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Research Article

Liuwei Dihuang Wan Inhibits Osteoclastogenesis and Ovariectomy-Induced Bone Loss by Suppressing NF-κB Signaling

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DOI:

10.3791/72270

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July 28th, 2026

 ,  ,  ,  , 
1School of Medicine, Nankai University, 2School of Basic Medical Sciences, Zhejiang Chinese Medical University, 3Emergency Medical Center, Ningbo Hospital of Integrated Traditional Chinese and Western Medicine, 4The Second People's Hospital of Changzhou, the Third Affiliated Hospital of Nanjing Medical University

* These authors contributed equally

In This Article

Summary

This study demonstrated that Liuwei Dihuang Wan (LWDHW) suppresses osteoclast differentiation and ovariectomy-induced bone loss by inhibiting the NF-κB signaling pathway, as revealed through LC-MS/MS analysis, network pharmacology, and in vivo and in vitro validation.

Abstract

Liuwei Dihuang Wan (LWDHW), a classic traditional Chinese medicine formula, is widely used to treat various diseases associated with kidney deficiency and has shown satisfactory clinical efficacy in postmenopausal osteoporosis (PMOP). However, its pharmacological mechanisms remain unclear. To address this, we first analyzed the chemical components of LWDHW using LC-MS/MS, which identified 1,615 components. Network pharmacology then predicted that inflammation and the NF-κB signaling pathway are potential therapeutic mechanisms underlying LWDHW’s effects on PMOP. Subsequently, 27 female C57BL/6J mice were randomly assigned to the Sham, Model, and LWDHW groups. Mice in the latter two groups underwent bilateral ovariectomy (OVX), and those in the LWDHW group received oral LWDHW at 9.75 g/kg/day for 8 weeks. Serum and femur samples were collected for Enzyme-Linked Immunosorbent Assay (ELISA), Micro-Computed Tomography (µCT), histological examination, and immunohistochemistry (IHC). Primary bone marrow-derived macrophages (BMMs) were treated with LWDHW and then subjected to TRAP staining and qRT-PCR. In OVX mice, LWDHW significantly alleviated bone loss and deterioration of bone microarchitecture, accompanied by reduced serum β‑CTX levels and decreased osteoclast numbers in femoral tissues. IHC staining revealed notably lower expression of TNF-α, IL-1β, and p-P65 in femoral tissues of LWDHW-treated mice compared to the model group. In vitro, TRAP staining demonstrated that LWDHW inhibited RANKL/M-CSF-induced osteoclast differentiation of BMMs and downregulated the expression of Ctsk, MMP9, and Nfatc1. Western blot analysis further showed that protein levels of P65, p-P65, IκBα, and p-IκBα were significantly decreased in BMMs following LWDHW treatment. Collectively, LWDHW alleviates postmenopausal osteoporosis, possibly by suppressing NF-κB signaling‑mediated osteoclastogenesis.

Introduction

Postmenopausal osteoporosis (PMOP) is a prevalent metabolic bone disease characterized by bone mass loss and deterioration of bone microarchitecture1. With the growing aging population, the increasing incidence of PMOP has become a global public health issue that seriously threatens the health of postmenopausal women2. In China, the overall prevalence of PMOP is as high as 32.1%3. Affected individuals are more susceptible to severe complications such as chronic back pain, spinal deformities, and fragility fractures4,5. Currently, FDA-approved medications for osteoporosis include bisphosphonates6, parathyroid hormone67 and denosumab8, which are widely used in clinical practice. However, their long-term application is often limited by various adverse effects9. Therefore, there is a pressing need to develop safer and more effective therapeutic agents for PMOP. Among the potential alternatives, Chinese herbal medicines have garnered increasing attention due to their recognized efficacy and favorable safety profile10.

Liuwei Dihuang Wan (LWDHW) is a classical formula in traditional Chinese medicine (TCM), first documented in the ancient text Key to Therapeutics of Children's Diseases during the Song Dynasty. For thousands of years, LWDHW has been widely used in clinical practice to treat various diseases attributed to kidney deficiency11,12,13,14. According to the TCM theory of “kidney governing bones”, bone mass loss and bone deterioration in PMOP patients are primarily caused by insufficient nourishment from kidney essence15. Consequently, LWDHW is commonly applied in the clinical management of PMOP and has demonstrated definite therapeutic efficacy16,17. However, although animal and clinical studies have revealed that LWDHW exerts multiple pharmacological effects, including antioxidative18, anti-inflammatory19 and antifibrotic activities14, its underlying mechanism against PMOP remains largely unknown.

