NMR Assignments of Six Asymmetrical N-Nitrosamine Isomers Determined in an Active Pharmaceutical Ingredient by DFT Calculations.

N-nitrosamines, which are well-known pro-mutagens, are found in drugs, pickled food and tobacco. Therefore, controlling their concentrations is very important. When an HPLC, GC or NMR analysis is conducted to investigate certain asymmetrical N-nitrosamines, two sets of signals attributed to the asymmetric N-nitrosamine isomers are usually observed. However, few reports on the NMR assignment of asymmetrical N-nitrosamine isomers have been published. In this study, we investigated the NMR assignments of the Z/E isomers of six asymmetrical N-nitrosamines by means of density functional theory (DFT) calculations. The configuration of the major isomer of asymmetrical N-nitrosamine 3 was the Z-configuration. The configuration of the major isomers of asymmetrical N-nitrosamines 4-7 was the E-configuration. Then, we determined the Z/E ratios of these asymmetrical N-nitrosamines by means of variable temperature (VT) and room temperature (RT) 1H-NMR experiments. The ratios of the Z/E isomer 3 quickly increased beyond 100% in the VT 1H NMR experiments. The ratios of Z/E isomers 4-7 were increased in the range of 10-60% in the VT 1H NMR experiments. The results of this study indicate that identifying the isomers of asymmetrical N-nitrosamine is necessary to control the quality of N-nitrosamines for active pharmaceutical ingredients (APIs).

[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.

[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.

Fructus Ligustri Lucidi modulates estrogen receptor expression with no uterotrophic effect in ovariectomized rats.

BACKGROUND: Accumulating evidence suggests that Fructus Ligustri Lucidi (FLL) plays a beneficial role in preventing the development of osteoporosis. However, the effects of FLL on estrogen receptor (ER) α and ERß expressions remain unknown. Therefore, in the current study we attempted to probe into the effects of FLL on ERα and ERß expressions in femurs, tibias and uteri of ovariectomized (OVX) rats. METHODS: The OVX rats were orally administrated with FLL water extract (3.5 g/kg/day) for 12 weeks. The uteri, femurs, tibias and serum were harvested from rats. The serum levels of estrogen (E2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by ELISA. The expressions of ERα and ERß in the femurs and tibias as well as uteri were analysed by western blot and immunohistochemical staining. RESULTS: FLL treatment did not increase uterus relative weight in OVX rats. Further, FLL treatment increased ERα expression in the femurs and tibias, and enhanced ERß expression in the uteri of OVX rats. However, the resulted expression of ERα was stronger than that of ERß in OVX rats in response to FLL treatment. Meanwhile, administration with FLL to OVX rats increased FSH and LH but did not increase E2 level in the serum. CONCLUSION: FLL treatment shows tissue selection on ERα and ERß expressions in the femurs and tibias as well as uteri of OVX rats without uterotrophic effect, which may offer the scientific evidence of the efficiency and safety of its clinical application.

Radix Salviae miltiorrhizae improves bone microstructure and strength through Wnt/ß-catenin and osteoprotegerin/receptor activator for nuclear factor-κB ligand/cathepsin K signaling in ovariectomized rats.

Although radix Salviae miltiorrhizae (RSM) is reported to exhibit the antiosteoporotic effect in preclinical study, the underlying mechanism is unclear. To this end, ovariectomized (OVX) rats were employed with administration of RSM (5 g/kg) for 14 weeks. The disturbed serum levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), tartrate-resistant acid phosphatase, and receptor activator of nuclear factor-κB ligand (RANKL) in OVX rats were improved by RSM treatment. Furthermore, supplement of RSM to OVX rats resulted in an increase in femoral bone mineral density and bone strength as well as an improvement in bone microstructures. Moreover, the decreased expression of phosphor (p)-LRP6, insulin-like growth factor-1(IGF-1), ALP, and OPG, as well as increased expression of RANKL and cathepsin K in the tibias and femurs of OVX rats were shifted by RSM treatment. Additionally, RSM reversed the decreased ratio of p-glycogen synthase kinase 3ß (GSK3ß) to GSK3ß and increased ratio of p-ß-catenin to ß-catenin in OVX rats. Altogether, it is suggestive that RSM improves bone quantity and quality by favoring Wnt/ß-catenin and OPG/RANKL/cathepsin K signaling pathways in OVX rats thereby suggesting the potential of this herb to be a novel source of antiosteoporosis drugs.

Diabetes Perturbs Bone Microarchitecture and Bone Strength through Regulation of Sema3A/IGF-1/ß-Catenin in Rats.

PURPOSE: Increasing evidence supported that semaphorin 3A (Sema3A), insulin-like growth factor (IGF)-1 and ß-catenin were involved in the development of osteoporosis and diabetes. This study is aimed to evaluate whether Sema3A/IGF-1/ß-catenin is directly involved in the alterations of bone microarchitecture and bone strength of diabetic rats. METHODS: Diabetic rats were induced by streptozotocin and high fat diet exposure. Bone microarchitecture and strength in the femurs were evaluated by micro-CT scanning, three-point bending examination and the stainings of HE, alizarin red S and safranin O/fast green, respectively. The alterations of lumbar spines microarchitecture were also determined by micro-CT scanning. Western blot and immunohistochemical analyses were used to examine the expression of Sema3A, ß-catenin, IGF-1, peroxisome proliferator-activated receptor γ (PPARγ) and cathepsin K in rat tibias. RESULTS: Diabetic rats exhibited decreased trabecular numbers and bone formation, but an increased trabecular separation in the femurs and lumbar spines. Moreover, the increased bone fragility and decreased bone stiffness were evident in the femurs of diabetic rats. Diabetic rats also exhibited a pronounced bone phenotype which manifested by decreased expression of Sema3A, IGF-1 and ß-catenin, as well as increased expression of cathepsin K and PPARγ. CONCLUSIONS: This study suggests that diabetes could perturb bone loss through the Sema3A/IGF-1/ß-catenin pathway. Sema3A deficiency in bone may contribute to upregulation of PPARγ and cathepsin K expression, which further disrupts bone remodeling in diabetic rats.

