الملخص
Objective:To study the main active components and potential mechanism of the treatment of ovarian cancer by Scutellariae Barbatae Herba - Cremastrae Pseudobulbus Pleiones Pseudobulbus based on network pharmacology and in vitro cell experiments.Methods:TCMSP and SwissTargetPrediction databases were used to retrieve and collect the active components and targets of Scutellariae Barbatae Herba - Cremastrae Pseudobulbus Pleiones Pseudobulbus, and four databases including GeneCards, OMIM, DrugBank and DisGeNET were used to search the targets related to ovarian cancer. After screening the common potential targets of Scutellariae Barbatae Herba - Cremastrae Pseudobulbus Pleiones Pseudobulbus and ovarian cancer, the STRING database was used to realize the construction of PPI network interacting with the proteins of the two common targets. DAVID database was used for GO and KEGG enrichment analysis. Cytoscape 3.9.0 software was used to construct the "active component-target-pathway" network. Finally, the key targets and pathways were validated in vitro. MTT method was used to detect the proliferation inhibition rate of human ovarian cancer SKOV3 cells with different concentrations of Quercetin and sitosterol. Human ovarian cancer SKOV3 cells were divided into control group, Quercetin group and sitosterol group according to the random number table method. After corresponding drug intervention for 48 hours, the effects of Quercetin and sitosterol on SKOV3 cell cloning ability were detected by clone formation experiment, the levels of Akt1, VEGFA, Caspase-3 mRNA in each group were detected by PCR, and the protein expressions of PI3K, Akt, p-Akt, Caspase-3, and VEGFA in cells were detected by Western blot.Results:Through screening, 29 effective chemical components in Scutellariae Barbatae Herba and 3 effective chemical components in Cremastrae Pseudobulbus Pleiones Pseudobulbus were obtained, including 2 components in total. 291 drug-related targets, 1969 potential targets of ovarian cancer were acquired. There were 137 common targets of "Scutellariae Barbatae Herba - Cremastrae Pseudobulbus Pleiones Pseudobulbus" and the drug-disease intersection of ovarian cancer. Through GO functional analysis, 930 items were obtained, involving 722 biological processes, 76 cellular components and 132 molecular functions. KEGG enrichment analysis involved 169 signaling pathways, including tumor pathway, PI3K-Akt signaling pathway, P-53 signaling pathway, IL-17 signaling pathway, etc. In vitro experiment results showed that cell proliferation inhibition rate increased and cell cloning ability decreased in Quercetin and sitosterol groups; The levels of Akt1 and VEGFA mRNA in Quercetin group and sitosterol group decreased ( P<0.01 or P<0.05), the expression of Caspase-3 mRNA and protein increased ( P<0.01), and the expressions of PI3K, p-Akt and VEGFA protein decreased ( P<0.01). Conclusion:Scutellariae Barbatae Herba - Cremastrae Pseudobulbus Pleiones Pseudobulbus inhibits the occurrence and development of ovarian cancer through multi target and multi pathway synergistic effects, and can induce SKOV3 cell apoptosis by regulating the expressions of key genes such as AKT1, VEGFA, Caspase-3, and PI3K/Akt pathway.
الملخص
ObjectiveTo screen out the main targets and related signaling pathways of the herbal pair Cremastrae Pseudobulbus-Rhapontici Radix in treating breast cancer based on network pharmacology and verify their action mechanism in in vitro experiments. MethodThe main chemical components and related targets of Cremastrae Pseudobulbus-Rhapontici Radix were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， and the target genes related to breast cancer from GeneCards. Following the screening of the common targets of Cremastrae Pseudobulbus-Rhapontici Radix and breast cancer using Venn， the Cremastrae Pseudobulbus-Rhapontici Radix-breast cancer network and protein-protein interaction （PPI） network were constructed. The effective targets were then subjected to gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） pathway enrichment analysis. The resulting outcomes were then verified by cell counting kit （CCK）-8 assay， flow cytometry， and Western blot. ResultThe screening yielded seven effective components and 61 targets of Cremastrae Pseudobulbus-Rhapontici Radix， among which 55 targets were involved in breast cancer. The GO analysis revealed 832 entries， which were mainly enriched in the biological processes. According to KEGG pathway enrichment analysis， 85 signaling pathways were obtained， including tumor suppressor p53， vascular endothelial growth factor （VEGF）， epidermal growth factor receptor （EGFR）， and phosphatidylinositol 3-kinase （PI3K）/protein kinase B（Akt）. It was verified in in vitro experiments that the alcohol extract of Cremastrae Pseudobulbus-Rhapontici Radix inhibited the proliferation of human breast cancer MDA-MB-231 cells and induced their apoptosis. Compared with the blank control group and the dimethyl sulfoxide （DMSO， 0.1% solvent） group， the medication groups exhibited obviously decreased absorbance in MDA-MB-231 cells （P<0.01） and increased apoptosis rate （P<0.01）. The results of Western blot demonstrated that compared with the blank control group and the DMSO group， each medication significantly reduced the phosphorylated （p）-PI3K/PI3K and p-Akt/Akt in cells （P<0.05）. ConclusionThe ethanol extract of Cremastrae Pseudobulbus-Rhapontici Radix effectively inhibits the proliferation of human breast cancer MDA-MB-231 cells and induces their apoptosis， which may be related to the inhibition of the activation of PI3K/Akt signaling pathway.