Integrating Network Pharmacology and In Vitro Experiments for Assessing the Anti-Tumor Effects of Phyllanthus Urinaria L Anti-neoplastic Decoction in Hepatocellular Carcinoma.

Background: In China, traditional Chinese medicine (TCM) is an important part of the comprehensive treatment of hepatocellular carcinoma (HCC), and Chinese herb formulas with the effect of "yiqi jianpi jiedu huayu" (replenishing qi, strengthening spleen, and removing toxicity and blood stasis) are the common and efficient treatments for HCC. However, the mechanism of these formulas in treating HCC remain unclear. Objective: In this paper, our goal is to explore the potential mechanism of Phyllanthus urinaria L anti-neoplastic decoction (PAD), the representative formula of "yiqi jianpi jiedu huayu", in treating HCC. Design: The research team performed the network pharmacology and in vitro experiment (preparation of PAD aqueous extract, cell cultures and MTT assay, cell apoptosis assay, wound healing assay, transwell assays, western blot). Setting: The study took place in the Department of Hepatology, the Fourth Clinical Medical College of Guangzhou University of Chinese Medicine (Shenzhen Traditional Chinese Medicine Hospital), China. Outcome Measures: The active components and targets of PAD and HCC targets were screened by five Chinese herbs and two disease databases respectively. The network pharmacology was utilized to construct the relationship network between PAD and HCC, and the mechanism was predicted by pathway enrichment analysis. The experiment was performed to verify the intervention effect of PAD on HCC and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway. Results: The relationship network between PAD and HCC suggested that PAD mainly regulated the potential therapeutic targets of HCC by key active components such as quercetin, luteolin, calycosin, wogonin, and pinocembrin. Pathway analysis demonstrated PAD could play an anti-HCC effect via multiple pathways (e.g., PI3K/Akt). Results of the experiment showed that PAD could effectively inhibit the proliferation and migration of HCC cells, and promote HCC cells apoptosis in a concentration-dependent behavior. Additionally, PAD could decrease the protein expression of phosphorylated PI3K/Akt. Conclusion: PAD mainly exerts an anti-HCC effect through multiple active components represented by quercetin and multiple pathways represented by the PI3K/Akt pathway. This study provided an experimental basis for the clinical application of PAD.

Investigation of folate-modified EGCG-loaded thermosensitive nanospheres inducing immunogenic cell death and damage-associated molecular patterns in hepatocellular carcinoma.

BACKGROUND: The systemic treatment of advanced hepatocellular carcinoma is currently facing a bottleneck. EGCG, the primary active compound in green tea, exhibits anti-tumor effects through various pathways. However, there is a lack of study on EGCG-induced immunogenic cell death (ICD) in hepatocellular carcinoma. METHODS: In a previous study, we successfully synthesized folate-modified thermosensitive nano-materials, encapsulated EGCG within nanoparticles using a hydration method, and established the EGCG nano-drug delivery system. The viability of HepG2 cells post-EGCG treatment was assessed via the MTT and EdU assays. Cell migration and invasion were evaluated through wound healing experiments, Transwell assays, and Annexin V-FITC/PI assay for apoptosis detection. Additionally, the expression levels of damage-associated molecular patterns (DAMPs) were determined using immunofluorescence, ATP measurement, RT-qPCR, and Western Blot. RESULTS: The drug sensitivity test revealed an IC50 value of 96.94 µg/mL for EGCG in HepG2 cells after 48 h. EGCG at a low concentration (50 µg/mL) significantly impeded the migration and invasion of HepG2 cells, showing a clear dose-dependent response. Moreover, medium to high EGCG concentrations induced cell apoptosis in a dose-dependent manner and upregulated DAMPs expression. Immunofluorescence analysis demonstrated a notable increase in CRT expression following low-concentration EGCG treatment. As EGCG concentration increased, cell viability decreased, leading to CRT exposure on the cell membrane. EGCG also notably elevated ATP levels. RT-qPCR and Western Blot analyses indicated elevated expression levels of HGMB1, HSP70, and HSP90 following EGCG intervention. CONCLUSION: EGCG not only hinders the proliferation, migration, and invasion of hepatocellular carcinoma cells and induces apoptosis, but also holds significant clinical promise in the treatment of malignant tumors by promoting ICD and DAMPs secretion.

Determining the Traditional Chinese Medicine (TCM) Syndrome with the Best Prognosis of HBV-Related HCC and Exploring the Related Mechanism Using Network Pharmacology.

