms underlying
   the activity of P7C3 in bone remodeling and provide a basis for further
   pharmacological studies of this compound.

Materials and Methods

Study approval

   All animal and human experimental procedures followed were reviewed and
   approved by the Ethics Committee of Soochow University (Approval No.
   SUDA20220701A02).

Mice and reagents

   C57BL/6 mice (female, 6–8 weeks old) were purchased from the Laboratory
   Animal Center of Soochow University. During the experimental period,
   the mice were housed in ventilated cages in a specific pathogen‐free
   facility at room temperature (22–24°C) and kept on a 12‐h light/dark
   cycle, with water ad libitum and standard chow. P7C3 was obtained from
   MedChemExpress (Monmouth Junction, NJ, USA) and stored in dimethyl
   sulfoxide (DMSO) at −20°C. Recombinant mouse macrophage‐colony
   stimulating factor (M‐CSF) and RANKL were purchased from R&D Systems
   (Minneapolis, MN, USA). Soluble, recombinant human M‐CSF, RANKL and
   tumor growth factor β (TGF‐β) were obtained from PeproTech (London,
   UK). Anti‐Akt, anti‐phospho‐Akt, anti‐ERK, anti‐phospho‐ERK, anti‐p38,
   anti‐phospho‐p38, anti‐JNK, anti‐phospho‐JNK, anti‐NFATc1, anti‐CTSK,
   anti‐MMP‐9, and anti‐GAPDH antibodies were purchased from Cell
   Signaling Technology (Beverly, MA, USA). Anti‐TRAF‐6, anti‐IκB,
   anti‐p65‐NF‐κB, and anti‐phospho‐p65‐NF‐κB antibodies were purchased
   from Abcam (Cambridge, MA, USA). Alpha‐minimum essential medium
   (α‐MEM), fetal bovine serum (FBS), streptomycin, and penicillin were
   obtained from Gibco BRL (Grand Island, NY, USA).

Cell proliferation and viability

   The cytotoxicity of P7C3 on precursor cells of the osteoclastic and
   osteogenic lineages was determined with
   3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT)
   assay. Bone marrow–derived macrophages (BMMs), RAW264.7, bone
   mesenchymal stem cells (BMSCs), and MC3T3‐E1 cells were seeded in
   96‐well plates and proliferated in full ɑ‐MEM medium (α‐MEM containing
   10% FBS, 1% penicillin and streptomycin) at a density of 5 × 10^3
   cells/well. With the indicated concentrations of P7C3 added for 1or
   2 days, the medium containing MTT (5 mg/mL) was replaced in each well
   and incubated for an additional 2 h at 37°C. Then, the crystals were
   dissolved in DMSO and detected at 490 nm (OD490) by a Bio‐Tek
   microplate reader (BioTek, Winooski, VT, USA). Compared with the
   absorbance values of untreated controls, cell viability was calculated.

In vitro osteoclastogenesis assay

   Six to eight‐week‐old female C57BL/6J mice were euthanized to prepare
   BMMs as described.^( [89]^36 ^) Briefly, bone marrow cells (BMCs) were
   obtained by flushing the marrow cavities of the femur and tibia with
   α‐MEM and then cultured in full α‐MEM medium (α‐MEM containing 10% FBS,
   1% penicillin and streptomycin) at 37°C/5% CO[2] overnight. Nonadherent
   cells were harvested and further cultured in complete α‐MEM containing
   30 ng/mL M‐CSF (R&D Systems) for 3 days to generate BMMs. To generate
   osteoclasts, BMMs were plated at a density of 1 × 10^4 cells/well in a
   96‐well plate in full medium containing 30 ng/mL M‐CSF and 50 ng/mL
   RANKL (R&D Systems) for 5–7 days with the indicated concentrations of
   P7C3. After the cells were fixed with 4% paraformaldehyde for
   20 minutes, an acid phosphatase leukocyte kit (Sigma‐Aldrich,
   Darmstadt, Germany) was used for TRAP staining. TRAP^+ multinucleated
   cells with three or more nuclei were counted as osteoclasts under an
   Olympus (Waltham, MA, USA) microscope.

   For human osteoclast differentiation, peripheral blood mononuclear
   cells (PBMCs) of normal healthy authors were obtained from
   ethylenediamine tetraacetic acid (EDTA)‐blood using Ficoll (Lymphoflot;
   Bio‐Rad Laboratories, Hercules, CA, USA) density gradient
   centrifugation. PBMCs were cultured in a complete medium containing
   30 ng/mL of M‐CSF, 3 ng/mL of RANKL, and 1 ng/mL TGF‐β (all PeproTech,
   Rocky Hill, NJ, USA) with the indicated concentrations of P7C3 for
   7 days to generate osteoclasts.

In vitro bone resorption assay

   BMMs were cultured on 24‐well plates coated with hydroxyapatite
   (Corning Inc., Corning, NY, USA) in full medium containing
   30 ng/mL M‐CSF and 50 ng/mL RANKL with the indicated concentrations of
   P7C3. The medium was changed every 2 days. After 5 days, the cells were
   cleaned with 5% sodium hypochlorite and the resorption pits were
   photographed by a microscope. The bone resorption area was analyzed and
   quantified using the software ImageJ (NIH, Bethesda, MD, USA;
   [90]https://imagej.nih.gov/ij/).

