Abstract

This study aimed to explore the mechanism of Rehmanniae Radix Praeparata–Cistanches Herba (RRP‑CH) herb pair in treating steroid‑induced avascular necrosis of the femoral head (SANFH) using network pharmacology and in vivo animal experiments. Active components and potential targets of RRP‑CH were screened via the TCMSP database. Disease targets for SANFH were collected from GeneCards, OMIM, DisGeNET, and DrugBank. The overlapping targets were applied to construct a herb‑component‑disease‑target interaction network, perform protein‑protein interaction (PPI) analysis, and conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Molecular docking was performed to verify the binding affinity between core components and hub targets. A SANFH rat model was established using lipopolysaccharide (LPS) combined with methylprednisolone sodium succinate to validate the therapeutic effects and underlying pathways.

Network pharmacological analysis identified 5 core components and 36 shared targets for RRP‑CH against SANFH. The hub targets included GSK‑3β, TNF, IL‑6, RUNX2, and β‑catenin. KEGG pathway enrichment highlighted the Wnt/β‑catenin signaling pathway as the most critical pathway. Molecular docking confirmed that quercetin, stigmasterol, and syringaresinol dimethyl ether stably bound to these key proteins (binding energy ≤ −5.0 kcal/mol). Animal experiments showed that RRP‑CH significantly alleviated femoral head injury: the empty lacunae rate, osteoclast count, cell necrosis, and adipocyte number were reduced; bone trabecular thickness and bone mineral density (BMD) were improved. RRP‑CH inhibited the protein expression of TNF‑α and IL‑6, and upregulated the expression of LRP6, Wnt3a, β‑catenin, RUNX2, and the p‑GSK‑3β/GSK‑3β ratio.

In conclusion, the RRP‑CH herb pair may treat SANFH by activating the Wnt/β‑catenin signaling pathway, suppressing inflammatory responses, and promoting osteogenic differentiation. These findings support the development of Cistanche‑based botanical therapies for hormone‑induced osteonecrosis.

Keywords: Steroid‑induced avascular necrosis of the femoral head; Rehmanniae Radix Praeparata; Cistanches Herba; Network pharmacology; Wnt/β‑catenin signaling pathway; Herbal formula for hormone‑induced osteonecrosis; Cistanche tubulosa extract

 

 

 

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1. Introduction

Steroid‑induced avascular necrosis of the femoral head (SANFH) is a disabling orthopedic disorder caused by long‑term or high‑dose glucocorticoid administration, which impairs femoral head blood supply and induces progressive osteocyte death. It is insidious onset, rapidly progressive, and often leads to femoral head collapse and joint dysfunction in young and middle‑aged adults. Current Western treatments include pharmacotherapy, weight‑bearing reduction, and total hip arthroplasty. However, medical therapy is limited by inconsistent efficacy and side effects, while joint replacement imposes high economic costs and long‑term complication risks.

Traditional Chinese Medicine (TCM) often uses kidney‑tonifying, essence‑replenishing, and marrow‑strengthening strategies for osteonecrosis. Rehmanniae Radix Praeparata (Shu Di Huang) and Cistanches Herba (Rou Cong Rong) form a classic herb pair that harmonizes Yin and Yang and tonifies the kidney, widely used in formulas such as Zuogui Wan and Jinkui Shenqi Wan. Modern pharmacological studies indicate that Rehmanniae Radix Praeparata improves bone remodeling, reduces bone marrow adiposity, and mitigates inflammation. Cistanches Herba ("Desert Ginseng") promotes osteoblast proliferation and differentiation, enhances bone density, and improves microcirculation; its major active constituents include echinacoside, verbascoside, and phenylethanoid glycosides from Cistanche tubulosa, which are widely developed as anti‑fatigue, anti‑aging, liver‑protective, and immunomodulatory extracts.

Despite clinical efficacy, the molecular mechanism of RRP‑CH against SANFH remains unclear. This study integrated network pharmacology, molecular docking, and animal experiments to reveal the key components, targets, and signaling pathways of RRP‑CH in treating SANFH, with a focus on Cistanche‑derived ingredients to support the research and development of botanical therapies for hormone‑induced femoral head necrosis.

2. Materials and Methods

2.1 Materials

Fifty SPF‑grade male Sprague–Dawley rats (200 ± 20 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (SCXK9 (Liao) 2020‑0001). All animal procedures were approved by the Animal Ethics Committee of Changchun University of Chinese Medicine (Approval No. 2022522).

Reagents: methylprednisolone sodium succinate (Pfizer), LPS (Solaribo), alendronate sodium (Merck Sharp & Dohme). Antibodies: GAPDH, Wnt3a, LRP6, β‑catenin, p‑GSK‑3β, GSK‑3β, RUNX2, TNF‑α, IL‑6 (Proteintech). ELISA kits for BALP, OCN, PINP, IL‑6, TNF‑α (Jiangsu Meimian Industrial Co., Ltd.).