Bone homeostasis relies on the balance between bone resorption by osteoclasts and bone formation by osteoblasts20. This balance is disrupted as osteoclast differentiation and activity are enhanced under inflammatory conditions, leading to accelerated bone resorption21. In this process, the NF-κB pathway serves as a core regulator. Inflammatory factors such as TNF-α and IL-1β can induce phosphorylation and degradation of IκBα, triggering the nuclear translocation of phosphorylated p65 (p-P65)22. Consequently, downstream osteoclast-specific genes, including Ctsk, Mmp9, and Nfatc1, are activated to promote osteoclastogenesis23,24.

Network pharmacology is a cross-disciplinary field that integrates computer science, pharmacology, and bioinformatics25. It exhibits distinct advantages in comprehensively exploring the pharmacological mechanisms underlying the therapeutic effects of Chinese herbs26. In the present study, network pharmacology was employed to predict the potential biological processes and signaling pathways involved in the anti-osteoporotic effects of LWDHW. Concurrently, the chemical components of LWDHW were analyzed using LC-MS/MS. Furthermore, in vivo validation using an OVX mouse model and in vitro experiments with primary bone marrow-derived macrophages (BMMs) were conducted to further investigate the pharmacological mechanism of LWDHW against PMOP. The workflow is presented in Figure 1.

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Protocol

All animal experiments were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (IACUC-202402-07).

Preparation and LC-MS/MS analysis of LWDHW

The LWDHW formula consists of six Chinese herbs: Rehmannia glutinosa (Gaertn.), Cornus officinalis (Sieb.), Paeonia suffruticosa (Andr.), Dioscoreae opposite (Thunb.), Poria cocos (Schw.), and Alisma orientale (Sam.). Detailed information on these herbs is listed in Table 1. All raw herbs were supplied by the First Affiliated Hospital of Zhejiang Chinese Medical University (Hangzhou, China) and boiled in purified water at an herb-to-water ratio of 0.1 kg/L. The resulting aqueous extract was filtered and concentrated to a working solution at 0.5 g/mL for subsequent LC-MS/MS analysis and animal experiments. Furthermore, the working solution was processed into freeze-dried powder for cell experiments.

In LC-MS/MS analysis, the liquid chromatography conditions were as follows: mobile phase A (aqueous) and mobile phase B (organic) consisted of 0.1% formic acid in water and acetonitrile, respectively, for positive ion mode, or 5 mM ammonium formate in water and acetonitrile, respectively, for negative ion mode. The total runtime was 8 min, with a flow rate of 0.3 mL/min, a column temperature of 40 °C, and an injection volume of 2 µL. For mass spectrometry, data were acquired in Full MS-ddMS2 mode, with spray voltages set at +3.50 kV (positive) and –2.50 kV (negative).

Bioactive ingredients and targets prediction of LWDHW

The individual names of six constituent herbs were entered into the Traditional Chinese Medicine System Pharmacology (TCMSP) database to obtain the full ingredient list for LWDHW. Bioactive ingredients were subsequently screened using the pharmacokinetic parameters of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.1827,28,29,30. The corresponding proteins of the bioactive ingredients were identified and submitted to UniProt to retrieve their standardized gene names, which were defined as the final gene targets of LWDHW.

Overlapping targets between LWDHW and PMOP

Gene targets associated with PMOP were retrieved by searching the keyword "postmenopausal osteoporosis" in both the GeneCards and DisGeNET databases on October 2, 2024. Targets were filtered using score thresholds of >0.1 in DisGeNET and >10 in GeneCards, and duplicate entries were removed to obtain a non-redundant set of PMOP-related genes. Subsequently, the herb-derived targets of LWDHW and the PMOP-associated targets were imported into the Venny online tool to generate a Venn diagram depicting the overlapping targets between LWDHW and PMOP.

Gene Ontology (GO) and signaling pathway enrichment analysis

The overlapping targets between LWDHW and PMOP were subject to GO enrichment analysis using the DAVID database. The retrieved GO terms were classified into three categories, including cellular component, molecular function, and biological process (BP). After filtering FDR < 0.01, the top 30 BP terms were listed in a bubble diagram in descending order of fold enrichment.

Next, the overlapping targets were entered into the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Metascape databases for signaling pathway enrichment analysis. The enriched pathways from the KEGG and Metascape databases were ranked by the number of corresponding targets in descending order, and the top 25 pathways from each database were displayed as bar charts.