NF‐κB activation and nuclear translocation assay

   RAW264.7 cells were cultured with complete medium, 50 ng/mL RANKL or
   50 ng/mL RANKL plus 10μM P7C3 for 24 hours. Fixing fluid was added to
   the cells after the culture medium was removed. Then, the cells were
   washed three times with washing solution and blocked for 1 hour in
   blocking buffer. Following overnight incubation with the primary
   antibody at 4°C, cells were incubated with a secondary antibody
   conjugated to Cy3 for 1 hour at room temperature.
   4′,6‐diamidino‐2‐phenylindole (DAPI) was added to stain nuclei for
   5 minutes and then washed three times. After that, confocal microscopy
   was used to visualize the samples. All steps above were carried out
   according to the instructions contained in the NF‐κB activation and
   nuclear translocation assay kit (Beyotime Biotech, Shanghai, China).

In vitro osteoblast differentiation and mineralization

   BMSCs were isolated from C57BL/6 mice (female, 6–8 weeks old) as in our
   previous works.^( [91]^37 , [92]^38 ^) The cells were cultured in
   12‐well plates in osteogenic medium (complete DMEM, 10mM
   β‐glycerophosphate, 50 μg/mL ascorbic acid, and 10nM dexamethasone)
   with or without P7C3. For alkaline phosphatase (ALP) staining, a
   BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Biotech,
   Shanghai, China) was used to stain cells following osteogenic induction
   for 14 days. For Alizarin red staining (ARS), the cells were stained
   with Calcium Stain Kit (Modified Alizarin Red S Method) (Solarbio,
   Beijing, China) after 21 days of osteogenic differentiation. The ALP
   and ARS staining regions were then visualized under a microscope and
   measured using the software ImageJ.

Quantitative real‐time PCR (qPCR)

   Total RNA was extracted with TRIzol reagent (Biosharp, Hefei, China)
   according to the manufacturer's instructions. The cDNA was obtained by
   reverse transcription of total RNA using NovoScript®Plus All‐in‐one 1st
   Strand cDNA Synthesis SuperMix (gDNA Purge). Then, the qRT‐PCR product
   was amplified with the NovoStart® SYBR Quantitative real‐time PCR
   (qPCR) SuperMix Plus (Novoprotein, Shanghai, China). The expression of
   target genes was normalized to that of glyceraldehyde‐3‐phosphate
   dehydrogenase (GAPDH). A list of specific primer sequences is provided
   in Table [93]S1.

Western blot analyses

   To extract the protein, the cells were washed with phosphate‐buffered
   saline (PBS) and lysed on ice in a radioimmunoprecipitation assay
   (RIPA) buffer containing protease inhibitors. The supernatants were
   then obtained by centrifugation at 12,000g for 15 minutes and
   quantified with a bicinchoninic acid (BCA) protein assay kit. An equal
   amount of protein was separated using sodium dodecyl
   sulfate–polyacrylamide gel electrophoresis (SDS/PAGE) gels and
   transferred onto polyvinylidene difluoride (PVDF) membranes (Bio‐Rad,
   Hercules, CA, USA). With 5% nonfat milk, the membranes were blocked and
   incubated with relevant primary antibodies at 4°C overnight. The next
   day, the membranes were incubated with appropriate horseradish
   peroxidase (HRP)‐conjugated secondary antibodies and visualized with
   chemiluminescent substrate (ECL). The gray bands of the proteins were
   quantized using the software ImageJ. The original data of Western blots
   is provided in Fig. [94]S4.

RNA sequencing

   MC3T3‐E1 cells were cultured in six‐well plates in osteogenic medium
   for 3 days in the presence or absence of P7C3. Total RNA from the
   harvested cells was extracted with TRIzol reagent and assessed by
   Agilent Bioanalyzer (Agilent, Waldbronn, Germany). RNA libraries were
   produced using NEB Next® Ultra II RNA Library Prep Kit for Illumina and
   then sequenced on an Illumina HiSeq 4000 platform (Illumina, San Diego,
   CA, USA). Low‐quality reads and adapter sequences were removed from raw
   reads. Then, reads were aligned against the mouse genome with
   v.GRCm38.91. Based on this matrix, differential genes expressed between
   two groups were identified (R v3.5.1, DESeq2 v1.20). A gene expression
   that changes more than 1.3‐fold and p < 0.05 was regarded as
   significantly different. The genes obtained were further analyzed, for
   instance through Kyoto Encyclopedia of Genes and Genomes (KEGG)
   enrichment analysis.

Animal model

   Female C57BL/6 mice, 8 weeks old, were divided randomly and evenly into
   three groups: sham, Ti, and Ti plus P7C3. With the exception of the
   sham group, 30 mg Ti particles were embedded beneath the periosteum, at
   the midpoint of the sutural line. To evaluate the therapeutic effect of
   P7C3 on Ti particle‐induced bone destruction, the mice were treated
   with vehicle (sham and Ti group) or P7C3 (20 mg/kg in Ti plus P7C3
   group) every day through intraperitoneal injection. After 14 days, the
   calvariae were collected for further μCT and histological analysis.

