RESUMO
This study explored the phytoestrogen-like effect of Siwu Decoction(SWD) and the estrogen receptor(ER)-mediated molecular mechanism based on network pharmacology and in vivo experiment. The active components and targets of SWD were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and related targets of "estrogen" from GeneCards and Online Mendelian Inheritance in Man(OMIM). Cytoscape and STRING were employed to construct the protein-protein interaction(PPI) network and "chemical component-target-disease" network and core targets were identified, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the core targets by R software. For the in vivo experiment, the 22-day-old SD female rats were treated(ig) with SWD for 4 days. Via hematoxylin-eosin(HE) staining, the morphological changes of rat uterus were observed. Reverse transcriptase-polymerase chain reaction(RT-PCR) was performed to detect mRNA expression of ER subtypes, estrogen-related targets, and the main regulatory factors in the estrogen signaling pathway. The results indicated 74 targets of SWD exerted phytoestrogen-like effect. KEGG pathway enrichment result suggested that estrogen signaling pathway was closely related to the phytoestrogen-like effect of SWD. Rats in SWD group demonstrated significantly thickened endometrium and significantly decreased expression of ERα, ERß, and G protein-coupled estrogen receptor(GPER) mRNA in ovarian tissue. In addition, significant lowering of ERα and ERß mRNA expression and significant rise of GPER mRNA expression in uterine tissue were observed in the SWD group. The expression of mitogen-activated protein kinase(MAPK) p38, MEK1/2 and extracellular signal-regulated kinase(ERK)1/2 mRNA was significantly low while that of epidermal growth factor receptor(EGFR) mRNA was significantly high in both ovarian and uterine tissues of SWD group compared with those in the control group. In conclusion, the phytoestrogen-like effect of SWD is closely related to the estrogen signaling pathway. The result lays a basis for revealing molecular mechanism of SWD in the treatment of gynecological diseases.
Assuntos
Medicamentos de Ervas Chinesas , Animais , Medicamentos de Ervas Chinesas/farmacologia , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Estrogênios/farmacologia , Feminino , Humanos , Medicina Tradicional Chinesa , Simulação de Acoplamento Molecular , Farmacologia em Rede , Fitoestrógenos , RNA Mensageiro , Ratos , Receptores de Estrogênio/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANT: Yangjing Zhongyu decoction (YJZYD) is a recipe from a Chinese classic medical work and has been empirically used in female infertility for hundreds of years, but the mechanisms of YJZYD on facilitating ovarian granulosa cells remain unfold. AIM OF THE RESEARCH: The purpose of the study is to determine the rewarding effects of YJZYD on H2O2-induced KGN cells, involving mitochondrial activity, estradiol biosynthesis, and energy metabolism. MATERIALS AND METHODS: The ingredients of YJZYD were investigated by UPLC-ESI-MS/MS analysis. The effects of YJZYD and H2O2 on cell viability were determined by CCK-8. Intracellular ROS were assessed by DCFH-DA. Intracellular Ca2+ was detected using Fura-4 AM. Mitochondrial membrane potential (MMP) was measured by JC-1. The production of energy was assessed by ATP. Apoptosis rate was analyzed by Annexin V-FITC/PI. Western blotting was used to evaluate the expression of proteins related to energy metabolism, apoptosis, mitochondrial mitophagy, and estrogen biosynthesis. E2 levels were measured by ELISA. RESULTS: 121 compounds were identified in YJZYD by UPLC-ESI-MS/MS analysis. YJZYD could enhance mitochondrial activity by suppressing intracellular ROS and Ca2+, and increasing MMP and ATP content. YJZYD stimulated the expression of anti-apoptosis protein Bcl-2 and lowered the early apoptosis rate and the expression of Bax. Besides, YJZYD rescued E2 secretion and improved the expression of FSHR, CYP19A1, and the ratio of p-CREB/CREB. In addition, YJZYD weakened H2O2-induced mitophagy by compromising the expression of PINK1, Parkin, Beclin1 and P62. Moreover, YJZYD strengthened energy metabolism by increasing ATP generation and the expression of SIRT1, PGC1α, NRF1, and COX IV. The combination of YJZYD and autophagy inhibitor had a stronger protective effect on energy metabolism. CONCLUSION: This study evaluated the protective effects of YJZYD on H2O2-induced KGN cells. YJZYD could enhance mitochondrial activity, E2 biosynthesis, and energy metabolism. These results strongly indicated that YJZYD might play a role in preserving ovarian granulosa cells and female fecundity.
