# Immunomodulatory Mechanisms of Rehmanniae Radix Praeparata–Achyranthes Root–Chinese Angelica Root Combination in Nontraumatic Osteonecrosis of the Femoral Head: A Comprehensive Network Pharmacology and Molecular Docking Study Focusing on Immunological Pathways

## TL;DR

This study explores how a traditional Chinese herbal combination may treat nontraumatic osteonecrosis by modulating the immune system, particularly through suppressing harmful immune responses.

## Contribution

The study introduces a novel immunological hypothesis for the treatment of NONFH using a specific herbal combination, supported by network pharmacology and molecular docking.

## Key findings

- The herbal combination SND targets 46 immune-related pathways, including those involved in neutrophil extracellular trap formation.
- Molecular docking confirmed strong binding between SND components and key immune targets like HCK, with a binding energy of −8.7 kcal/mol.
- SND is proposed to alleviate NONFH by modulating immune cell activity, especially neutrophils and osteoclasts.

## Abstract

Nontraumatic osteonecrosis of the femoral head (NONFH) is a devastating condition characterised by immune dysregulation and sterile inflammation, which are increasingly acknowledged as central pathogenic mechanisms. This study aims to identify the core herbal combination for NONFH and systematically explore its immunomodulatory effects and underlying pharmacological mechanisms, with a focus on immune system interactions.

The core combination ‘Rehmanniae radix praeparata (SDH)‐achyranthes root (NX)‐Chinese angelica root (DG)’ (SND) was identified via data mining of clinical literature using the Traditional Chinese Medicine Inheritance Support System (TCMISS) V2.5 platform. Its chemical constituents were characterised by ultrahigh performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MSE) technology, yielding 127 identified compounds, 47 of which were selected as bioactive components based on drug‐likeness screening. The potential molecular targets of SND and NONFH were predicted and intersected, with a focus on immune‐related targets. Subsequent comprehensive enrichment analysis was performed, emphasising immune pathway involvement, along with immune infiltration profiling. A support vector machine (SVM) model was constructed to identify key immune‐related targets, and interactions were validated via molecular docking and molecular dynamics (MD) simulations.

SND shared 46 candidate targets with NONFH. Enrichment analysis revealed these targets were significantly enriched in immune‐inflammatory pathways, especially those related to immune cell activation and regulation. Notably, pathways involved in neutrophil extracellular trap (NET) formation and other innate immune responses were prominent. Machine learning identified five key targets: ACP1, NDUFAF3, haematopoietic cell kinase (HCK), CXCR2 and platelet‐activating factor receptor (PTAFR)—all of which play critical roles in the modulation, signalling and activation of immune cells, particularly neutrophils and macrophages. Subsequent immune infiltration analysis demonstrated a strong correlation between these key targets (e.g. HCK, CXCR2 and PTAFR) and neutrophil abundance in NONFH. Molecular docking (MD) and molecular dynamics simulations (MDS). MD confirmed stable binding between the active components and these targets, with the HCK‐chrysophanic acid complex exhibiting the strongest affinity (binding energy: −8.7 kcal/mol).

Our integrated analysis suggests that SND alleviates NONFH primarily through multi‐target immunomodulation, explicitly involving suppression of NET formation and regulation of immune cell activity, especially neutrophils and osteoclasts. This study presents a novel, immunologically explicit hypothesis for the mechanism of SND against NONFH, providing a solid theoretical foundation for future experimental validation and clinical application.