   To study the effects of P7C3 on ovariectomy (OVX)‐induced osteoporosis
   model, 8‐week‐old female C57BL/6 mice were randomly divided into three
   groups: sham, OVX, and OVX plus P7C3. With the exception of the Sham
   group, the female mice were bilaterally ovariectomized. The uterus
   weight and weight development were assessed to confirm successful OVX.
   Six weeks after surgery, P7C3 (20 mg/kg) was intraperitoneally
   administered to the OVX mice once a day. Two weeks later, serum and
   bone tissue samples were collected for subsequent experiments.

μCT analysis and bone histomorphometry

   μCT analysis (SkyScan 1174; SkyScan, Aartselaar, Belgium) was performed
   with the consistent parameters (voxel size, 10.3 μm; X‐ray voltage,
   50 kV; electric current, 810 μA; rotation step, 0.5 degrees). The
   three‐dimensional (3D) models of the calvaria and distal femur were
   reconstructed using the analysis program CTvol software. For
   quantitative assessment, static parameters such as bone mineral density
   (BMD), bone volume/tissue volume (BV/TV %), porosity percentage (%),
   bone surface/tissue volume (BS/TV), trabecular separation (Tb. Sp), and
   trabecular bone number (Tb.N) of each sample were measured.

   Following μCT analysis, the bones were decalcified for sectioning and
   staining. To evaluate the bone trabecular structure and identify
   osteoclasts, 5‐μm‐thick sections were stained with hematoxylin and
   eosin (H&E) or TRAP. The parameters of bone tissues were quantified
   with the OsteoMeasure software (OsteoMetrics Inc., Decatur, GA, USA).
   To analyze the bone segment with immunofluorescence, 8‐μm‐thick
   longitudinally oriented sections were stained with anti‐Osterix (Abcam;
   ab209484). The relative expression levels were quantized by ImageJ.

Blood biochemicals analysis

   Blood samples were allowed to clot for 30 minutes and centrifuged at
   3500g for 10 minutes. Aliquots of serum were prepared and stored at
   −80°C. Enzyme‐linked immunosorbent (ELISA) was employed to measure
   mouse serum RANKL and OPG according to the manufacturer's protocols
   (R&D Systems).

Cytotoxicity evaluation in vivo

   To evaluate the toxicity of the treatment, the main organs of treated
   mice were separated for H&E staining. Additionally, serum
   concentrations of aspartate aminotransferase (AST), alanine
   aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine were
   determined.

Statistical analysis

   A minimum of three experiments were conducted, with quantitative data
   presented as the mean ± standard deviation (SD). The statistical
   analysis was conducted using the GraphPad Prism 8 software (GraphPad
   Software, Inc., La Jolla, CA, USA) with one‐way analysis of variance
   (ANOVA) or Student two‐tailed t tests. In the graphs, statistical
   significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001,
   ****p < 0.0001. No significance is indicated by “ns.”

Results

P7C3 inhibited RANKL‐induced osteoclast differentiation and bone resorption

   P7C3 is a small‐molecular compound with neuroprotective effects. To
   test whether it can also modulate bone metabolism, we investigated its
   effects on osteoclast differentiation in vitro. As assessed by TRAP
   staining and bone resorption pit assay, P7C3 treatment led to a
   significant decrease in the number and size of osteoclasts in a
   concentration‐dependent manner (Fig. [95]1A–E ). With the concentration
   at 10μM, P7C3 significantly reduced the number of osteoclasts and the
   area of bone resorption pits, compared to the control group
   (vehicle‐treated cells). Notably, P7C3 did not affect cell survival or
   proliferation (Fig. [96]S1A,B), indicating that its effect on
   osteoclast differentiation was not due to cytotoxicity or growth
   inhibition.

Fig. 1.

   Fig. 1
   [97]Open in a new tab

   P7C3 inhibits osteoclast differentiation and bone resorption in vitro.
   (A) TRAP staining of osteoclasts derived from bone marrow cells treated
   with various concentrations of P7C3. (B) Quantification of the number
   of osteoclasts in A. (C) Quantification of the TRAP‐positive area in A.
   (D) Bone resorption assay of osteoclasts induced with different
   concentrations of P7C3. (E) Quantification of the area of bone
   resorption in D. (F) mRNA expression of osteoclast‐specific genes with
   and without P7C3 treatment, as measured by qPCR. (G) Western blot
   analysis of osteoclast‐specific proteins with and without P7C3
   treatment. (H–J) Relative quantification of the proteins in G. (K) TRAP
   staining of osteoclasts derived from peripheral blood mononuclear cells
   treated with various concentrations of P7C3. Data are presented as
   mean ± SD; Statistical significance was calculated by one‐way ANOVA.
   *p < 0.05, **p < 0.01, ***p < 0.001. n = 3.

