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.

Si-Wu-Tang attenuates liver fibrosis via regulating lncRNA H19-dependent pathways involving cytoskeleton remodeling and ECM deposition.

Liver fibrosis is a dynamic wound-healing response characterized by the agglutination of the extracellular matrix (ECM). Si-Wu-Tang (SWT), a traditional Chinese medicine (TCM) formula, is known for treating gynecological diseases and liver fibrosis. Our previous studies demonstrated that long non-coding RNA H19 (H19) was markedly upregulated in fibrotic livers while its deficiency markedly reversed fibrogenesis. However, the mechanisms by which SWT influences H19 remain unclear. Thus, we established a bile duct ligation (BDL)-induced liver fibrosis model to evaluate the hepatoprotective effects of SWT on various cells in the liver. Our results showed that SWT markedly improved ECM deposition and bile duct reactions in the liver. Notably, SWT relieved liver fibrosis by regulating the transcription of genes involved in the cytoskeleton remodeling, primarily in hepatic stellate cells (HSCs), and influencing cytoskeleton-related angiogenesis and hepatocellular injury. This modulation collectively led to reduced ECM deposition. Through extensive bioinformatics analyses, we determined that H19 acted as a miRNA sponge and mainly inhibited miR-200, miR-211, and let7b, thereby regulating the above cellular regulatory pathways. Meanwhile, SWT reversed H19-related miRNAs and signaling pathways, diminishing ECM deposition and liver fibrosis. However, these protective effects of SWT were diminished with the overexpression of H19 in vivo. In conclusion, our study elucidates the underlying mechanisms of SWT from the perspective of H19-related signal networks and proposes a potential SWT-based therapeutic strategy for the treatment of liver fibrosis.

Prognostic model construction and target identification of Si-Wu-Tang against breast cancer.

The targets and mechanisms of Si-Wu-Tang (SWT) against (Breast cancer) BRCA were identified and a survival model and nomogram was construted by network pharmacology, bioinformatic analysis and in vitro experiments. A total of 72 anti-breast cancer SWT targets were selected, among which eleven genes (MAOA、SQLE、CACNA2D1、GLI1、RORB、ITGB3、TACR1、NR3C2、CA3、RBP4 and PTK6) were used to construct a novel prognostic model of breast cancer. The anti-breast cancer activity of SWT was related to the modulation of the receptor tyrosine kinases signaling pathways. Moreover, two compounds, mairin and senkyunone were found to bind directly to ITGB3 and RORB proteins. Finally, mRNA and protein expression of ITGB3 and RORB was observed to be significantly down-regulated after incubation of MCF-7 cells with SWT. Overall, our results indicated that mairin and senkyunone were the key ingredients present in SWT, and ITGB3 as well as RORB proteins were the major targets affected by SWT. The prognostic model can be used to predict the outcome of BRCA patients.

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.

Erxian decoction alleviates cisplatin-induced premature ovarian failure in rats by reducing oxidation levels in ovarian granulosa cells.