Network construction

The "Formula-Herbs-Ingredients-Targets-Disease" and "Targets-Pathways" networks were constructed in Cytoscape to visualize the interrelationships among components. A protein-protein interaction (PPI) network was generated by inputting the overlapping targets into the STRING database. Hub targets within the network were identified by screening based on topological features, including degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC).

Animal grouping, OVX-induced osteoporosis model and drug intervention

A total of 27 female C57BL/6J mice (2 months old, weighing 22 ± 2 g) were provided by the Animal Experiment Center of Zhejiang Chinese Medical University (Hangzhou, Zhejiang). All animals were housed under specific pathogen-free (SPF) conditions with a 12 h light/dark cycle, ambient temperature maintained at 25 °C, and humidity between 40% and 60%, with free access to food and water. After 1 week of adaptive feeding, the mice were randomly assigned to three groups (n = 9 per group) using a random number table: Sham, Model, and LWDHW. Mice in the Model and LWDHW groups underwent bilateral ovariectomy (OVX) to establish the PMOP model, while the Sham group underwent a sham procedure involving the resection of an equivalent volume of periovarian adipose tissue. Starting two days post-surgery, mice in the LWDHW group received daily oral gavage of LWDHW working solution at a dose of 9.75 g/kg/day (converted from the human equivalent dose based on body surface area), whereas the Model and Sham groups received an equal volume of saline. Biological specimens, including serum and femoral tissues, were collected 8 weeks following administration for subsequent analysis. No unexpected death or mortality occurred in any of the groups throughout the experimental period.

Serum enzyme-linked immunosorbent assay (ELISA)

Whole blood was collected from the retro-orbital plexus and centrifuged at 1000 x g for 15 min at 4 °C to obtain serum. Serum concentrations of osteocalcin (OCN) and β-C-terminal telopeptide of type I collagen (β-CTX) were measured using the corresponding ELISA kits according to the manufacturer's protocols.

µCT and biomechanical analysis

Femoral samples were scanned using a high-resolution micro-computed tomography (µCT) equipment at an isotropic resolution of 10 µm. The three-dimensional (3D) microstructure of the distal femur was reconstructed from the acquired images using NRecon software. Subsequently, the following bone microarchitecture parameters, including bone mineral density (BMD, g/mm3), bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, mm), and trabecular number (Tb.N, 1/mm), were quantified within the region of interest (ROI) of the distal femur.

The biomechanical properties of femoral samples (n = 3 per group) were evaluated via a three-point bending test using an Axial-Torsion Fatigue Testing System. A compressive load was applied to the mid-diaphysis at a constant displacement rate of 2 mm/min until structural failure. The maximum load (N) was automatically recorded by the system's software.

Histomorphology, immunohistochemistry (IHC), and immunofluorescence (IF) staining

Following fixation with 4% paraformaldehyde, decalcification in 14% EDTA solution, and dehydration through a graded ethanol series, femoral samples were processed into paraffin sections of 3 µm-thick as previously described31. The sections were stained with Alcian Blue Hematoxylin (ABH)/Orange G for morphological evaluation. Quantitative analysis of the trabecular bone area in the distal femoral metaphysis was performed using OsteoMetrics software.

Liver and kidney specimens were fixed, embedded in paraffin, and cut into 4 µm sections. After routine deparaffinization and rehydration, the sections were stained with hematoxylin and eosin. Histological examination was performed under a light microscope.

IHC and IF assays were performed on paraffin-embedded sections according to established procedures. Briefly, antigen retrieval was carried out by incubating sections in 0.01 M citrate buffer at 60 °C for 4 h. Sections were then incubated overnight at 4 °C with primary antibodies including ALP (1:300), IL‑1β (1:500), TNF‑α (1:500), and p‑P65 (1:300). After incubation with corresponding secondary antibodies for 20 min, sections were subjected to diaminobenzidine (DAB) solution and hematoxylin counterstaining for IHC or stained with DAPI for nuclear labeling in IF. For positive staining quantification, four randomly selected fields were captured from each of three tissue sections per sample. In IHC staining, the mean optical density was defined as the ratio of the integrated optical density (IOD) to the corresponding cavity area, as determined using Image‑Pro Plus software. In IF staining, the positive staining area was normalized to the field area and expressed as a percentage, and the quantification was performed with the same software.