   The expression levels of osteoclast‐specific genes and proteins further
   confirmed the inhibitory effect of P7C3 on osteoclast differentiation.
   As shown in Fig. [98]1F , P7C3 treatment significantly reduced the mRNA
   expression levels of osteoclast‐specific genes, including CTSK, CTR,
   V‐ATPase, TRAP, DC‐STAMP, NFATc1, and c‐FOS, 3 days after co‐incubation
   with the osteoclastogenic factor RANKL. Consistently, P7C3 treatment
   also decreased the protein expression levels of NFATc1, CTSK, and MMP‐9
   (Fig. [99]1G–J ). These results suggest that P7C3 may interfere with
   the early differentiation process of osteoclasts, leading to a decrease
   in their fusion and activity.

   We further evaluated the effect of P7C3 on osteoclast differentiation
   induced by human peripheral blood cells. As shown in Fig. [100]1K and
   Fig. [101]S2A, P7C3 also inhibited the differentiation of osteoclasts
   in a concentration‐dependent manner. Intriguingly, the inhibitory
   effect of P7C3 on human osteoclast differentiation was more pronounced
   than that on mice, indicated by the significantly lower number and size
   of human osteoclasts. These suggest that P7C3 may have a potential
   translational value in the treatment of bone diseases in humans.

P7C3 suppressed RANKL‐induced Akt‐NF‐κB‐NFATc1 signaling

   To investigate the mechanism underlying the inhibitory effect of P7C3
   on osteoclast differentiation, we performed Western blot analysis to
   examine the key signaling proteins involved in RANKL‐induced
   osteoclastogenesis. We found that treatment of P7C3 did not affect the
   expression levels of MAPK pathway proteins (Erk, JNK, p38)
   (Fig. [102]2A and Fig. [103]S2B–D). However, the protein levels of p65,
   a key component of the NF‐kB signaling pathway, were significantly
   decreased after P7C3 treatment (Fig. [104]2B–D ). Moreover, P7C3
   treatment was associated with a decrease in the phosphorylation level
   of p65 and its nuclear translocation, consistent with the inhibitory
   effect of P7C3 on osteoclastogenesis (Fig. [105]2E ).

Fig. 2.

   Fig. 2
   [106]Open in a new tab

   P7C3 inhibits osteoclast differentiation via the Akt‐NF‐κB‐NFATc1
   pathway. (A) Effects of P7C3 (10μM) on MAPK signaling pathways (ERK,
   JNK, p38) in osteoclast differentiation. (B) Effects of P7C3 (10μM) on
   NF‐κB pathway in osteoclast differentiation. (C,D) Relative
   quantitative analysis of proteins involved in the NF‐κB pathway in B.
   (E) Immunofluorescence staining of p65 nuclear translocation in
   RAW264.7 cells induced with or without P7C3 (10μM) during osteoclast
   differentiation. (F) Effects of P7C3 (10μM) on Akt phosphorylation
   during osteoclast differentiation, as measured by Western blot
   analysis. (G) Effects of Akt phosphorylation activator SC79 on
   downstream NF‐κB pathway activation inhibited by P7C3 (10μM). (H)
   Docking analysis of small molecule P7C3 with Akt protein, showing
   binding energy of −9.328 kcal/mol. Data are presented as mean ± SD;
   statistical significance was calculated by one‐way ANOVA. *p < 0.05,
   **p < 0.01. n = 3.

   In order to investigate whether the NF‐κB pathway is indirectly
   targeted by P7C3, we examined the expression levels of Akt, a known
   upstream regulator of NF‐κB signaling in P7C3‐treated cells. P7C3
   treatment decreased the phosphorylation level of Akt (Fig. [107]2F and
   Fig. [108]S2E), suggesting that P7C3 may regulate NF‐κB signaling in an
   Akt‐dependent manner. To validate this hypothesis, we pretreated the
   cells with a phospho‐Akt activator, SC79, which could activate Akt and
   promote its downstream signaling. As expected, SC79 treatment rescued
   the inhibitory effect of P7C3 on the phosphorylation level of p65 and
   the expression level of NFATc1, which is a key transcription factor
   involved in osteoclast differentiation (Fig. [109]2G and
   Fig. [110]S2F–H). These results indicate that the Akt‐NF‐kB‐NFATc1
   signaling pathway is involved in the inhibitory effect of P7C3 on
   osteoclast differentiation.

   Next, we performed molecular docking simulations using AutoDock
   software to further explore the molecular basis of the interaction
   between P7C3 and Akt protein. The results showed that P7C3 can form
   hydrogen bonds with important residues in the active site of Akt,
   potentially stabilizing the Akt‐P7C3 complex (Fig. [111]2H ). These
   findings support the hypothesis that P7C3 may directly interacts with
   Akt and modulates its activity and downstream signaling. Overall, we
   indicate that P7C3 inhibits osteoclast differentiation by targeting the
   Akt‐NF‐kB‐NFATc1 pathway, likely through direct interaction with Akt.

