RESUMO
BACKGROUND: Apigenin, as a natural flavonoid, has low intrinsic toxicity and has potential pharmacological effects against hepatocellular carcinoma (HCC). However, the molecular mechanisms involving microRNAs (miRNAs) and their target genes regulated by apigenin in the treatment of HCC have not been addressed. OBJECTIVE: In this study, the molecular mechanisms of apigenin involved in the prevention and treatment of HCC were explored in vivo and in vitro using miRNA transcriptomic sequencing to determine the basis for the clinical applications of apigenin in the treatment of HCC. METHODS: The effects of apigenin on the proliferation, cell cycle progression, apoptosis, and invasion of human hepatoma cell line Huh7 and Hep3B were studied in vitro, and the effects on the tumorigenicity of Huh7 cells were assessed in vivo. Then, a differential expression analysis of miRNAs regulated by apigenin in Huh7 cells was performed using next-generation RNA sequencing and further validated by qRT-PCR. The potential genes targeted by the differentially expressed miRNAs were identified using a curated miRTarBase miRNA database and their molecular functions were predicted using Gene Ontology and KEGG signaling pathway analysis. RESULTS: Compared with the control treatment group, apigenin significantly inhibited Huh7 cell proliferation, cell cycle, colony formation, and cell invasion in a concentration-dependent manner. Moreover, apigenin reduced tumor growth, promoted tumor cell necrosis, reduced the expression of Ki67, and increased the expression of Bax and Bcl-2 in the xenograft tumors of Huh7 cells. Bioinformatics analysis of the miRNA transcriptome showed that hsa-miR-24, hsa-miR-6769b-3p, hsa-miR-6836-3p, hsa-miR-199a-3p, hsa-miR-663a, hsa-miR-4739, hsa-miR-6892-3p, hsa-miR-7107-5p, hsa-miR-1273g-3p, hsa-miR-1343, and hsa-miR-6089 were the most significantly up-regulated miRNAs, and their key gene targets were MAPK1, PIK3CD, HRAS, CCND1, CDKN1A, E2F2, etc. The core regulatory pathways of the up-regulated miRNAs were associated with the hepatocellular carcinoma pathway. The down-regulated miRNAs were hsa-miR-181a-5p and hsa-miR-148a-3p, and the key target genes were MAPK1, HRAS, STAT3, FOS, BCL2, SMAD2, PPP3CA, IFNG, MET, and VAV2, with the core regulatory pathways identified as proteoglycans in cancer pathway. CONCLUSION: Apigenin can inhibit the growth of HCC cells, which may be mediated by up-regulation or down-regulation of miRNA molecules and their related target genes.
RESUMO
BACKGROUND AND AIM: Huashi Baidu Decoction (HSBD) is a novel complex prescription which has positive effects on severe COVID-19. This study was aimed to discover key Chinese materia medica, main active compounds, hub therapeutic target proteins and core signal pathways in the potential therapeutic mechanism of HSBD on severe COVID-19 through integrating network pharmacological methods. EXPERIMENTAL PROCEDURE: TCMSP, TCMID and STITCH databases were used to screen out active compounds and target proteins of HSBD. GeneCards database was used to screen out disease genes of severe COVID-19. The potential therapeutic targets of HSBD on severe COVID-19 were used to construct protein-protein interaction network through STRING database and the hub target proteins were discovered. Next, GO and KEGG enrichment analysis were carried out to discover core signal pathways. Finally, the network diagram of "Chinese materia medica-active compounds-therapeutic target proteins" was built, then key Chinese materia medica and main active compounds were selected. RESULTS AND CONCLUSION: HSBD might treat severe COVID-19 through 45 potential target genes, among them, there were 13 hub target genes: RELA, TNF, IL6, IL1B, MAPK14, TP53, CXCL8, MAPK3, MAPK1, IL4, MAPK8, CASP8, STAT1. Meanswhile, GO_BiologicalProcess and KEGG signaling pathways analysis results showed that the core signal pathways were inflammation and immune regulation pathways. Finally, 4 key Chinese materia medica and 11 main active compounds were discovered in the HSBD. In conclusion, the therapeutic mechanism of HSBD on severe COVID-19 might involve its pharmacological effects of anti-inflammation and immune regulation via acting on 45 disease-related proteins of severe COVID-19. TAXONOMY CLASSIFICATION BY EVISE: Viral Pneumonia, COVID-19, Acute Respiratory Distress Syndrome, Septic Shock, Chinese Herbal Medicine.
RESUMO
BACKGROUND: Alcoholic fatty liver disease (AFLD) is the first stage of the alcoholic liver disease course. Yin-Chen-Hao-Tang (YCHT) has a good clinical effect on the treatment of AFLD, but its molecular mechanism has not been elucidated. In this study, we tried to explore the molecular mechanism of YCHT in improving hepatocyte steatosis in AFLD mice through network pharmacology and RNA sequencing (RNA-Seq) transcriptomics. METHODS: Network pharmacological methods were used to analyze the potential therapeutic signaling pathways and targets of YCHT on AFLD. Then, the AFLD mice model was induced and YCHT was administered concurrently. Liver injury was measured by serum alanine aminotransferase (ALT) activity and liver tissue H&E staining, and liver steatosis was determined by serum triglyceride (TG) level and liver tissue Oil Red staining. The molecular mechanism of YCHT on prevention and treatment of mice AFLD was investigated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differential expression genes data obtained by liver tissue RNA-Seq. Finally, ethanol-induced AFLD AML12 hepatocyte model was established, YCHT with or without PPARα agonist pemafibrate or PPARγ inhibitor GW9662 was administered, Nile Red fluorescent staining was used to evaluate steatosis levels in AML12 hepatocytes, and qRT-PCR was used to detect PPARα and PPARγ gene expression. RESULTS: The results of network pharmacology analysis showed that YCHT may exert its pharmacological effect on AFLD through 312 potential targets which are involved in many signaling pathways including the PPAR signaling pathway. AFLD mice experiments results showed that YCHT markedly decreased mice serum ALT activity and serum TG levels. YCHT also significantly improved alcohol-induced hepatic injury and steatosis in mice livers. Furthermore, KEGG pathway enrichment results of RNA-Seq showed that the PPAR signaling pathway should be the most relevant pathway of YCHT in the prevention and treatment of AFLD. AFLD hepatocyte model experiment results showed that YCHT could remarkably reduce hepatocyte steatosis through reducing PPARγ expression and increasing PPARα expression. CONCLUSIONS: Our study discovered that PPARγ and PPARα are the key targets and the PPAR signaling pathway is the main signaling pathway for YCHT to prevent and treat AFLD.