Primary bone marrow macrophages (BMMs) culture and cell counting assay

Bone marrow cells were flushed out from the femurs and tibiae of 2-month-old C57BL/6 mice with phosphate-buffered saline (PBS). The resulting cell suspension was filtered through a 70 µm strainer and centrifuged at 200 x g for 5 min. The cell pellet was resuspended in α-MEM complete medium containing 10% fetal bovine serum. After 24 h of incubation at 37 °C in a 5% CO₂ atmosphere, non-adherent cells were removed. To induce macrophage differentiation, adherent cells were cultured in medium containing 50 ng/mL macrophage colony-stimulating factor (M-CSF) and 40 ng/mL RANKL for 5–7 days until mature BMMs were observed.

BMMs were plated at 3 x 104 cells/well in 96-well plates. After 24 h of treatment with gradient concentrations of LWDHW, BMMs were incubated with 10% Cell Counting Kit-8 (CCK-8) working solution at 37 °C for 1 h. The absorbance of each well was measured at 450 nm using a microplate reader.

Quantitative gene expression analysis on primary BMMs

BMMs were seeded into 6 cm dishes at a density of 3 x 106 cells per dish. After treatment with 500 and 1,000 µg/mL LWDHW for 24 h, total RNA was extracted from BMMs using the RNA-Quick Purification Kit according to the manufacturer’s instructions. Following quantification by spectrophotometry, 500 ng of total RNA was reverse-transcribed into cDNA. Quantitative real-time PCR (qRT-PCR) was performed using gene-specific primers (Table 2), and the PCR cycling conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Gene expression levels were analyzed using the 2ΔΔCt method, with GAPDH as the internal reference gene.

Western blot analysis on primary BMMs

After being plated at 3 x 106 cells per dish in 6 cm dishes, BMMs were treated with various interventions and then lysed on ice with RIPA lysis buffer. Total protein was collected and quantified with a BCA Protein Assay Kit. Protein samples (30 µg per lane) were separated by SDS-PAGE on 12.5% polyacrylamide gels and transferred onto 0.2 µm PVDF membranes. After blocking for 20 min, the membranes were incubated overnight at 4 °C with the following primary antibodies: P65 (1:1000), p-P65 (1:1000), IκBα (1:1000), p-IκBα (1:1000), and β-actin (1:1000). The next day, membranes were incubated with secondary antibody for 1 h at room temperature. Protein bands were visualized using an ECL Chemiluminescence Detection Kit and captured using an Automatic Chemiluminescence Image Analysis System.

TRAP staining of femoral tissues and BMMs

Femoral tissue sections and BMMs plated in 24‑well plates at 1 x 105 cells/well were stained using a Tartrate-Resistant Acid Phosphatase (TRAP) staining kit following the manufacturer’s protocol. The osteoclast-covered trabecular bone surface (Oc.S/BS) in tissue sections and the osteoclast formation area in BMM cultures were quantified using Image-Pro Plus software.

Statistical analysis

All data were presented as mean ± standard deviation. A one-way ANOVA followed by Dunnett’s test was used for statistical analysis in SPSS. *p < 0.05 and **p < 0.01 were considered statistically significant.

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Results

Chemical components of LWDHW analyzed by LC-MS/MS

The chemical profile of the LWDHW aqueous solution was characterized by LC-MS/MS analysis. A total of 1615 components were identified, comprising 968 detected in positive ionization mode and 647 in negative ionization mode (Figure 2A). The details of the top 10 components from each ionization mode are listed in Figure 2B,C.

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Discussion

With global population aging, PMOP has become a serious worldwide health concern, affecting millions of women annually36. Given the side effects associated with current anti-osteoporotic drugs, it is imperative to develop safer and more effective therapeutic strategies. LWDHW is a classic kidney-tonifying formula in TCM and serves as an alternative medicine for treating various bone diseases37,38. Clinical evidence has demonstrated its eff...

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Disclosures

All authors declare no competing financial or non-financial interests.