P7C3 stimulated osteoblast differentiation

   Osteoblasts and osteoclasts work together to ensure proper bone
   remodeling. In the previous sections, we verified P7C3 is capable of
   inhibiting osteoclast differentiation and bone resorption. We further
   investigated its effect on osteoblast differentiation and function to
   explore the effect of P7C3 on bone remodeling. First, we confirmed that
   P7C3 was not cytotoxic to osteoblasts and showed good biocompatibility
   within the used concentration range (Fig. [112]S1C,D). Then, we
   investigated the effect of P7C3 on osteoblast differentiation and
   function. As shown in Fig. [113]3A–D , P7C3 treatment triggered a
   significant increase in ALP activity and mineralized nodule formation,
   suggesting that P7C3 promotes osteoblast differentiation and
   mineralization. Moreover, we analyzed the expression levels of several
   osteoblast‐specific genes and markers by real‐time PCR analysis to
   confirm this observation at the molecular level. As shown in
   Fig. [114]3E,F , at day 3 after the induction of osteogenic
   differentiation, P7C3 treatment has already resulted in a significant
   upregulation of the expression of early osteoblasts differentiation
   markers, such as ALP and RUNX2. Besides, the expression levels of late
   markers of osteoblast differentiation including OPN and OCN
   (Fig. [115]3G,H ), were significantly elevated at day 7 after
   osteogenic induction, indicating that P7C3 may enhance both early‐ and
   late‐stage osteoblast differentiation. These results demonstrate that
   P7C3 can promote osteoblast differentiation and maturation in vitro.

Fig. 3.

   Fig. 3
   [116]Open in a new tab

   P7C3 promotes osteoblast differentiation and mineralization via the
   Wnt/β‐catenin pathway. (A) ALP staining of osteoblasts induced with or
   without P7C3 (10μM) during osteogenic differentiation. (B) Relative
   quantification of ALP staining area in A. (C) Alizarin red staining of
   mineralized nodules formed in osteoblasts induced with or without P7C3
   (10μM) during osteogenic differentiation. (D) Relative quantification
   of Alizarin red staining area in C. (E–H) mRNA expression of
   osteoblast‐specific genes with and without P7C3 (10μM) treatment,
   measured by qPCR. (I) Volcano plot of differentially expressed genes
   identified by RNA sequencing analysis of osteoblasts induced with or
   without P7C3 during osteogenic differentiation. (J) The number of
   upregulated and downregulated genes identified by RNA sequencing
   analysis in I. (K) KEGG pathway enrichment analysis of differentially
   expressed genes in I. (L) Bubble plot showing specific signaling
   pathways identified by KEGG enrichment analysis in K. (M) Western blot
   analysis of β‐catenin expression in osteoblasts induced with or without
   P7C3 (10μM) during osteogenic differentiation. Data are presented as
   mean ± SD; statistical significance was calculated by Student t test.
   *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3.

   To further explore the mechanisms underlying the effects of P7C3 on
   osteoblast differentiation, we used RNA sequencing to examine the
   transcriptomic changes in osteoblast precursor cells treated with P7C3.
   We found that P7C3 treatment induced significant changes of 500 genes
   expression, including 270 upregulated and 230 downregulated genes
   (Fig. [117]3I,J ). KEGG pathway enrichment analysis revealed that the
   differentially expressed genes were highly enriched in signaling
   pathways (Fig. [118]3K ). Further pathway enrichment analysis focusing
   on bone‐related functions showed that the Wnt/β‐catenin signaling
   pathway was significantly upregulated by P7C3 treatment (Fig. [119]3L
   ). To validate the upregulation of the Wnt/β‐catenin signaling pathway
   by P7C3, we examined the protein level of β‐catenin, the central
   component of the pathway. As shown in Fig. [120]3M and Fig. [121]S2I,
   P7C3 treatment resulted in a significant increase in the protein level
   of β‐catenin, indicating P7C3 could activate the Wnt/β‐catenin
   signaling pathway. Taken together, we have demonstrated that P7C3 could
   promote osteoblast differentiation and mineralization without
   cytotoxicity. The transcriptomic changes induced by P7C3 suggest that
   its promotion effect on osteoblast differentiation might be mediated by
   the activation of the Wnt/β‐catenin signaling pathway.

P7C3 suppressed Ti particle–induced calvarial osteolysis in vivo

   To test the potential therapeutic value of P7C3 for bone diseases, we
   conducted animal experiments using a Ti particle–induced bone
   destruction model and an OVX‐induced osteoporosis model to explore the
   potential therapeutic value of P7C3 for bone diseases (Fig. [122]4A ).
   By μCT analysis, we assess the effect of P7C3 on bone destruction in Ti
   particle–challenged mice. As shown in Fig. [123]4B–E , P7C3 treatment
   significantly inhibited bone erosion, as evidenced by increased bone
   mineral density and bone volume compared with the Ti particle control
   group. Hematoxylin and eosin (H&E) staining also confirmed the
   protective effect of P7C3 on bone tissue and resulted in a marked
   increase in new bone formation areas compared with the Ti particle
   control group (Fig. [124]4F and Fig. [125]S2J). Furthermore, TRAP
   staining of bone slices and whole skull bones showed that P7C3
   treatment significantly inhibited the activation of osteoclasts, as
   evidenced by reduced TRAP‐positive cells (Fig. [126]4F–I ).

Fig. 4.

