The Extract of Ilex cornuta Bark Promotes Bone Healing by Activating Adenosine A2A Receptor

Abstract

Introduction: Bone fracture is a common reason causing human disability. The delay union and nonunion rates are approximately 5-10% despite patients receiving active treatment. Currently, there is a limited number of drugs directly accelerating bone healing, especially direct extracts from plants. Moreover, the pharmacological effects of Ilex cornuta bark are still unknown. This study aimed to explore the effects and mechanisms of Ilex cornuta bark in bone healing.

Methods and results: First, the promoting effects of Ilex cornuta bark on bone healing were verified by the mice femur fracture model as Ilex cornuta bark increased the callus formation and enhanced the biomechanical stability during the bone healing process. Second, the target gene of Ilex cornuta bark in bone healing identified by bioinformatics analysis and immunofluorescence validation was ADORA2A. Third, 410 main compound compositions of Ilex cornuta bark were explored by a non-target metabolomic analysis, where 190 of them were neg ion mode, and 220 were pos ion mode. Molecular docking was used to predict the regulatory effect of the compounds on adora2a (adenosine A2A receptor), and ursonic acid had the lowest binding energy with adora2a. Finally, nfkb1 was the transcription factor (TF) of adora2a, and ursonic acid also had the lowest binding energy by bioinformatic analysis and molecular docking.

Conclusion: Overall, Ilex cornuta bark water extract was a new plant extract on promoting bone healing; in addition, the mechanism of it might be activating adora2a though Nfkb1.

Keywords: Ilex cornuta bark; Nfkb1; adenosine A2A receptor; bioinformatic analysis; bone healing; non-target metabolomic analysis.

Figure 1

The Ilex cornuta bark and a schematic diagram. (A) The Ilex cornuta bark and its water extracts. (B) The animal experiments design. 30 mice with femur fracture were received daily intragastric administration treatment. The observation time points were 7,14,21 days.

Figure 2

The different views of μCT in 7, 14 days and 21 days. The images showed that mice received Ilex cornuta bark treatment had more callus in the coronal view, sagittal view, axial view in 14 days and 21 days.

Figure 3

Ilex cornuta bark on callus formation in 7, 14 days and 21 days. (A) the three-dimensional reconstruction images of femur and fracture site. Bone tissue was colored as purple and callus was yellow. Ilex cornuta group mice had more callus and mineralized tissue in fracture site at 14.21 days. (B) The bar chart revealed the difference between groups, # p<0.05. n=5 at each timepoint.

Figure 4

Ilex cornuta bark on biomechanical strength of bone healing process. (A) The equivalent stress and strain maps. Mice received Ilex cornuta bark treatment had less stress and strain at 21 days. (B) The bar chart revealed the difference between groups, # p<0.05. n=5 at each timepoint.

Figure 5

The target genes of Ilex cornuta bark on bone healing by bioinformatics analysis. (A) The line chart of -log10FDR and log2(P-value) value of the 206 target genes. (B) The PPI networks of Ilex cornuta bark target genes. The target 206 genes were ranked by betweenness value as yellow to dark purple. Adora2a was the key gene of the PPI network and had 19 target genes.

Figure 6

The GO enrichment results of adora2a and related target genes. The scatter diagram showed the top 10 enriched terms of biological process, cellular component and molecular function.

Figure 7

The KEGG enrichment and non-target metabolomics analysis results. (A) The scatter diagram showed the top 10 enriched pathways. cAMP signaling pathway and neuroactive ligand–receptor interaction had the most involved genes. (B) The images showed the main negative modes compound compositions of Ilex cornuta bark. (C) The images showed the main positive modes compound compositions of Ilex cornuta bark.

Figure 8

Molecular docking of the top 10 compounds on adora2a. (A) The binding condition images. (B) The binding site and the bioactive pocket. (C) The binding energy scatter diagram revealed that ursonic acid had the lowest binding energy with adora2a.

Figure 9

The Immunofluorescence images of the fracture site. (A) adora2a immunofluorescent staining. Ilex cornuta bark treatment promoted adora2a expression at 7,14 days. The contrast and brightness were evenly adjusted for each picture. Immunofluorescent images, ×100; original scale bar, 50 μm. (B) The bar chart revealed the difference between groups, # p<0.05. n=5 at each timepoint.

Figure 10

The TF of adora2a and molecular docking of the top 10 compounds on nfkb1. (A) The TRRUST database and JASPAR 2022 showed that nfkb1 was the only one TF of adora2a and the binding sequence might be GGGAATTTCC. (B) The binding condition images with nfkb1. (C) The binding site and the bioactive pocket with nfkb1. (D) The binding energy scatter diagram revealed that ursonic acid had the lowest binding energy with nfkb1.