 

Table 1. Shortlisted Components for Network Pharmacology Analysis of Jian Gu Fu Zhi Capsules

Mol ID	Molecule name	Chinese name	Molecular weight (MW)	OB (%)	DL
MOL000359	sitosterol	Sitosterol	414.79	36.91	0.75
MOL000449	stigmasterol	Stigmasterol	412.77	43.83	0.76
MOL000358	beta-sitosterol	β-Sitosterol	414.79	36.91	0.75
MOL005320	arachidonate	Arachidonic acid	304.52	45.57	0.20
MOL005384	suchilactone	Senerol	368.41	57.52	0.56
MOL007563	yangambin	Caulic resin dimethyl ether	446.54	57.53	0.81
MOL000098	quercetin	Quercetin	302.25	46.43	0.28
MOL008871	marckine	Macaine	475.69	37.05	0.69

Fig. 1 Venn Diagram of Common Target Points Between SANFH (Rehmannia glutinosa) and RRP-CH

 

 

2.2 Screening of Active Components and Targets of RRP‑CH

TCMSP database was used to retrieve components of RRP‑CH. Criteria: OB ≥ 30% and DL ≥ 0.18. Corresponding targets were standardized to gene symbols using the UniProt database (Homo sapiens).

 

Table 2. Effects of SANFH on Histopathological Indicators of Rat Femoral Head Tissue (xˉ±s\bar{x} \pm sxˉ±s, n = 10)

Group	Dose (g/kg)	Empty lacunae rate (%)
Control group	-	4.12 ± 1.92
Model group	-	23.50 ± 4.52*
Positive control group	7.3×10−37.3 \times 10^{-3}7.3×10−3	12.83 ± 2.90**##
High-dose group	1	12.42 ± 4.32**##
Low-dose group	0.5	18.56 ± 3.56#

Notes (as shown in the figure): Compared with the control group: ** P<0.01P<0.01P<0.01. Compared with the model group: # P<0.05P<0.05P<0.05, ## P<0.01P<0.01P<0.01.

Fig. 2 PPI Network Analysis and Core Target Screening for RRP-CH in Treating SANFH

 

 

2.3 Identification of SANFH‑Related Targets

SANFH targets were retrieved from GeneCards (Relevance score > 0.5), OMIM, DisGeNET (Score‑gda > 0.1), and DrugBank. After deduplication and standardization, the intersection of drug targets and disease targets was identified as potential therapeutic targets using Venny 2.1.0.

 

Table 3. Effects of RRP-CH on Bone Mineral Density Parameters in SANFH Rat Femurs (xˉ±s\bar{x} \pm sxˉ±s, n = 10)

Group	Dose (g/kg)	Bone mineral density (g/cm2^22)
Control group	-	0.6889 ± 0.0226
Model group	-	0.2613 ± 0.0916*
Positive control group	7.3×10−37.3 \times 10^{-3}7.3×10−3	0.5082 ± 0.0342**##
High-dose group	1	0.4357 ± 0.0622**##
Low-dose group	0.3386 ± 0.0189#

Notes (as shown in the figure): Compared with the control group: ** P<0.01P<0.01P<0.01. Compared with the model group: # P<0.05P<0.05P<0.05, ## P<0.01P<0.01P<0.01.

Fig. 3 GO and KEGG Enrichment Analysis of RRP-CHin Preventing SANFH

Fig. 4 Heatmap and visualisation of molecular docking binding energies; Note: B-P denotes quercetin, stigmasterol, and Yangambin ether with RUNX2; Quercetin, stigmasterol, and Yangambinether with GSK3-β; quercetin, stigmasterol, and Yangambinether with TNF-α; quercetin, stigmasterol, and Yangambin ether with IL-6; Visualisation of molecular docking between quercetin, stigmasterol, Yangambin ether and β-catenin

Fig. 5 H&E staining of femoral heads in rats from different groups (×200 magnification)

 

2.4 Network Construction and Topological Analysis

The RRP‑CH‑SANFH‑component‑target network was built and visualized in Cytoscape 3.9.1. Core components and hub targets were screened by degree, betweenness, and closeness centrality.

Table 4. Effects of RRP-CH on Serum Levels of BALP, PINP, OCN, IL-6, and TNF-α in Rats with SANFH (xˉ±s\bar{x} \pm sxˉ±s, n = 10)

Group	BALP (ng/mL)	PINP (ng/mL)	OCN (ng/mL)	IL-6 (pg/mL)	TNF-α (pg/mL)
Control group	35.44 ± 5.05	36.97 ± 2.38	46.36 ± 1.10	45.84 ± 9.21	34.92 ± 11.08
Model group	17.12 ± 4.7*	18.20 ± 1.65*	24.20 ± 1.96*	102.14 ± 15.73*	120.62 ± 17.25*
Positive control group	27.97 ± 4.93**##	28.04 ± 2.40**##	38.30 ± 1.87**##	68.04 ± 8.20**##	82.13 ± 12.25**##
High-dose group	26.41 ± 5.92**##	24.49 ± 1.21**##	39.01 ± 0.08**##	75.10 ± 17.32**##	80.51 ± 18.28**##
Low-dose group	24.05 ± 4.55#	21.03 ± 1.85#	27.06 ± 2.20##	83.59 ± 9.32#	101.88 ± 8.47#

Notes (as shown in the figure): Compared with the control group: ** P<0.01P<0.01P<0.01. Compared with the model group: # P<0.05P<0.05P<0.05, ## P<0.01P<0.01P<0.01.