   Fig. 4
   [127]Open in a new tab

   P7C3 suppresses Ti particle‐induced calvarial osteolysis. (A) Study
   design of in vivo animal experiments. (B) 3D reconstruction of
   calvarial bones by μCT. (C–E) Quantitative analysis of μCT images,
   including bone mineral density (BMD), bone volume/total volume (BV/TV),
   and porosity percentage. (F) H&E and TRAP staining of calvarial bone
   tissue, showing the new bone formation and osteoclasts, respectively.
   (G) Relative quantification of TRAP‐positive cells in F. (H) TRAP
   staining of osteoclast distribution in whole calvarial bone (n = 3).
   (I) Quantification of TRAP‐positive staining area in H. (J–M) qPCR
   analysis of osteoclast‐specific genes and osteoblast‐specific genes in
   calvarial bone tissue (n = 3). (N–P) ELISA assay of serum levels of
   RANKL and OPG, as well as their ratio. Data are presented as mean ± SD;
   statistical significance was calculated by one‐way ANOVA. *p < 0.05,
   **p < 0.01, ***p < 0.001. n = 5.

   To explore the molecular basis of the in vivo effects of P7C3, we
   performed real‐time PCR analysis of bone tissues to examine the
   expression levels of osteoblast‐ and osteoclast‐specific genes. As
   shown in Fig. [128]4J,K , P7C3 treatment significantly inhibited the
   expression of osteoclast‐specific genes such as CTSK and CTR,
   indicating that P7C3 could suppress osteoclast differentiation and
   activity in vivo. In contrast, the expression levels of
   osteoblast‐specific genes such as ALP and RUNX2 were significantly
   upregulated in P7C3‐treated mice, suggesting that P7C3 could promote
   osteoblast differentiation and bone formation in vivo (Fig. [129]4L,M
   ).

   In addition, we also examined the serum levels of RANKL and OPG, which
   were two key factors involved in regulating osteoclast differentiation
   and activation. As shown in Fig. [130]4N–P , P7C3 treatment led to a
   significant decrease in the serum level of RANKL and an increase in the
   level of OPG in Ti particle–challenged mice. Furthermore, the ratio of
   RANKL to OPG was significantly reduced by P7C3 treatment, indicating
   that P7C3 may regulate bone remodeling by modulating the RANKL/OPG
   signaling axis. Taken together, these results provide direct evidence
   that P7C3 can inhibit Ti particle–induced bone erosion, promote new
   bone formation, and regulate bone remodeling by modulating osteoclast
   and osteoblast activity.

P7C3 improved bone remodeling in the OVX‐induced osteoporosis model

   To further evaluate the therapeutic potential of P7C3 in bone diseases,
   we also examined its effects on the development of postmenopausal
   osteoporosis using an OVX‐induced osteoporosis model. Mice that
   underwent OVX surgery showed significant weight gain and uterine
   degeneration compared with the sham‐operated mice, consistent with the
   characteristics of postmenopausal osteoporosis (Fig. [131]S3A–C).

   As shown in Fig. [132]5A–H , μCT imaging analysis of the femurs from
   OVX‐induced mice treated with P7C3 revealed a significant increase in
   bone mass, as evidenced by increased trabecular bone mineral density,
   trabecular bone volume, bone surface area, and trabecular number, as
   well as reduced trabecular separation compared with the untreated OVX
   group. Moreover, histological analysis of bone tissues revealed that
   P7C3 treatment significantly increased the number of trabeculae and
   reduced the severity of bone loss in OVX‐induced mice (Fig. [133]5I and
   Fig. [134]S3D). TRAP staining of bone slices showed that P7C3 treatment
   significantly inhibited osteoclast activity in OVX‐induced mice which
   is consistent with the in vitro findings (Fig. [135]5J , and
   Fig. [136]S3E,F). We also examined the expression levels of RANKL and
   OPG in the serum of OVX‐induced mice treated with P7C3. P7C3 treatment
   led to a significant decrease in the level of RANKL and an increase in
   the level of OPG (Fig. [137]5K,L ).

Fig. 5.

   Fig. 5
   [138]Open in a new tab

   P7C3 improves bone mass in OVX‐induced osteoporosis models. (A) 2D
   cross‐section of mouse femurs by μCT. (B) 3D reconstruction of femurs
   by μCT. (C–H) parameters, including trabecular bone mineral density,
   cortical bone mineral density, bone volume/total volume (BV/TV), bone
   surface area/total volume (BS/TV), trabecular number (Tb.N), and
   trabecular separation(Tb.Sp). (I) H&E staining of bone tissue showing
   trabecular bone distribution. (J) TRAP staining of bone tissue sections
   showing osteoclasts. (K,L) ELISA assay of serum levels of RANKL and
   OPG. (M) Immunofluorescence staining of osteoblast‐specific protein
   osterix expression in bone tissue. (N) Relative quantification of
   osterix protein expression in M. Data are presented as mean ± SD;
   statistical significance was calculated by one‐way ANOVA. *p < 0.05,
   **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5.