ETHNOPHARMACOLOGICAL RELEVANT: Erxian Decoction (EXD) has been used empirically for more than 70 years to treat premature ovarian failure (POF), but more research is needed to understand how it works. AIM OF THE RESEARCH: The study aims to ascertain both in vivo and in vitro rewards of EXD. MATERIALS AND METHODS: EXD is composed of Curculiginis Rhizoma, Epimedii Folium, Morindae Officinalis, Angelicae Sinensis, Anemarrhenae Rhizoma, and Phellodendri Chinensis Cortex. UPLC/MS analysis was used to investigate the components of EXD. Using a POF model created by administering cisplatin to rats intraperitoneally, the pharmacodynamic effects of EXD were investigated. Three dose groups of EXD were garaged into rats: high (15.6 g/kg), medium (7.8 g/kg), and low (3.9 g/kg). By using a vaginal smear, the impact of EXD on the rat estrous cycle was evaluated. An ELISA test was used to measure the anti-Mullerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the serum of rats. By using HE stains, pathological alterations in the ovaries may be seen. MDA and SOD levels in ovarian samples were used to measure the degree of ovarian oxidation. TUNEL labeling of ovarian sections was used to find apoptosis levels. By using ATP, energy production was evaluated. The relative expression of proteins connected to aging and the RAGE pathway was assessed using Western blot. Then, using H2O2, a model of senescent human ovarian granulosa cells (KGN) was created in vitro. The impact of EXD and H2O2 on cellular senescence was discovered using-galactosidase staining. Cell apoptosis levels were found using PI/Hoechest33342. By using DCFH-DA, intracellular ROS was examined. MDA and SOD concentrations were used to measure the degree of cellular oxidation. RAGE-related mRNA and protein expression were evaluated using RT-qPCR and western blotting. RESULTS: Using UPLC/MS analysis, 39 chemicals in EXD were found. Rats' estrous cycles were enhanced by EXD, which increased ovarian index and follicle count and reduced the proportion of atretic follicles in the rats. EXD reduced LH and FSH output while restoring AMH and E2 secretion. In ovarian tissues, EXD reduced the amount of apoptosis and MDA while raising SOD activity and ATP levels. The protein levels of p16, p21, p53, and Lamin A/C were among the senescence-related proteins that EXD lowered, along with the levels of RAGE, PI3K, BAX, and CASPASE 3. Anti-apoptotic protein BCL-2 was also raised in the RAGE pathway. Senescence, apoptosis, ROS, and MDA levels in the KGN cells were lowered in vitro by EXD. Additionally, EXD increased the anti-apoptotic potential by changing the expression of CAT, SOD2, and SIRT1. RAGE, BAX, BCL-2, CASPASE 3, and p38 expression levels were altered by EXD, enhancing its anti-apoptotic capability. CONCLUSION: EXD boosted the ovary's antioxidant and anti-apoptotic capabilities while enhancing the estrous cycle and hormone output. These findings strongly suggested that EXD may contribute to the alleviation of POF and ovarian granulosa cells senescence.

Chemical Stability and Bioactivity of tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone in in vitro test systems.

Danshen Si Wu is a Traditional Chinese Medicine used for menopausal complains. Beside tanshinone IIA (Tan IIA), Danshen also contains tanshinone I (Tan I), cryptotanshinone (CT) and dihydrotanshinone (DT). The aim of this study was to compare the biological activity of these tanshinones and to determine their cytotoxicity and genotoxicity. Purities and stabilities of the substances were analyzed by LC-DAD and LC-MS analyses. DT and CT concentrations decreased rapidly in dimethylsulfoxide and were converted to Tan I and Tan IIA, respectively. In aqueous solution concentration of all tanshinones decreased after 24 h. Tan I and Tan IIA showed dose-dependent bioactivity mediated by ERα and ERß. No cytotoxic and genotoxic effects for Tan I and Tan IIA were detected. In a yeast transactivation assay Tan I and Tan IIA showed antiandrogenic activity. A significant anabolic activity in C2C12 cells could be detected for Tan I and Tan IIA. In conclusion our data provide evidence that Tan I and Tan IIA are the most relevant bioactive tanshinones in Danshen. Our finding that all tanshinones display a certain instability in aqueous solutions is relevant when discussing their potential therapeutic benefits in humans.

[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.

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.

[Mechanism of traditional Chinese medicine balancing Yin-Yang by targeting ERα/ERß and its application in treatment of menopausal syndrome].

