Metabolomics and integrated network pharmacology analysis reveal SNKAF decoction Suppresses Cell Proliferation and Induced Cell Apoptisis in Hepatocellular Carcinoma via PI3K/Akt/P53/FoxO Signaling Axis
Authors: Guo Wei , Yao Xiaohui , Lan Siyuan , Zhang Chi , Li Hanhan , Chen Zhuangzhong , Yu Ling , Liu Guanxian , Lin Yuan , Liu Shan , Chen Hanrui 
Published: February 2022

Abstract
Background: Since there is no comprehensive method on the treatment of HCC, R&D still focused on the systemic therapies about drug As alternative medicine rises, new measures for post HCC treatment have been offered gradually. SNKAF decoction, a classic Chinese herbal prescription, has been smoothly treated liver cancer in the clinic. Nevertheless, there is no research on the core active component and target of SNKAF decoction. Here, the possible active ingredients related to HCC in the SNKAF decoction have been examined. Methods: Mouse models were established to measure the anticancer effect of SNKAF decoction on HCC. Further we investigated the SNKAF decoction on inhibiting the hepatoma cells proliferation by cell viability, cloning and invasion assays in vitro, also the induced apoptosis effects on human HCCLM3 and MHCC97H cells by Western blotting, TUNEL/Hoechst staining and flow cytometric. Secondly, the SNKAF’s components were collected from the TCMSP database and TCM@Taiwan database. The HCC-related genes were obtained from GeneCards, and the protein-protein interaction (PPI) data of SNKAF’s targets and HCC genes were obtained from the String database. After that, the DAVID platform was applied for Gene Ontology (GO) enrichment analysis and KEGG pathway enrichment analysis. Metabolomic analysis was used to identify the potential genes and pathways in hepatocellular carcinoma treated with SNKAF decoction. Then, the expression of PI3K、Akt、P53、FoxO proteins of potential signal pathway were detected by Western blot. Results: The animal experiments showed that SNKAF decoction can inhibited tumor growth ( P < 0.05)and induced no weight loss on the body weight of mice during the period. In vitro data showed that HCCLM3 and MHCC97H cell proliferation was inhibited by SNKAF serum in a time- and concentration dependent manner, the ability of cell migration and invasion was also significantly inhibited when the SNKAF serum concentration were ranged 5-20%, also the cells accumulated in S and G2/M Phase and apoptosis was increased in both cell lines by SNKAF serum. Based on the experimental study in vitro, the SNKAF serum at concentration of 10% showed good biological activity so as to facilitate further deeper research. The network pharmacology analysis results identified 196 candidate compounds and 2537 potential targets were yielded by SFI and they shared 234 targets associated with HCC, further combined analysis with metabonomics showed that 217 target genes overlapped. The core target genes include BCL2, MCL1, Myc, PTEN, gsk3b, CASP9, CREB1, MDM2, pt53 and CCND1, cancer-associated pathways greatly took part in the mechanisms of SNKAF including p53, FoxO, phosphoinositide 3-kinase (PI3K)/Akt signaling pathways which closely related to tumor include apoptosis, cell cycle. In addition, west bolting verified that 10% SNKAF serum had significant effect on the main proteins of PI3K/Akt/P53/FoxO signaling pathway in both cell lines. Conclusion: SNKAF decoction-containing serum inhibited HCCLM3 and MHCC97H cell proliferation, migration, invasion, induced apoptosis, and regulated the expression of tumor-related proteins. we confirmed that SNKAF decoction can promise alternative treatments for HCC patients.