   Finally, we also examined the expression levels of osteoblast‐specific
   markers in the bone tissue of OVX‐induced mice treated with P7C3. As
   shown in Fig. [139]5M,N , immunofluorescence staining of bone tissue
   using an osterix antibody revealed that P7C3 treatment significantly
   increased the expression of this key osteoblast‐specific marker in bone
   tissue from OVX‐induced mice. This finding suggests that P7C3 may
   promote new bone formation and improve bone density in vivo by
   promoting osteoblast differentiation and activity. Taken together, our
   results provide direct evidence that P7C3 can effectively attenuate
   bone loss and improve bone density in OVX‐induced osteoporosis models.

P7C3 showed excellent biological safety properties

   To assess the safety and toxicity of P7C3 in vivo, we performed a
   comprehensive analysis of the major organs in mice treated with P7C3.
   Compared with the sham‐operated group, P7C3‐treated mice showed no
   significant signs of toxicity or cellular damage (Fig. [140]6A ).
   Furthermore, we also measured the levels of indicators of liver and
   kidney function, including ALT, AST, BUN, and creatinine. As shown in
   Fig. [141]6B–E , the levels of ALT and AST in P7C3‐treated mice were
   within the normal range, indicating that P7C3 treatment did not
   compromise liver function. Similarly, the levels of BUN and creatinine
   in P7C3‐treated mice were not significantly different from those in the
   control group, suggesting that P7C3 did not induce renal damage or
   affect kidney function. Hence, P7C3 treatment is safe and
   well‐tolerated in mice, with neglectable toxicity and adverse effects.
   These findings support the clinical development of P7C3 as a potential
   therapeutic agent for bone diseases.

Fig. 6.

   Fig. 6
   [142]Open in a new tab

   Biological safety of P7C3 in vivo. (A) H&E staining of major organs
   (heart, liver, spleen, lung, and kidney) in mice treated with P7C3 or
   vehicle control. (B–E) Serum levels of ALT, AST, BUN, and creatinine in
   mice treated with P7C3 or vehicle control. Data are presented as
   mean ± SD; n = 5.

Discussion

   In the present study, we investigated the effects of a small molecule
   compound P7C3 on bone metabolism and its underlying mechanisms. Our
   findings demonstrate that P7C3 was able to inhibit osteoclast formation
   while promoting osteoblast differentiation at the same time.
   (Fig. [143]7) These effects suggest that P7C3 holds great potential as
   a therapeutic agent for the treatment of bone diseases, including
   osteoporosis. Current therapies for osteoporosis employ a range of
   strategies targeting either bone resorption or bone formation. In
   addition, most of these drugs carry their safety concerns.
   Bisphosphonates can cause gastrointestinal side effects, including
   esophageal irritation, and may increase the risk of osteonecrosis of
   the jaw.^( [144]^4 , [145]^39 ^) Denosumab has been linked to an
   increased risk of fractures risk after discontinuing the drug.^(
   [146]^1 , [147]^40 ^) Teriparatide and abaloparatide may increase the
   risk of osteosarcoma, a type of bone cancer.^( [148]^4 , [149]^41 ^)
   Recently, romosozumab, a monoclonal antibody that targets sclerostin
   has been shown to both reduce bone resorption and increase bone
   formation. Nevertheless, safety issues such as an increased risk of
   cardiovascular events have limited its wide use.^( [150]^4 , [151]^42
   ^) A new therapeutic agent for osteoporosis is needed to be developed
   that can prevent bone resorption and enhance bone formation, without
   causing undesirable side effects.

Fig. 7.

   Fig. 7
   [152]Open in a new tab

   Schematic model outlining the actions of P7C3 on bone. The molecular
   mechanism of P7C3 in inhibiting osteoclast differentiation and
   promoting osteoblast differentiation (left) and regulating bone
   remodeling by suppressing bone resorption and promoting bone formation
   to increase bone mass (right) have been demonstrated.

   Previous studies indicated that P7C3 exerts its broad‐spectrum
   neuroprotective effects in different species of animals through various
   cellular mechanisms.^( [153]^26 , [154]^32 , [155]^43 , [156]^44 ^) It
   has been shown to promote neuronal survival and increase neurite
   outgrowth, the process by which neurons extend their processes to
   communicate with other cells.^( [157]^30 , [158]^32 ^) It has also been
   demonstrated to suppress microglia‐mediated neuroinflammation by
   modulating the NF‐κB pathway.^( [159]^45 ^) In this study, we further
   discovered that P7C3 can inhibit osteoclast differentiation signals by
   suppressing the upstream phosphorylation of Akt, thereby inhibiting the
   downstream NF‐κB signal. The inhibition of the Akt‐NF‐κB‐NFATc1
   signaling pathway by P7C3 provides a clear molecular mechanism for its
   anti‐osteoclastic activity. This pathway has been reported to be
   responsible for osteoclast differentiation and is also closely related
   to pathologies such as osteoarthritis.^( [160]^46 ^) Previous studies
   have shown that inhibition of Akt‐NF‐κB‐NFATc1 pathway components can
   reduce bone resorption and prevent bone loss in animal models.^(
   [161]^47 , [162]^48 ^) Our results support this finding, and
   demonstrate the value of targeting this pathway in developing novel
   therapies for osteoporosis and other bone‐related disorders.

