3. Discussion

In traditional Chinese medicine (TCM), steroid-induced avascular necrosis of the femoral head (SANFH) is categorized as Kidney Impotence (Gu Wei) or Bone Erosion (Gu Shi) based on its clinical manifestations and progressive features. Its core pathogenesis involves deficiency of the liver and kidney and impaired blood circulation. Prolonged or high-dose glucocorticoid administration is a leading cause of SANFH, yet glucocorticoids remain irreplaceable in clinical practice. Endogenous glucocorticoids functionally resemble kidney yang, serving as a vital force for storing and activating innate body energy. Supraphysiological exogenous glucocorticoids act like excessive fire (Zhuang Huo), which consumes vital qi and induces yin-yang deficiency, damaging kidney function and eventually triggering osteonecrosis.

 

 

 

 

 

TCM interventions for SANFH mainly promote angiogenesis and osteogenesis, suppress local inflammation, relieve hip pain, improve femoral head perfusion, and enhance bone repair. The Rehmanniae Radix Praeparata–Cistanches Herba (RRP-CH) herb pair exerts anti-inflammatory and osteogenic effects and is widely used in orthopedic disorders. Its pharmacological activities are attributed to active components including stigmasterol and quercetin. However, mechanistic studies on RRP-CH for osteonecrosis remain limited. This study combined network pharmacology and experimental validation to identify active components, potential targets, and underlying mechanisms of RRP-CH against SANFH.

 

 

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Network pharmacology identified the top three active components: quercetin, stigmasterol, and syringaresinol dimethyl ether. Quercetin is a flavonoid; stigmasterol is a phytosterol abundant in vegetable oils, nuts, and vegetables; syringaresinol dimethyl ether is a key lignan from clove oil. All three possess potent antioxidant, anti-inflammatory, and immunomodulatory properties, inhibiting pro-inflammatory cytokine production and release to mitigate steroid-induced inflammation. Quercetin also scavenges free radicals and reduces oxidative stress, protecting bone cells from steroid‑induced damage.

Protein–protein interaction (PPI) analysis highlighted GSK‑3β, IL‑6, TNF‑α, β‑catenin, and RUNX2 as high‑degree hub targets. TNF‑α and IL‑6 are pivotal pro‑inflammatory cytokines that induce osteocyte and osteoblast apoptosis via death receptor pathways, driving cellular loss and structural destruction in the femoral head. ELISA results showed that RRP‑CH significantly reduced serum TNF‑α and IL‑6 levels and elevated bone formation markers (BALP, PINP, OCN), indicating enhanced osteoblast function and activated repair of steroid‑disturbed bone metabolism.

Hematoxylin–eosin staining revealed that RRP‑CH markedly improved bone trabecular alignment and integrity, reduced empty lacunae rate (P < 0.01), increased trabecular area, and decreased intramedullary adipocytes. KEGG enrichment analysis linked RRP‑CH action to the Wnt/β‑catenin signaling pathway, which balances osteogenesis and osteoclastogenesis to counteract inflammation and osteogenic suppression-core pathological features of SANFH. Activation of this pathway restores bone metabolic homeostasis, promotes bone repair, and slows disease progression. Molecular docking confirmed strong binding between quercetin, stigmasterol, syringaresinol dimethyl ether and β‑catenin, GSK‑3β, and RUNX2.

Western blot analysis demonstrated that high‑dose RRP‑CH significantly upregulated LRP6, Wnt3a, β‑catenin, p‑GSK‑3β, and RUNX2 in the Wnt/β‑catenin pathway. Wnt3a binding to LRP6 inhibits GSK‑3β, preventing β‑catenin phosphorylation and degradation, enabling its nuclear translocation to activate RUNX2 and drive osteogenic gene transcription. These findings indicate that RRP‑CH promotes osteogenic differentiation by activating the Wnt/β‑catenin pathway.

This study demonstrates that RRP‑CH effectively regulates bone metabolism and enhances bone repair in SANFH, providing experimental and theoretical support for its clinical application. Notably, Cistanches Herba (Cistanche tubulosa), rich in echinacoside, verbascoside, phenylethanoid glycosides, and flavonoids, serves as a key ingredient in this herbal formula and shows great potential for developing botanical therapies for hormone‑induced osteonecrosis (source: https://www.xjcistanche.com/products). Limitations include reliance on in silico target prediction; future studies should characterize key pharmacodynamic constituents and their precise mechanisms.

 

 

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XJCistanche. Cistanche tubulosa extract products: echinacoside, verbascoside, phenylethanoid glycosides. https://www.xjcistanche.com/products. Accessed 2026‑03‑27.