 

2.5 PPI Network Analysis

PPI network was constructed using the STRING database (confidence score ≥ 0.9, disconnected nodes hidden). Core targets were ranked by topological parameters.

2.6 GO and KEGG Enrichment Analyses

Metascape was used for GO biological process (BP), cellular component (CC), molecular function (MF), and KEGG pathway enrichment (Homo sapiens, P < 0.05).

2.7 Molecular Docking

Crystal structures of hub targets were downloaded from PDB. Molecular docking was performed using MOE software. Binding energy ≤ −5.0 kcal/mol was defined as strong binding.

2.8 Preparation of RRP‑CH Extract

RRP (20 g) and CH (15 g) were powdered, soaked in purified water for 30 min, refluxed for 1 h, filtered, and concentrated to 1.0 g crude drug per mL.

2.9 Animal Grouping, Model Establishment, and Intervention

Rats were randomly divided into control, model, alendronate (positive control), RRP‑CH low‑dose (1.08 g/kg), and high‑dose (2.16 g/kg) groups. The SANFH model was induced by LPS (10 μg/kg, i.p., 2 days) plus methylprednisolone (100 mg/kg, i.m., 3 times/week for 4 weeks). Drugs were administered intragastrically for 8 weeks.

2.10 Sample Collection

After treatment, blood was collected from the abdominal aorta, and serum was stored at −80 °C. Right femurs were dissected and stored at −80 °C or in 4% paraformaldehyde.

2.11 Hematoxylin–Eosin (H&E) Staining

Femoral heads were decalcified, sectioned, and stained. Histological changes and empty lacunae rate were observed and quantified.

2.12 Bone Mineral Density Measurement

BMD of the right femur was measured using a dual‑energy X‑ray absorptiometry detector (GE Prodigy Pro).

2.13 ELISA

Serum levels of BALP, OCN, PINP, IL‑6, and TNF‑α were measured by ELISA.

2.14 Western Blot Analysis

Femoral tissue proteins were extracted, quantified, and subjected to SDS‑PAGE. Membranes were incubated with primary antibodies against Wnt3a, LRP6, β‑catenin, p‑GSK‑3β, GSK‑3β, RUNX2, TNF‑α, and IL‑6, followed by HRP‑conjugated secondary antibodies. Protein bands were visualized using ECL and analyzed with ImageJ.

2.15 Statistical Analysis

Data are presented as mean ± standard deviation. One‑way ANOVA followed by post‑hoc tests were used for multiple comparisons. P < 0.05 was considered statistically significant.

Fig. 6 Effects of RRP-CH on WB Protein Expression in Rat Femoral Bone Tissue **P < 0.01, *P < 0.05

 

3. Results

3.1 Network Pharmacology and Molecular Docking Results

3.1.1 Active Components and Targets of RRP‑CH

Eight active components met OB and DL criteria, corresponding to 167 non‑redundant targets.

3.1.2 SANFH Targets and Overlapping Targets

A total of 727 SANFH‑related targets were collected. The Venn diagram identified 36 overlapping targets between RRP‑CH and SANFH.

3.1.3 Core Components and Hub Targets

The top 3 active components were quercetin, stigmasterol, and syringaresinol dimethyl ether. The top 5 hub targets were GSK‑3β, IL‑6, TNF‑α, β‑catenin, and RUNX2.

3.1.4 GO and KEGG Analyses

GO terms were enriched in inflammatory regulation, osteoblast differentiation, and angiogenesis. KEGG analysis highlighted the Wnt/β‑catenin signaling pathway as the most significantly enriched pathway.

3.1.5 Molecular Docking Validation

All three core components showed strong binding affinity (≤ −5.0 kcal/mol) with the five hub targets, supporting stable interactions.

3.2 Effects of RRP‑CH on Pathological Injury of Femoral Head

H&E staining showed that RRP‑CH treatment alleviated bone trabecular disruption, reduced empty lacunae rate, osteoclast number, and adipocyte accumulation. The high‑dose group exhibited significant improvement (P < 0.01).

3.3 Effects on BMD

Compared with the model group, BMD was significantly increased in the RRP‑CH high‑dose group (P < 0.01).

3.4 Effects on Serum Biomarkers

RRP‑CH dose‑dependently increased serum BALP, OCN, and PINP (osteogenic markers) and decreased IL‑6 and TNF‑α (pro‑inflammatory cytokines).

3.5 Effects on Wnt/β‑Catenin Pathway and Inflammatory Proteins

Western blot showed that RRP‑CH significantly upregulated LRP6, Wnt3a, β‑catenin, RUNX2, and p‑GSK‑3β/GSK‑3β, and downregulated TNF‑α and IL‑6 in a dose‑dependent manner.