   The promotion of osteoblast differentiation by P7C3 is also a crucial
   finding. The Wnt/β‐catenin signaling pathway has been regarded as an
   important regulator of osteoblastogenesis and bone formation.^(
   [163]^49 , [164]^50 , [165]^51 ^) We provide informative evidence that
   P7C3 can activate this pathway and promote osteoblast differentiation.
   This is a promising finding, as a decline in osteoblast differentiation
   and bone formation is a hallmark of bone diseases such as osteoporosis
   and osteogenesis imperfecta.^( [166]^52 ^)

   More importantly, P7C3 has shown remarkable safety and biocompatibility
   in animal models, with neglectable apparent adverse effects during
   extensive investigations for neurodegenerative disorders in previous
   and current studies.^( [167]^23 , [168]^26 , [169]^31 ^) These
   properties make P7C3 an attractive candidate for the development of new
   therapies for the treatment of osteoporosis.

   In conclusion, our study has explored the mechanism and extended the
   use of P7C3 in bone disease treatment. The findings of this study offer
   a promising outlook for the therapeutic potential of P7C3 in the
   treatment of bone‐related disorders such as osteoporosis, which is a
   prevalent medical condition affecting millions of people worldwide.
   Furthermore, given its neuroprotective properties, P7C3 has potential
   therapeutic benefits for age‐related neurodegenerative diseases.^(
   [170]^23 , [171]^26 , [172]^53 ^) As both osteoporosis and
   neurodegenerative diseases are age‐related diseases,^( [173]^54 ^) the
   potential for a single drug to treat both conditions could have
   significant clinical implications. Thus, P7C3 warrants further
   investigation to establish its safety, efficacy, and optimal
   therapeutic dosing in human clinical trials. The potential for P7C3 to
   be used in combination with existing treatments for osteoporosis and
   other bone diseases also merits further exploration.

Author Contributions

   Bo Tian: Conceptualization; data curation; formal analysis;
   investigation; methodology; writing – original draft. Jinyu Bai:
   Conceptualization; data curation; formal analysis; funding acquisition;
   investigation; methodology. Lei Sheng: Data curation; formal analysis;
   investigation; methodology. Hao Chen: Investigation. Wenju Chang:
   Formal analysis. Yue Zhang: Investigation; methodology. Chenlu Yao:
   Methodology; writing – review and editing. Chenmeng Zhou:
   Investigation; methodology. Xiaoyu Wang: Formal analysis. Huajian Shan:
   Methodology; project administration. Qirong Dong: Conceptualization;
   funding acquisition; methodology; supervision. Chao Wang:
   Conceptualization; funding acquisition; supervision; writing – original
   draft; writing – review and editing. Xiaozhong Zhou: Conceptualization;
   funding acquisition; methodology; supervision; writing – review and
   editing.

Peer Review

   The peer review history for this article is available at
   [174]https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/j
   bm4.10811.

Disclosures

   All authors report no conflicts of interest.

Supporting information

   Fig. S1. Effects of P7C3 on cell proliferation and viability. (A, B)
   MTT assay showing the viability of osteoclast precursor cells treated
   with P7C3 for 24 or 48 h. (C, D) MTT assay showing the viability of
   osteoblast precursor cells treated with P7C3 for 24 or 48 h. Data are
   presented as mean ± SD; n = 6.

   Fig. S2. Quantitative analysis. (A) Quantitative analysis of
   Fig. [175]1K. (B–D) Relative protein expression quantification of
   Fig. [176]2A. (E) Relative protein quantification analysis of
   Fig. [177]2F. (F–H) Relative protein expression quantification of
   Fig. [178]2G. (I) Relative protein quantification analysis of
   Fig. [179]3M. (J) Morphological quantification analysis of H&E‐stained
   new bone formation area in Fig. [180]4F (n = 5). Data are presented as
   mean ± SD; Statistical significance was calculated by one‐way ANOVA and
   Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
   n = 3.

   Fig. S3. Confirmation of OVX‐induced osteoporotic animal model and
   quantitative analysis of bone morphology. (A) Body weight changes of
   mice after OVX surgery. (B) Uterine atrophy at 8 weeks post‐surgery.
   (C) Quantification of uterine weight in (B). (D) Morphometric
   measurement analysis of bone in Fig. [181]5I. (E) and (F) Relative
   quantification analysis of TRAP‐stained osteoclast formation on bone
   surface in Fig. [182]5J. BV/TV, bone volume per tissue volume; OC.S/BS,
   osteoclast surface area per bone surface; OC.N/BS, osteoclast number
   per bone surface. Data are presented as mean ± SD; Statistical
   significance was calculated by Student's t test and one‐way ANOVA.
   *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5.

   Fig. S4. Original data of western blots in the paper. (A) Original
   western blot image for Fig. [183]1G. (B) Original western blot image
   for Fig. [184]2A. (C) Original western blot image for Fig. [185]2B. (D)
   Original western blot image for Fig. [186]2F. (E) Original western blot
   image for Fig. [187]2G. (F) Original western blot image for
   Fig. [188]3M.

   Table S1. Specific Primer Sequences for qPCR Analysis
   [189]Click here for additional data file.^ (2.4MB, docx)

Acknowledgments