The coordination and unification of Yin and Yang are the basis of normal human life activities. Along with the age growth and aging of the body, women will suffer from menopausal syndrome during menopause. In addition to the significant changes in the genital system, there are also pathological manifestations in estrogen target points including bone, nerve and cardiovascular systems, due to the imbalance of Yin and Yang. Besides the insufficiency of estrogen, the main cause of menopausal syndrome is the changes in the response of target organs to estrogen. In other words, the biological effects mediated by estrogen receptor(ER) alpha and beta subtypes in target cells are often different or even opposite; the changes of expression level and ratio of ERα and ERß are also important causes for the abnormal estrogenic effects in target organs and the imbalance of Yin and Yang of the body. Therefore, on one hand, the therapeutic mechanism of drugs is ER-mediated estrogenic effect. On the other hand, the drugs have a regulatory effect on ER subtype expression in target cells and Yin-Yang state in target organs and even organisms, so as to cause further changes in the response of target cells to estrogen or estrogenic components, and exert its therapeutic effects. This paper reviews the pharmacological mechanism of gynecological traditional Chinese medicine in harmonizing Yin and Yang in estrogen-positive target cells and the clinical efficacy in the following aspects, including estrogen and its mechanism, the estrogenic effect of ER in traditional Chinese medicine and the mechanism of ER subtype in balancing Yin and Yang and mediating and regulating the main target tissues in menopausal syndrome treatment.

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.

[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.

ER-mediated anti-tumor effects of shikonin on breast cancer.

Estrogen receptor (ER) is expressed in most Breast cancer (BC) patients. G protein-coupled estrogen receptor (GPER), which is a membrane-bound estrogen receptor, is associated with the tumor development and progression in BC. Shikonin (SK) is a natural compound that is known to have anti-tumor effects. This study aims to assess the effects of shikonin on the cell proliferation, cell cycle and cell apoptosis of BC and whether the effects are related to ER/GPER signaling pathway. The results demonstrated that shikonin inhibited the cellular proliferation of MCF-7 BC cells via G0/G1 arrest and apoptosis in concentration-dependent manner. The anti-proliferative effect of SK on SK-BR-3 BC cells was associated with apoptosis. Both ERα and GPER were expressed in MCF-7 cells, while ERα were negative and GPER were positive in SK-BR-3 cells. Furthermore, shikonin downregulated the expression of ERα and GPER, and this effect was not affected by the estrogen environment. In addition, shikonin downregulated the EGFR and p-ERK expression in MCF-7 and SK-BR-3, which was also not affected by the estrogen environment. EGFR and p-ERK were still suppressed by co-treatment with the selective GPER against G1 or antagonist G15. In conclusion, these results suggest that shikonin shows anti-tumor effects on MCF-7 and SK-BR-3 cells. The effects seem to be associated with EGFR/p-ERK downregulation via ERα and GPER inhibition.

Anabolic effect of the traditional Chinese medicine compound tanshinone IIA on myotube hypertrophy is mediated by estrogen receptor.

Skeletal muscle loss during menopause is associated with a higher risk of developing diabetes type II and the general development of the metabolic syndrome. Therefore, strategies combining nutritional and training interventions to prevent muscle loss are necessary. Danshen Si Wu is a traditional Chinese medicine used for menopausal complains. One of the main compounds of Danshen Si Wu is tanshinone IIA. Physiological effects of tanshinone IIA have been described as being mediated via the estrogen receptor. Therefore, it was the aim of this study to determine its tissue specific ERα- and ERß-mediated estrogenic activity, to investigate its antiestrogenic properties, and, particularly, to study estrogen receptor-mediated biological responses to tanshinone IIA on skeletal muscle cells. The purity of tanshinone IIA was analyzed by LC-DAD-MS/MS analysis. ERα/ERß-mediated activity was dose-dependently analyzed in HEK 239 cells transfected with ERα or ERß expression vectors and respective reporter genes. Androgenic, antiandrogenic, and antiestrogenic properties of tanshinone IIA were analyzed in a yeast reporter gene assay. The effects of tanshinone IIA on proliferation and cell cycle distribution were investigated in ERα positive T47D breast cancer cells. The ability of tanshinone IIA to stimulate estrogen receptor-mediated myotube hypertrophy was studied in C2C12 myoblastoma cells. Our data show that tanshinone IIA is quite potent at stimulating ERα and ERß reporter genes with comparable efficacy. Tanshinone IIA displayed antiestrogenic and also antiandrogenic properties in a yeast reporter gene assay. It inhibited the growth of T47D breast cancer cells by suppressing proliferation and arresting the cells in G0/G1. Tanshinone IIA also stimulated the hypertrophy of C2C12 myotubes via an estrogen receptor-mediated mechanism. Summarizing our results, tanshinone IIA can be characterized as an estrogen receptor partial agonist with antiandrogenic properties. It seems to inhibit ERα-mediated cell proliferation but induces ERß-related biological responses like hypertrophy of myotubes. These findings are interesting with respect to the treatment of a variety of complains of postmenopausal females, including muscle wasting.

