Objective:

2

To explore the mechanism of action of asperosaponin Ⅵ (AⅥ) in the treatment of rheumatoid arthritis (RA) and validate it in

ex vivo

experiments using network pharmacology and molecular docking methods.

Methods:

2

The predicted targets of AⅥ were obtained from PharmMaper

UniProt and SwissTarget Prediction platforms

the disease targets were collected from Online Mendelian Inheritance in Man

Therapeutic Target Database and GeneCards databases

the intersection targets of AⅥ and RA were obtained from Venny 2.1.0

and the protein-protein interaction (PPI) network was obtained from STRING database

which was analyzed by Cytoscape software and screened to obtain the core targets. Cytoscape software was used to analyze PPI network and screen the core targets. Based on the Database for Annotation

Visualization and Integrated Discovery database

Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed

and Cytoscape software was used to construct the "Disease-Pathway-Target-Drug" network

which was finally verified by molecular docking and animal experiments.

Results:

2

Network pharmacological studies showed that AⅥ was able to modulate 289 targets

with 102 targets for the potential treatment of RA

with the core pathway being the AKT/PI3K signaling pathway

and the core targets being the epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9). Molecular docking results showed that AⅥ could produce strong binding with both of the 2 core targets.

In vitro

cellular experiments showed that AⅥ reduced nitric oxide

prostaglandin E

2

tumor necrosis factor-alpha (TNF-α)

interleukin-6 (IL-6)

and IL-1β levels (

P

<

0.05) and inhibited cyclooxygenase-2

nitric oxide synthase

EGFR

MMP9

phosphorylated phosphoinositide 3-kinase (p-PI3K)

and phosphorylated serine-threonine kinase (p-AKT) proteins (

P

<

0.05). The results of

in vivo

studies showed that AⅥ improved RA score and foot swelling thickness and decreased TNF-α

IL-6

p-PI3K and p-AKT levels in RA rats (

P

<

0.05).

Conclusion:

2

AⅥ exerts anti-inflammatory and anti-RA effects which might be related to the EGFR/MMP9/AKT/PI3K pathway.