Acknowledgements

This work was supported by the Natural Science Foundation of Zhejiang Province (ZCLQN25H2701).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.01 M citrate bufferSolarbioC1010For antigen retrieval of femoral sections for IHC and IF staining in Protocol Step 10
0.2 μm PVDF membranes BioRad1620177For electrotransfer of proteins from SDS-PAGE to membrane in Protocol Step 13
12.5% polyacrylamide gelsEpizyme BiotechPG113For SDS-PAGE separation of proteins in Protocol Step 13
ALP antibody ARIGOARG57422For IHC detection in femoral sections in Protocol Step 10
Automatic Chemiluminescence Image Analysis System Tanon 5200For Western blot band visualization in Protocol Step 13
Axial-Torsion Fatigue Testing System Instronmodel 5569R1412For biomechanical testing of femoral samples in Protocol Step 9
BCA Protein Assay KitBeyotimeP0010SFor protein quantification prior to Western blot in Protocol Step 13
CCK-8BeyotimeC0039For cell viability measurement of BMMs in Protocol Step 11
CytoscapeNot applicableNot applicableVersion 3.8.0
DAVID databaseNot applicableNot applicableVersion 6.8; https://david.ncifcrf.gov/home.jsp
DisGeNET databaseNot applicableNot applicablehttps://www.disgenet.org/
ECL Chemiluminescence Detection KitVazymeE411-04For chemiluminescent detection of Western blot signals in Protocol Step 13
ELISA kit of OCNMEIMIANMM-0551M1For measurement of serum osteocalcin levels in Protocol Step 8
ELISA kit of β-CTXMEIMIANMM-44858M1For measurement of serum β-CTX levels in Protocol Step 8
GeneCards databaseNot applicableNot applicablehttps://www.genecards.org/
Green Premix Ex Taq II FASTTakaraCN830AFor qRT-PCR amplification in Protocol Step 12
High-resolution micro-computed tomography (μCT) equipmentBrukerSkyscan 1176For high-resolution μCT imaging of femoral bone microstructure in Protocol Step 9
IL-1β antibody BIOSSbs6319RFor IHC detection in femoral sections in Protocol Step 10
IκBαantibodyCST4812For Western blot detection in Protocol Step 13
Kyoto Encyclopedia of Genes and Genomes (KEGG) database Not applicableNot applicablehttps://www.kegg.jp/
M-CSFAMIZONAAMK1001-S1For inducing BMMs differentiation in Protocol Step 11
Metascape databaseNot applicableNot applicablehttps://metascape.org/
Orbitrap Exploris 120 Thermo Fisher ScientificBRE725531For LC-MS/MS analysis of LWDHW in Protocol Step 2
P65 antibodyCST8242For Western blot detection in Protocol Step 13
p-IκBα antibodyCST2859For Western blot detection in Protocol Step 13
p-P65 antibodyCST3033SFor Western blot detection in Protocol Step 13
p-P65 antibodySuzhou Ruiying RLP0187For IHC detection in femoral sections in Protocol Step 10
PrimeScript RT Reagent Kit TakaraRR047AFor reverse transcription of total RNA in BMMs for qRT-PCR analysis in Protocol Step 12
QuantStudio 5 systemApplied BiosystemsA28573For qRT-PCR analysis of osteoclast marker genes in Protocol Step 12
RANKLAMIZONAAMK1001-S1For inducing BMMs differentiation in Protocol Step 11
RIPA lysis bufferEpizyme BiotechPC101For total protein extraction from BMMs in Protocol Step 13
RNA-Quick Purification Kit TransGen BiotechER501-01-V2For extraction of total RNA in BMMs in Protocol Step 12
SPSS software IBMversion 26For statistical analysis of experimental data in Protocol Step 15
STRING database Not applicableNot applicablehttps://string-db.org/
Tartrate-Resistant Acid Phosphatase (TRAP) staining kitAMIZONAAMK1001-S1For TRAP staining to identify and quantify osteoclasts in Protocol Step 14
Thermo Vanquish UPLC systemThermo Fisher ScientificTSLC-002For LC-MS/MS analysis of LWDHW in Protocol Step 2
TNF-αantibodySuzhou Ruiying RLM3472For IHC detection in femoral sections in Protocol Step 10
Traditional Chinese Medicine System Pharmacology (TCMSP) database Not applicableNot applicablehttp://lsp.nwu.edu.cn/tcmsp.php
UniProt database Not applicableNot applicablehttps://www.uniprot.org/
UPLC ColumnWatersACQUITY HSS T3For LC-MS/MS analysis of LWDHW in Protocol Step 2
Venny online tool Not applicableNot applicablehttps://bioinfogp.cnb.csic.es/tools/venny/index.html
β-actin antibodyAbcamab8227For Western blot detection in Protocol Step 13

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Osteoclastogenesis InhibitionNF-KappaB SignalingPostmenopausal OsteoporosisOvariectomy Bone LossBone Marrow MacrophagesTRAP StainingMicro-Computed TomographyWestern BlotNetwork Pharmacology
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