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ORIGINAL RESEARCH ARTICLE

Unraveling the mechanism of Si-Ni-San against non-alcoholic fatty liver disease: An integration of network pharmacology and experimental verification

Yue Hu1 ,  Bowen Yang1 ,  Yishu Wang1 ,  Chengyao Xiao1 ,  Tingting Wang1 ,  Cancan Guo1 ,  Yunfei Li1 ,  Jingxia Du*1
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1 Department of Pharmacy, College of Basic Medicine and Forensic Medicine, Henan University of Science and Technology, Luoyang, Henan, China
GPD, 026030002 https://doi.org/10.36922/GPD026030002
Received: 15 January 2026 | Revised: 24 April 2026 | Accepted: 30 April 2026 | Published online: 28 May 2026
© 2026 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

The global prevalence of non-alcoholic fatty liver disease (NAFLD) is rising. Si-Ni-San (SNS), a classical herbal formula for chronic liver disease, has unclear mechanisms against NAFLD. This study integrated network pharmacology with in vivo validation to investigate its mechanisms. Potential SNS and NAFLD targets were retrieved from public databases. A protein-protein interaction network was identified, followed by Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. For in vivo experiments, NAFLD was induced in mice by feeding a high-fat diet, with SNS administered orally for therapeutic intervention. Liver Oil Red O staining was evaluated, and biochemical parameters, including total cholesterol (TC) and triglyceride (TG) in serum, were assessed. Furthermore, molecular techniques were used to examine the expression levels of inflammatory and oxidative stress-related factors predicted by network pharmacology. A total of 157 overlapping potential targets between SNS and NAFLD were identified, yielding the top 10 hub targets including IL-6, TNF-α, HIF-1α, MMP-9, AKT1, STAT3, Caspase-3, JUN, PTGS2, and TGF-β1. Additional oxidative stress-related targets (NRF2, HO-1, NQO1) were also examined. KEGG/GO analyses indicated involvement of inflammation and oxidative stress pathways. Molecular docking further demonstrated strong binding affinities of its active components with STAT3 and NRF2. In vivo, SNS reduced liver weight index, lipid droplets, serum TC, and TG levels. Western blot analysis showed that SNS treatment led to a marked inhibition of JAK2 and STAT3 phosphorylation. SNS downregulated the expression of pro-inflammatory cytokines, such as Il6 and Il1b, while upregulating endogenous antioxidant proteins Sestrin2 and NRF2, thereby enhancing the expression of key downstream antioxidant genes, including Nqo1 and Hmox1. In conclusion, integrated network pharmacology and in vivo validation demonstrated that SNS ameliorates NAFLD by activating the Sestrin2/NRF2/HO-1 pathway to enhance antioxidant capacity and inhibiting the JAK2/STAT3 pathway to alleviate inflammatory injury.

Keywords
Si-Ni-San
Non-alcoholic fatty liver disease
Network pharmacology analysis
JAK2/STAT3
Sestrin2/NRF2/HO-1
Funding
The present study was supported by the Henan Province Scientific and Technology Research Project (Grant No. 262102310193) and the College Student Innovation and Entrepreneurship Training Program of Henan Province (Grant No. S202510464081).
Conflict of interest
The authors declare no competing interests regarding the publication of this manuscript.
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