Curcumin treatment suppresses CCR7 expression and the differentiation and migration of human circulating fibrocytes.

BACKGROUND/AIM: Recent studies have demonstrated that circulating fibrocytes contribute to the formation and development of fibrosis. Curcumin, a polyphenolic compound isolated from turmeric, has been shown to have anti-fibrotic effects in various organs. We and others have demonstrated that curcumin beneficially affects the development of fibrosis. However the effect of curcumin on circulating fibrocytes has not been reported. METHODS: Human circulating fibrocytes were isolated from leukocyte concentrates of healthy human donors and identified based on the expression of CD34, CD45, collagen I (COLI), and chemokine receptor CCR7 (CCR7) via flow cytometry. Cell Counting Kit-8 was used to evaluate cell viability. The effect of curcumin on the differentiation and migration of human circulating fibrocytes was evaluated by immunofluorescence staining, flow cytometry and a transwell migration assay. Transforming growth factor (TGF)-ß1 secretion was examined by ELISA. RESULTS: Curcumin treatment (72 h; 20 µM) significantly decreased the expression of COL I, α-SMA and CCR7, as well as TGF-ßl secretion, in human circulating fibrocytes. The inhibitory effect of curcumin on the differentiation and migration of human circulating fibrocytes is likely via regulating the CCR7/CCL21 signaling pathway, in particular by reducing CCR7 expression. These observed effects may be beneficial in resolving fibrosis by suppressing TGF-ß1 secretion. CONCLUSION: Our results suggest that curcumin has the potential to suppress the differentiation and migration of circulating fibrocytes, which would provide new explanation for curcumin's application in the development of fibrosis in various organs.

[Effect of carnosol against proliferative activity of breast cancer cells and its estrogen receptor subtype's mediation and regulation mechanisms].

Carnosol has been proved to have anti-breast cancer effect in previous research. But its ER subtype's specific regulation and mediation mechanisms remain unclear. The aim of this study is to observe the effect of carnosol on cell proliferation and its estrogen receptor α and ß's specific regulation and mediation mechanisms with ER positive breast cancer T47D cell. With estrogen receptor α and ß antagonists MPP and PHTPP as tools, the MTT cell proliferation assay was performed to observe the effect of carnosol on T47D cell proliferation. The changes in the T47D cell proliferation cycle were detected by flow cytometry. The effect of carnosol on ERα and ERß expressions of T47D cells was measured by Western blot. The findings showed that 1 x 10(-5)-1 x 10(-7) mol x L(-1) carnosol could significantly inhibit the T47D cell proliferation, which could be enhanced by MPP or weakened by PHTPP. Meanwhile, 1 x 10(-5) mol x L(-1) or 1 x 10(-6) mol x L(-1) carnosol could significantly increase ERα and ERß expressions of T47D cells, and remarkably increase ERα/ERß ratio. The results showed that carnosol showed the inhibitory effect on the proliferation of ER positive breast cancer cells through target cell ER, especially ERß pathway. In the meantime, carnosol could regulate expressions and proportions of target cell ER subtype ERα and ERß.

Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone.