BACKGROUND: In traditional Chinese medicine (TCM), TCM syndrome is a key guideline, and Chinese materia medicas are widely used to treat hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC) according to different TCM syndromes. However, the prognostic value of TCM syndromes in HBV-related HCC patients has never been studied. METHODS: A retrospective cohort of HBV-related HCC patients at Shenzhen Traditional Chinese Medicine Hospital from December 2005 to October 2017 was analyzed. The prognostic value of TCM syndromes in HBV-related HCC patients was assessed by Kaplan-Meier survival curves and Cox analysis, and the TCM syndrome with the best prognosis of HBV-related HCC patients was determined. To further study the relevant mechanisms, key Chinese materia medicas (KCMMs) for the TCM syndrome with the best prognosis were summarized, and network pharmacology was also performed. RESULTS: A total of 207 HBV-related HCC patients were included in this research, and we found that HBV-related HCC patients with TCM excess syndrome had better OS. Then, a total of eight KCMMs for TCM excess syndrome were identified, whose crucial ingredients included quercetin, beta-sitosterol, kaempferol, luteolin, and XH-14, and KCMMs could play a therapeutic role through MAPK, JAK-STAT, Wnt, Hippo, and other pathways. Moreover, TP53, SRC, STAT3, MAPK3, PIK3R1, HRAS, VEGFA, HSP90AA1, EGFR, and JAK2 were determined as the key targets. CONCLUSION: We propose a new research method of "prognosis of TCM syndromes-KCMMs-network pharmacology" to reveal the prognostic value of TCM syndromes and the potential mechanism by which TCM syndromes affect prognosis.

Systematically Exploring the Antitumor Mechanisms of Core Chinese Herbs on Hepatocellular Carcinoma: A Computational Study.

OBJECTIVE: Chinese herbs play a positive role in the management of hepatocellular carcinoma (HCC) in China. However, it is not clear which of Chinese herbs are critical for the treatment of HCC. Besides, mechanisms of CCHs in the treatment of HCC remain unclear. Hence, our goal is to identify the core Chinese herbs (CCHs) for treating HCC and explore their antitumor mechanism. METHODS: Firstly, clinical traditional Chinese medicine (TCM) prescriptions for HCC were collected from Chinese National Knowledge Infrastructure (CNKI) database, and then, data mining software was used to identify CCHs. After that, bioactive compounds and corresponding target genes of CCHs were obtained using three TCM databases, and target genes of HCC were acquired from MalaCards and OMIM. Subsequently, common target genes of CCHs and HCC were screened. Moreover, biological functions and pathways were analyzed, and Cytoscape plugin cytoHubba was used to identify hub genes. Finally, prognostic values of hub genes were verified by survival analysis, and the molecular docking approach was utilized to validate the interactions between targets and bioactive compounds of CCHs. RESULTS: Eight CCHs were determined from 630 prescriptions, and 100 bioactive compounds (e.g., quercetin and luteolin) and 126 common target genes were screened. Furthermore, common target genes of CCHs and HCC were mainly enriched in cancer-associated pathways, and six hub genes with statistical significance in survival analysis were selected as key target genes for molecular docking. Additionally, molecular docking showed that the bioactive compounds docked well with the protein receptors of key target genes. CONCLUSION: By combining data mining, network pharmacology, molecular docking, and survival analysis methods, we found that CCHs may play a therapeutic role in HCC through regulating the target genes and pathways related to cancer occurrence and development, angiogenesis, metastasis, and prognosis.

Systematic Elucidation of the Potential Mechanisms of Core Chinese Materia Medicas in Treating Liver Cancer Based on Network Pharmacology.

OBJECTIVE: In this study, the data mining method was used to screen the core Chinese materia medicas (CCMMs) against primary liver cancer (PLC), and the potential mechanisms of CCMMs in treating PLC were analyzed based on network pharmacology. METHODS: Traditional Chinese medicine (TCM) prescriptions for treating PLC were obtained from a famous TCM doctor in Shenzhen, China. According to the data mining technique, the TCM Inheritance Support System (TCMISS) was applied to excavate the CCMMs in the prescriptions. Then, bioactive ingredients and corresponding targets of CCMMs were collected using three different TCM online databases, and target genes of PLC were obtained from GeneCards and OMIM. Afterwards, common targets of CCMMs and PLC were screened. Furthermore, a network of CCMMs bioactive ingredients and common target gene was constructed by Cytoscape 3.7.1, and gene ontology (GO) and signaling pathways analyses were performed to explain the mechanism of CCMMs in treating PLC. Besides, protein-protein interaction (PPI) analysis was used to identify key target genes of CCMMs, and the prognostic value of key target genes was verified using survival analysis. RESULTS: A total of 15 high-frequency Chinese materia medica combinations were found, and CCMMs (including Paeoniae Radix Alba, Radix Bupleuri, Macrocephalae Rhizoma, Coicis Semen, Poria, and Curcumae Radix) were identified by TCMISS. A total of 40 bioactive ingredients (e.g., quercetin, kaempferol, and naringenin) of CCMMs were obtained, and 202 common target genes of CCMMs and PLC were screened. GO analysis indicated that biological processes of CCMMs were mainly involved in response to drug, response to ethanol, etc. Pathway analysis demonstrated that CCMMs exerted its antitumor effects by acting on multiple signaling pathways, including PI3K-Akt, TNF, and MAPK pathways. Also, some key target genes of CCMMs were determined by PPI analysis, and four genes (MAPK3, VEGFA, EGF, and EGFR) were found to be correlated with survival in PLC patients. CONCLUSION: Based on data mining and network pharmacology methods, our results showed that the therapeutic effect of CCMMs on PLC may be realized by acting on multitargets and multipathways related to the occurrence and development of PLC.