Cong Han,1,* Jing Chen,2,* Chuanlin Shen,1 Qiuxia Liang,1 Ying An,1 Chaoyi Zhou,1 Kechun Liu,1 Qing Xia,1 Qiuxia He,1 Huazheng Zhang3 

1Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, Shandong, People’s Republic of China; 2Department of Tibetan Medicine, Tibetan Traditional Medicine College, Lhasa, Tibet, People’s Republic of China; 3Shandong Academy of Chinese Medicine, Jinan, Shandong, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Qing Xia, Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250103, People’s Republic of China, Email sdxq1021@163.com Huazheng Zhang, Shandong Academy of Chinese Medicine, Jinan, 250014, People’s Republic of China, Email zhanghuazheng1109@163.com

Purpose: Shiyiwei Golden Pill (SYW) is a classic traditional prescription used to treat mKhris-pa according to the theory of Tibetan medicine. At present, SYW is widely used to treat cholecystitis in Tibetan areas. However, the chemical constituents and anti-inflammatory mechanisms are still largely undiscovered. This study aimed to investigate the chemical composition and anti-inflammatory effects of SYW, as well as its potential mechanisms.
Methods: The components of SYW were identified using ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The anti-inflammatory effects of SYW were determined on zebrafish and RAW264.7 cell inflammation models. Additionally, we predicted the targets and pathways of SYW to confirm its anti-inflammatory effects using network pharmacology approaches. Finally, a quantitative real-time polymerase chain reaction (qRT–PCR) was performed to validate the expression of genes associated with anti-inflammatory pathways.
Results: We identified 94 compounds in SYW, mainly alkaloids, phenols, and flavonoids. SYW inhibited inflammatory cell proliferation and migration in the three zebrafish inflammation models. In the RAW264.7 cell model, SYW suppressed the levels of NO and pro-inflammatory cytokines. In addition, network pharmacology analysis revealed that ALB, IL6, TNF, AKT1, and EGFR were identified as the potential key targets of SYW. KEGG enrichment and qRT–PCR analysis showed that PI3K/Akt/FoxO signaling pathway was involved in the anti-inflammatory effects of SYW.
Conclusion: Herein, we identified 94 chemical constituents of SYW and demonstrated that SYW exerts anti-inflammatory effects by regulating the PI3K/Akt/FoxO signaling pathway.

Keywords: shiyiwei golden pill, inflammation, zebrafish, network pharmacology, PI3K/Akt/FoxO signaling pathway, cholecystitis