SCOPE: The phytoectysteroid ecdysterone (Ecdy) was reported to stimulate protein synthesis and enhance physical performance. The aim of this study was to investigate underlying molecular mechanisms particularly the role of ER beta (ERß). RESULTS: In male rats, Ecdy treatment increased muscle fiber size, serum IGF-1 increased, and corticosteron and 17ß-estradiol (E2) decreased. In differentiated C2C12 myoblastoma cells, treatment with Ecdy, dihydrotestosterone, IGF-1 but also E2 results in hypertrophy. Hypertrophy induced by E2 and Ecdy could be antagonized with an antiestrogen but not by an antiandrogen. In HEK293 cells transfected with ER alpha (ERα) or ERß, Ecdy treatment transactivated a reporter gene. To elucidate the role of ERß in Ecdy-mediated muscle hypertrophy, C2C12 myotubes were treated with ERα (ALPHA) and ERß (BETA) selective ligands. Ecdy and BETA treatment but not ALPHA induced hypertrophy. The effect of Ecdy, E2, and BETA could be antagonized by an ERß-selective antagonist (ANTIBETA). In summary, our results indicate that ERß is involved in the mediation of the anabolic activity of the Ecdy. CONCLUSION: These findings provide new therapeutic perspectives for the treatment of muscle injuries, sarcopenia, and cachectic disease, but also imply that such a substance could be abused for doping purposes.

The antioxidant effect of carnosol in bovine aortic endothelial cells is mainly mediated via estrogen receptor α pathway.

Antioxidant action is critical for maintaining the normal cardiovascular function and vascular endothelial cell is an important target of estrogen action through estrogen receptor (ER) pathway. This study is carried out to explore the antioxidant effect of carnosol in bovine aortic endothelial cells (BAECs) via ER pathway. The ER subtype specific estrogenic effect of carnosol was further demonstrated by luciferase reporter gene assay in human embryonic kidney (HEK) 293 cells. Carnosol was extracted from Chinese medicine Rosmarinus officinalis. ER positive BAECs were employed in cell proliferation assay and cell apoptosis tests. Oxidative stress by intracellular reactive oxygen species (ROS) were measured via 2'7'-dichlorofluorescein (DCF) production. ERα and ERß specific antagonists 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-yl]phenol (PHTPP) were employed as tools in the experiment. ER negative HEK 293 cells were employed in luciferase reporter gene assay. The results indicate that carnosol can effectively attenuate H(2)O(2) induced slowing down of cell growth and increasing of cell apoptosis. At the meantime, carnosol pretreating can also effectively reduce the H(2)O(2) induced intracellular ROS elevation in BAECs. ERα and ERß antagonist, especially ERα antagonist, can effectively decrease the above antioxidant effects of carnosol. The reporter gene analysis further demonstrates that the action of carnosol on inducing ERE dependent luciferase expression is realized via ER pathway. The conclusion is that carnosol can exert antioxidant effects towards oxidative stress induced by H(2)O(2) in BAECs. And such effects are realized via ER, especially ERα pathway. The results contribute to explain the mechanism of cardiovascular protective function of carnosol in postmenopausal women.

[Effect and mechanism of traditional Chinese medicine and their active constituents in postmenopausal osteoporosis].

Postmenopausal osteoporosis is one of the commonest systemic bone metabolism diseases among menopausal women, mainly caused by lowering internal estrogen. Although Hormone Replacement Therapy (HRT) is an effective method in clinical practice for years, it shows side-effect in increasing gynecological carcinoma. It has already been proved by clinical tests that multiple traditional Chinese medicine formulas and their monomer ingredients and phytoestrogen-like active constituents contained in traditional Chinese medicines are effective on treating osteoporosis with relatively less side-effects comparing with HRT. They show protective and therapeutic effects by acting on estrogen receptors of targeted tissues and targeted cells and then affecting expressions of bone metabolism-related regulatory proteins and factors in downstream signal conduct paths. Recent studies on estrogen related receptor (ERR) provide new possibilities and pathways for mechanism of traditional Chinese medicine and their active constituents in osteoporosis.