Multi-Target Mechanisms of Si-Ni-San on Anxious Insomnia: An Example of Network-pharmacology and Molecular Docking Analysis
Authors: Lin Chih Ting , Lin Hsin Yi , Peng Wen Huang , Wu Lung Yuan 
Published: October 2024

Background and objective:
Based on comprehensive network-pharmacology and molecular
docking analysis, this study was intended to unveil the multiple mechanisms of Si-Ni-San (SNS) in treating
anxious insomnia.

Methods:
The compounds of SNS were meticulously analyzed, selected and standardized with references to
their pharmacological attributes. The components included chaihu (Bupleurum chinense DC.), baishao
(Paeonia lactiflora Pall.), zhishi (Citrus aurantium L.) and gancao (Glycyrrhiza uralensis Fisch. ex DC.).
We used the Traditional Chinese Medicine System Pharmacology (TCMSP) Database, Traditional Chinese
Medicines Integrated Database (TCMID), GeneCards database, therapeutic target database (TTD) and comparative
toxicogenomic database (CTD) to construct the components-compounds-targets networks and used
Cytoscape 3.9.1 software to visualize the outcome. Afterwards, the STRING database and Cytoscape 3.9.1
software were utilized to construct and visualize the protein-protein interaction (PPI) network analysis. In
addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis were also conducted through the Database for Annotation, Visualization, and Integrated
Discovery (DAVID). The molecular docking program was carried out using AutoDock 4.2 software to understand
interactions between target receptors and compound ligands selected for study.

Results:
We thoroughly sorted and filtered 31 pharmacologically active compounds from SNS. Subsequently,
several potential target genes were predicted, of which there were 59 target genes distinctly associated
with anxious insomnia. The PPI analysis indicated that the core target proteins included AKT1, IL6, TNF,
SLC6A4, MAOA and GABRA2. The results of our study indicated that SNS potentially remediates anxious
insomnia by reducing inflammation, neurodegeneration, and cell apoptosis of neurons. In addition, GO and
KEGG enrichment analysis results indicated that SNS could modulate multiple aspects of anxious insomnia
through mechanisms related to pathways of neuroactive ligand-receptor interaction. These pathways include
various kinds of synaptic transmission pathways, and anti-inflammatory activity associated with response
pathways. When we compared the components-compounds-targets networks and the compounds-targets-synaptic
pathways networks, the five active compounds, including beta-Sitosterol, Kaempferol, Tetramethoxyluteolin,
Isorhamnetin and Shinpterocarpin, were selected to conduct molecular docking experiments.
Eleven target proteins, (AKT1, SLC6A4, ADRB2, MAOA, ACHE, ESR1, CYP3A4, CHRNA7, GABRA2,
HTR2A and NOS3), which also play significant roles in regulating serotonergic, cholinergic, dopaminergic
and GABAergic systems in the PPI network, were selected to act as receptors in molecular docking trials.
The results showed that docking pairs isorhamnetin-AKT1, isorhamnetin-SLC6A4, β-sitosterol-MAOA, β-
sitosterol-ACHE, isorhamnetin-CHRNA7 and shinpterocarpin-GABRA2 provided the most stable conformations
of ligand-receptor binding between key compounds and core target proteins in the SNS.

Conclusion:
In the study, we offer a computational result, revealing that SNS may alleviate sleep disorders
associated with anxiety through a “multi-compounds, multi-targets, and multi-pathways” mechanism. The
network-pharmacology and molecular docking outcomes could theoretically confirm the anti-anxiety and
anti-insomnia effects of SNS. Although this research is purely statistical and systematic without empirical
validation, it serves as a stepping stone and cornerstone for subsequent experimental investigations.