[Phytoestrogen-like effect of Siwu Decoction and molecular mechanism: based on network pharmacology and in vivo experiment].

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.

[Molecular mechanism of apoptosis of breast cancer SKBR-3 cells induced by cryptanshinone via G protein coupled estrogen receptor( GPER) mediated pathway].

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.

GmmiR156b overexpression delays flowering time in soybean.

Soybean [Glycine max (L.) Merr.] is an important crop used for human consumption, animal feed and biodiesel fuel. Wering time and maturity significantly affect soybean grain yield. In Arabidopsis thaliana, miR156 has been proposed to regulate the transition from the juvenile to the adult phase of shoot development, which is accompanied by changes in vegetative morphology and an increase in reproductive potential. However, the molecular mechanisms underlying miR156 function in soybean flowering remain unknown. Here, we report that the overexpression of GmmiR156b delays flowering time in soybean. GmmiR156b may target SPL orthologs and negatively regulate GmSPLs, thereby delaying flowering in soybean under LD and natural conditions. GmmiR156b down-regulates several known flowering time regulators in soybean, such as GmAP1 (a, b, c), GmLFY2, GmLFY2, GmFULs, GmSOC1s, GmFT5a, and GmmiR172. These data show that a similar miR156-SPL regulatory module was conserved in the soybean flowering pathway. However, GmFULs, GmSOC1a and GmSOC1b were significantly suppressed under LD conditions but not under SD conditions, which is different in Arabidopsis that these genes were down-regulated irrespective of photoperiod. In addition, GmmiR156b was up-regulated by E1, E2 (GmGI), E3 and E4, which control flowering time and maturity in soybean, and suppressed E1 (E1-Like) and E2 (E2-Like) genes under LD conditions. These data indicated that the miR156-SPL regulatory module was also with some degree of divergent in soybean flowering pathway.

GmCOL1a and GmCOL1b Function as Flowering Repressors in Soybean Under Long-Day Conditions.

CONSTANS (CO) has a central role in the photoperiod response mechanism in Arabidopsis. However, the functions of legume CO genes in controlling flowering remain unknown. Here, we analyze the expression patterns of E1, E2 and GmCOL1a/1b using near-isogenic lines (NILs), and we further analyze flowering-related genes in gmcol1b mutants and GmCOL1a-overexpressing plants. Our data showed that both E3 and E4 up-regulate E1 expression, with the effect of E3 on E1 being greater than the effect of E4 on E1. E2 was up-regulated by E3 and E4 but down-regulated by E1. GmCOL1a/1b were up-regulated by E1, E2, E3 and E4. Although the spatial and temporal patterns of GmCOL1a/1b expression were more similar to those of AtCOL2 than to those of AtCO, gmcol1b mutants flowered earlier than wild-type plants under long-day (LD) conditions, and the overexpression of GmCOL1a caused late flowering under LD or natural conditions. In addition, GmFT2a/5a, E1 and E2 were down-regulated in GmCOL1a-overexpressing plants under LD conditions. Because E1/2 influences the expression of GmCOL1a, and vice versa, we conclude that these genes may function as part of a negative feedback loop, and GmCOL1a/b genes may serve as suppressors in photoperiodic flowering in soybean under LD conditions.

Siwu decoction exerts a phytoestrogenic osteoprotective effect on postmenopausal osteoporosis via the estrogen receptor/phosphatidylinositol 3-kinase/serine/threonine protein kinase pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Siwu decoction (SWD) is widely used in gynecological diseases, such as peripheral menopause syndrome, premature ovarian failure, and menstrual disorder. However, the mechanism of SWD on postmenopausal osteoporosis (PMOP) remains unclear. AIM OF THE STUDY: To discover the phytoestrogenic osteoprotective effect of SWD on PMOP. MATERIALS AND METHODS: The potential mechanism of SWD on PMOP was filtered through network pharmacology research. The potential mechanism was verified in MC3T3-E1 cell lines in vitro. CCK8 assay was conducted to assess cell proliferation and the expressions of ER/PI3K/AKT pathway were analyzed using Western blot. Female F-344 rats were chosen to set up the PMOP model. The osteoprotective effect of SWD in vivo was evaluated using Hematoxylin-eosin staining, TRAP staining, Goldner staining and DXA. The potential mechanism was verified in vivo through Western blot and immunohistochemistry. RT-qPCR was conducted to unveil the expressions of osteogenesis genes. RESULTS: Network pharmacology research showed that ER/PI3K/AKT pathway may be the potential mechanism of SWD on PMOP. SWD promoted the proliferation of osteoblasts and regulated the protein expressions of ER/PI3K/AKT pathway in vitro. SWD improved the morphological structure, bone mineralization and bone mineral density of femurs and suppressed osteoclastogenesis in PMOP rat model via ER/PI3K/AKT pathway in vivo. In addition, SWD regulated the mRNA expressions of osteogenesis-related genes. CONCLUSIONS: SWD exerts a phytoestrogenic osteoprotective on PMOP by regulating ER/PI3K/AKT pathway, which marks it as a valuable medicine or supplement of PMOP.

