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Fuzheng Xiaozheng prescription (FZXZP), a traditional Chinese medicine, which was derived from the famous decoction, Sanjiasan, in the book of "Wenyilun" in Ming dynasty. Due to its function of invigorating the circulation of blood in Chinese medicine, it was usually used for treating the liver cirrhosis, hepatocellular carcinoma (HCC), etc. Clinical application found that FZXZP exhibited satisfactory therapeutic effects in HCC treatments. However, we still know little about the underlying mechanisms.In this study, we aim to gain a deeper insight into the inhibiting effects of FZXZP on HCC rats and preliminarily elucidate the underlying intervention effects.Two doses of FZXZP were adopted to evaluate the therapeutic effects on rat HCC, and then the intervention effects were evaluated from different aspects. High performance liquid chromatography (HPLC) was used for the active compounds prediction in FZXZP. Finally, the mRNA-Seq was conducted to reveal the intervention mechanisms and the mechanisms were further validated by quantitative Real-time PCR (qRT-PCR) and lipid contents analyses.The results showed that FZXZP significantly alleviated the serum biochemical indicators and improved the pathological characteristics of HCC rats. Mechanistically, FZXZP could regulate some lipid related metabolisms, including arachidonic acid, linoleic acid and retinol, as well as improving the steroid hormone biosynthesis, to improve the inflammatory statuses and restoring ability of HCC livers, and these were further confirmed by our following analyses on serum lipid contents and cytokine expressions. In addition, FZXZP could also negatively regulate four extracellular growth factors which could result in the blocking of two cancer-related signaling pathways, Ras/MAPK and Ras/PI3K-Akt.Our results suggested that FZXZP demonstrated significant inhibiting effects on rat HCC progresses, and these may be realized by improving the inflammatory statuses and blocking the Ras/MAPK and Ras/PI3K-Akt signaling pathways.

Bufonis (VB), an animal drug called Chansu in China, is the product of the secretion of Bufo gargarizans Cantor or B. melanostictus Schneider. As a traditional Chinese medicine (TCM) for a long time, it has been widely used in the treatment of heart failure, ulcer, pain, and various cancers. Cinobufaginn (CNB), the cardiotonic steroid or bufalene lactone extracted from VB, has the effects of detoxification, detumescence, and analgesia.The present study aimed to define the effects of CNB on non-small-cell lung cancer (NSCLC) and identify the potential molecular mechanisms.A549 cells were treated with cinobufagin and cell viability, apoptosis, migration, and invasion were then evaluated using Cell Counting Kit-8 (CCK8) assays, flow cytometry, and Transwell assays, respectively. Moreover, the levels of proliferating cell nuclear antigen (PCNA), cytokeratin8 (CK8), poly ADP-ribose polymerase (PARP), Caspase3, Caspase8, B-cell lymphoma/lewkmia-2(Bcl-2), Bcl2-Associated X(Bax), forkhead box O1 (FOXO1), and euchromatic histone-lysine N-methyltransferase2 (G9a, EHMT2) in A549 cells were evaluated using qRT-PCR and/or Western blot analysis (WB), Co-IP, immunofluorescence, and immunohistochemistry. An in vivo imaging system, TUNEL, Immunofluorescence, and immunohistochemistry were also used to detect proliferating cell nuclear antigen(PCNA), Ki67, E-Cadherin(E-Cad), FOXO1, and G9a in mouse xenograft model experiments.CNB suppressed cell proliferation, migration, and invasion but promoted apoptosis in A549 cells in a dose- and time-dependent manner, while cinobufagin had no cytotoxic effect on BEAS-2B cells. In vivo, cinobufagin inhibited the proliferation, migration, and invasion of A549 cells and promoted their apoptosis. The occurrence of the above phenomena was accompanied by an increase in FOXO1 expression and a decrease in G9a expression. In A549 cells, CNB did not reverse the changes in the proliferation, migration, invasion, and apoptosis of A549 cells after FOXO1 was successfully silenced.Our study provides the first evidence that cinobufagin suppresses the malignant biological behaviours of NSCLC cells in vivo and in vitro and suggests that mechanistically, this effect may be achieved by inhibiting the expression of the histone methyltransferase G9a and activating the tumour suppressor gene FOXO1. Taken together, our findings provide important insights into the molecular mechanism underlying cinobufagin's anticancer activity, and suggest that cinobufagin could be a candidate for targeted cancer therapy.

Ethnopharmacological relevance Fuzheng Xiaozheng prescription (FZXZP) is a traditional Chinese medicine (TCM) that was derived from Sanjiasan, a famous decoction documented in the book of Wenyilun in Ming dynasty. Based on our years' clinic application, FZXZP demonstrated satisfactory therapeutic effects in cirrhosis and hepatocellular carcinoma (HCC) treatments. However, the underlying mechanisms are still largely unknown. Aim of study In this study, we aim to systematically evaluate the intervention effects of FZXZP on rat HCC and deeply elucidate the underlying regulative mechanisms on rat HCC. Materials and methods The HCC rats were induced by using diethylnitrosamine (DEN) and two doses of FZXZP were adopted to treat the HCC rats. Liver phenotype, blood chemistry and liver histopathology were used to evaluate the intervention effects. High performance liquid chromatography (HPLC) was conducted to analyze the components of FZXZP. Finally, miRNA-Seq and mRNA-Seq were performed to investigate the regulative mechanisms of FZXZP on rat HCC and qRT-PCR was carried out to verify the accuracies of the two RNA-Seqs. Results Results of liver phenotypes, blood chemistry and liver histopathology demonstrated that FZXZP significantly alleviated the liver damage, inhibited the progresses of HCC. Nine potential components were identified from FZXZP, and anti-cancer prediction suggested that almost all of them were reported to show an anti-cancer effect. Mechanistically, FZXZP was found to promote the lipid related metabolisms, improve the anti-inflammation ability by activating PPAR signaling pathway, arachidonic acid metabolism, bile secretion, etc. CONCLUSION: our results suggested that FZXZP significantly alleviated the rat HCC, mechanistically by improving the anti-inflammation ability and promoting the lipid related metabolisms.