Assuntos
Peróxido de Hidrogênio , Espectrometria de Massas em Tandem , Feminino , Humanos , Trifosfato de Adenosina/metabolismo , Apoptose , Medicamentos de Ervas Chinesas , Células da Granulosa , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Si-Wu-Tang (SWT), a prestigious herbal formula from China, has been extensively used for centuries for female-related diseases. It has been documented that SWT has a significant inhibitory effect on non-triple-negative breast cancer (non-TNBC) cells. However, there has been limited comprehensive analysis of the targeted effects of the anticancer components of SWT and its exact biological mechanism. AIM OF THE STUDY: This study aims to uncover the mechanism by which SWT treats non-TNBC by applying a network pharmacological method combined with experimental validation. MATERIALS AND METHODS: First, SWT compounds were collected from the Traditional Chinese Medicines Systems Pharmacology database (TCMSP) and The Encyclopedia of Traditional Chinese Medicine (ETCM), and then the targets related to SWT were obtained from the TCMSP and SwissTarget databases. Second, a target data set of non-TNBC proteins was established by using the Online Mendelian Inheritance in Man (OMIM), GeneCards and Gene Expression Omnibus (GEO) databases. Third, based on the overlap of targets between SWT and non-TNBC, a protein-protein interaction (PPI) network was built to analyse the interactions among these targets, which focused on screening for hub targets by topology. On these hub genes, we conducted a meta-analysis and survival analysis to screen the best match targets, ESR1, PPARG, CAT, and PTGS2, which had a strong correlation with the ingredients of SWT in our verification by molecular docking. In vitro experiments further proved the reliability of the network pharmacology findings. Finally, FunRich software and the ClusterProfiler package were utilized for the enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) data. RESULTS: A total of 141 active ingredients and 116 targets of SWT were selected. GO enrichment analysis showed that the biological processes through which SWT acted against non-TNBC (FDR<0.01) mainly involved modulating energy metabolism and apoptosis. According to RT-qPCR and Western blotting, the mRNA and protein expression of ESR1, PPARG and PTGS2 were upregulated (P < 0.01), and the mRNA and protein levels of CAT were downregulated (P < 0.01), suggesting a multi-gene regulatory molecular mechanism of SWT against non-triple-negative breast cancer. CONCLUSIONS: This research explored the multi-gene pharmacological mechanism of action of SWT against non-TNBC through network pharmacology and in vitro experiments. The findings provide new ideas for research on the mechanism of action of Chinese medicine against breast cancer.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Apoptose/efeitos dos fármacos , Catalase/biossíntese , Catalase/genética , Ciclo-Oxigenase 2/genética , Bases de Dados de Compostos Químicos , Bases de Dados Genéticas , Metabolismo Energético/efeitos dos fármacos , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Medicina Tradicional Chinesa/métodos , Simulação de Acoplamento Molecular , PPAR gama/biossíntese , PPAR gama/genética , Ligação Proteica , Mapas de Interação de ProteínasRESUMO
BACKGROUND: Breast cancer is the most frequently diagnosed malignancy among women and the second leading cause of cancer death worldwide. Among which nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. Recently, membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor has been documented to take essential part in the development and treatment of breast cancer. The present study was designed to investigate the anti nER negative breast cancer effect of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen and its possible molecular pathway. METHODS: The following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay respectively. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT. RESULTS: The results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT could also exert antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G-1 and attenuated by G-15. Results of western blot and immunofluorescence showed that expression of PI3K and p-AKT could be downregulated by CPT and such effects were mediated by GPER which were further demonstrated by gene silence test. CONCLUSION: The current study showed that the antiproliferative action of CPT on SKBR-3 cells was realized by inhibition of GPER mediated PI3K/AKT pathway. These findings provide further validation of GPER serving as useful therapeutic target.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Fenantrenos/farmacologia , Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The study aimed to illuminate the role of G protein coupled estrogen receptor( GPER) and its mediated PI3 K/AKT signaling pathway in cryptotanshinone( CPT) induced apoptosis of breast cancer SKBR-3 cells,which is GPER positive and ER negative.The apoptosis rate of SKBR-3 cells was tested by Annexin V-FITC/PI staining and apoptosis effector caspase-3 was determined by Western blot. The key proteins in PI3 K/AKT signaling pathway mediated by GPER were detected by Western blot and immunofluorescence technique. Meanwhile,the agonist G1 and antagonist G15 of GPER and antagonist LY294002 of PI3 K were employed in the test to further clarify the effect of GPER and PI3 K/AKT pathway. The results indicated that the apoptosis rate was increased from 4. 7% to46. 1% and 69. 0% after treatment with 0,5,10 µmol·L~(-1) CPT for 48 h( P<0. 01). The expression of PI3 K,AKT and p-AKT were inhibited( P<0. 05 or P<0. 01),while caspase-3 level increased obviously after treatment with CPT( P<0. 01). Importantly,inhibitory effect of PI3 K/AKT signaling pathway by CPT was further enhanced by G1 and attenuated by G15. LY294002 also induced a further inhibition of expression of AKT and p-AKT. The mean fluorescence intensity of AKT and p-AKT could be decreased by CPT. Furthermore,CPT could downregulate GPER expression in SKBR-3 cells( P<0. 01),which could be inhibited by G1 and enhanced by G15.In conclusion,CPT could induce the apoptosis of ER negative and GPER positive breast cancer SKBR-3 cells and the molecular mechanism is related to its regulatory effect of GPER and its mediated PI3 K/AKT signaling pathway.