Tanshinone IIA Inhibits Triple-Negative Breast Cancer Cells MDA-MB-231 via G Protein-Coupled Estrogen Receptor- (GPER-) Dependent Signaling Pathway.

Due to the lack of classic estrogen receptors, there has been a shortage of targeted therapy for triple-negative breast cancer (TNBC), resulting in a poor prognosis. However, the newly discovered G protein-coupled estrogen receptor (GPER) has been found to be expressed in TNBC cells. Salvia miltiorrhiza (Danshen) is an essential Chinese medicine for gynecological disorders, and its component tanshinone IIA (Tan IIA) exerts an anticancer effect. Therefore, this study attempted to investigate whether GPER is involved in the inhibitory effect of Tan IIA on TNBC. We applied various databases and GO pathway analysis to predict the possible mechanism of Tan IIA. We identified 39 overlapping targets, including c-Jun, c-Fos, and caspase-3, and enriched cell cycle-related pathways. Next, we demonstrated the strong binding ability of Tan IIA to GPER by molecular docking assay. In the subsequent validation tests, Cell Counting Kit-8 (CCK8) assay showed that Tan IIA inhibited proliferation of MDA-MB-231 cells time and dose dependently without affecting normal cells. Using Transwell plate, flow cytometry, and Western blot assays, we showed that Tan IIA inhibited migration and induced apoptosis of MDA-MB-231 dose dependently. Importantly, protein expressions of GPER, epidermal growth factor receptor (EGFR), extracellular regulated protein kinases (ERK), c-Fos, and c-Jun were all decreased by Tan IIA dose dependently. Administration of GPER inhibitor partly abolished these effects. Furthermore, nuclear translocation of c-Fos and c-Jun as well as cell cycle-related proteins was downregulated by Tan IIA dose dependently. In summary, Tan IIA could inhibit the proliferation and migration of MDA-MB-231 cells and induce apoptosis, and the possible mechanism may be the regulation of GPER-mediated pathways, suggesting that GPER could be a therapeutic target for TNBC.

Yangjing Zhongyu decoction facilitates mitochondrial activity, estrogenesis, and energy metabolism in H2O2-induced human granulosa cell line KGN.

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.

Cryptotanshinone inhibits proliferation and induces apoptosis of breast cancer MCF-7 cells via GPER mediated PI3K/AKT signaling pathway.

G protein-coupled estrogen receptor (GPER) was reported to be a potential target in the breast cancer therapy. This study aimed to illuminate the function of GPER and its mediated PI3K/AKT pathway in cryptotanshinone (CPT) inducing cell apoptosis and antiproliferation effect on GPER positive breast cancer MCF-7 cells. Cell proliferation was tested by MTT assay. Apoptosis rates were tested by Annexin V-FITC/PI double staining and the cell cycle was researched by flow cytometry. Autodock vina was applied to make molecular docking between CPT or estradiol and GPER. siRNA technique and GPER specific agonist G-1 or antagonist G-15 were applied to verify the mediated function of GPER. Apoptosis and cell cycle related proteins, as well as the key proteins on PI3K/AKT signaling pathway were detected by western blot. The results indicated that CPT could exert antiproliferation effects by arresting cell cycle in G2/M phase and downregulating the expression of cyclin D, cyclin B and cyclin A. Besides, apoptosis induced by CPT was observed. CPT might be a novel GPER binding compounds. Significantly, suppression of PI3K/AKT signal transduction by CPT was further increased by G-1 and decreased by G-15. The study revealed that the effect of antiproliferation and apoptosis treating with CPT on MCF-7 cells might be through the downregulation of PI3K/AKT pathway mediated by activated GPER.

Virtual screening of the multi-gene regulatory molecular mechanism of Si-Wu-tang against non-triple-negative breast cancer based on network pharmacology combined with experimental validation.

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.

Inhibition of PI3K/AKT molecular pathway mediated by membrane estrogen receptor GPER accounts for cryptotanshinone induced antiproliferative effect on breast cancer SKBR-3 cells.

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.