Yuqing Xie,1,2,* Fengna Yan,1,2,* Xiaoli Liu,1,2 Lihua Yu,1,2 Huiwen Yan,1,2 Zimeng Shang,1,2 Yaxian Kong,3 Zhiyun Yang1,2 

1Center of Integrative Medicine, Beijing Ditan Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Capital Medical University Research and Translational Laboratory for Traditional Chinese Medicine in the Prevention and Treatment of Infectious Severe Hepatitis, Beijing, People’s Republic of China; 3Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Zhiyun Yang, Center of Integrative Medicine, Beijing Ditan Hospital, Capital Medical University, Beijing, People’s Republic of China, Email yangzhiyun2016@163.com Yaxian Kong, Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, People’s Republic of China, Email kongyaxian@ccmu.edu.cn

Purpose: Yangyin Fuzheng Jiedu Prescription (YFJP) is a traditional Chinese medicine (TCM) used for the treatment of hepatocellular carcinoma (HCC). However, the potential mechanisms remain unclear. The objective of this study is to clarify the mechanism of action of YFJP in treating HCC.
Methods: By constructing networks, the active components and molecular targets of YFJP in the treatment of HCC were explored. The TCGA database was utilized to analyze the correlation between the core target and the overall survival (OS) of patients with HCC. The regulatory effect of YFJP on T cell was evaluated by detecting samples from patients with HCC. The molecular mechanism of YFJP in treating HCC was validated through in vivo and in vitro experiments.
Results: Constructing networks and analyse indicated that the key targets of YFJP in the treatment of HCC is FoxO1. Analysis of the HCC patient cohort in the TCGA database demonstrated that FoxO1 is an independent protective factor for overall survival in patients with HCC. Pathway enrichment analysis enriched FoxO signaling pathway and Cellular senescence pathway. Prospectively collecting samples from patients with HCC suggested that YFJP treatment increased the proportion of CD8+ T cells. In vivo experiments showed that YFJP treatment ameliorated CD8+ T cell senescence in tumor-bearing mice. Western blot, flow cytometry and multi-color immunofluorescence co-staining showed that YFJP treatment increased the expression of FoxO1 in CD8+ T cells. The primary CD8+ T cells were sorted and co-cultured with an HCC cell line in vitro. Inhibiting the expression of FoxO1 in CD8+T cells confirmed that FoxO1 is a key target for YFJP to improve the senescence of CD8+ T cell.
Conclusion: FoxO1 is the key molecular target of YFJP in improving CD8+ T cell senescence in HCC. This study preliminarily clarified the mechanism of YFJP in regulating immunosenescence of HCC.

Keywords: hepatocellular carcinoma, senescence, T cell, network pharmacology, Yangyin Fuzheng Jiedu Prescription