Cinnamaldehyde in diabetes: A review of pharmacology, pharmacokinetics and safety.

Cinnamaldehyde, one of the active components derived from Cinnamon, has been used as a natural flavorant and fragrance agent in kitchen and industry. Emerging studies have been performed over the past decades to evaluate its beneficial role in management of diabetes and its complications. This review highlights recent advances of cinnamaldehyde in its glucolipid lowering effects, its pharmacokinetics, and its safety by consulting the Pubmed, China Knowledge Resource Integrated, China Science and Technology Journal, National Science and Technology Library, Wanfang Data, and the Web of Science Databases. For the inquiries, keywords such as Cinnamon, cinnamaldehyde, property, synthesis, diabetes, obesity, pharmacokinetics, and safety were used in various combinations. Accumulating evidence supports the notion that cinnamaldehyde exhibits glucolipid lowering effects in diabetic animals by increasing glucose uptake and improving insulin sensitivity in adipose and skeletal muscle tissues, improving glycogen synthesis in liver, restoring pancreatic islets dysfunction, slowing gastric emptying rates, and improving diabetic renal and brain disorders. Cinnamaldehyde exerts these effects through its action on multiple signaling pathways, including PPARs, AMPK, PI3K/IRS-1, RBP4-GLUT4, and ERK/JNK/p38MAPK, TRPA1-ghrelin and Nrf2 pathways. In addition, cinnamaldehyde seems to regulate the activities of PTP1B and α-amylase. Furthermore, cinnamaldehyde has the potential of metalizing into cinnamyl alcohol and methyl cinnamate and cinnamic acid in the body. Finally, there is a potential toxicity concern about this compound. In summary, cinnamaldehyde supplementation is shown to improve glucose and lipid homeostasis in diabetic animals, which may provide a new option for diabetic intervention. To this end, further scientific evidences are required from clinical trials on its glucose regulating effects and safety.

Fructus Ligustri Lucidi in Osteoporosis: A Review of its Pharmacology, Phytochemistry, Pharmacokinetics and Safety.

Background: Fructus Ligustri Lucidi (FLL) has now attracted increasing attention as an alternative medicine in the prevention and treatment of osteoporosis. This study aimed to provide a general review of traditional interpretation of the actions of FLL in osteoporosis, main phytochemical constituents, pharmacokinetics, pharmacology in bone improving effect, and safety. Materials and Methods: Several databases, including PubMed, China National Knowledge Infrastructure, National Science and Technology Library, China Science and Technology Journal Database, and Web of Science were consulted to locate publications pertaining to FLL. The initial inquiry was conducted for the presence of the following keywords combinations in the abstracts: Fructus Ligustri Lucidi, osteoporosis, phytochemistry, pharmacokinetics, pharmacology, osteoblasts, osteoclasts, salidroside. About 150 research papers and reviews were consulted. Results: FLL is assumed to exhibit anti-osteoporotic effects by improving liver and kidney deficiencies and reducing lower back soreness in Traditional Chinese Medicine (TCM). The data from animal and cell experiments demonstrate that FLL is able to improve bone metabolism and bone quality in ovariectomized, growing, aged and diabetic rats through the regulation of PTH/FGF-23/1,25-(OH)2D3/CaSR, Nox4/ROS/NF-κB, and OPG/RANKL/cathepsin K signaling pathways. More than 100 individual compounds have been isolated from this plant. Oleanolic acid, ursolic acid, salidroside, and nuzhenide have been reported to exhibit the anti-osteoporosis effect. The pharmacokinetics data reveals that salidroside is one of the active constituents, and that tyrosol is hard to detect under physiological conditions. Acute and subacute toxicity studies show that FLL is well tolerated and presents no safety concerns. Conclusions: FLL provides a new option for the prevention and treatment of osteoporosis, which attracts rising interests in identifying potential anti-osteoporotic compounds and fractions from this plant. Further scientific evidences are expected from well-designed clinical trials on its bone protective effects and safety.

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.

Bioequivalence Analysis of Terazosin Hydrochloride Tablets Based on Parallel Artificial Membrane Permeability Analysis.

Parallel artificial membrane permeability analysis (PAMPA) is used to determine the permeability of compounds through concentrated negatively charged phospholipid bilayer barriers. We employed MacroFlux (a scaled-up version of PAMPA) to test the permeation rate of terazosin hydrochloride (TH) tablets and predict in vivo bioequivalence. The dissolution profiles and permeability of one reference formulation, and seven generic TH tablets, were compared. The dissolution profiles of these generic tablets were equivalent to that of the reference drug in four different media. However, the flux and the total permeated amount of some generic TH tablets were below the lower limit of the confidence interval of the original acceptance range in MacroFlux, which implied risk in the bioequivalence test in vivo. We further evaluated potential factors responsible for this discrepancy by µFlux, including active pharmaceutical ingredient (API) permeability and excipient prescriptions. The analysis showed that different properties of API were a main factor leading to biological inequivalence in the MacroFlux assay, while excipient prescriptions did not have an impact on bioequivalence risk. These data indicated that the flux assay may be a helpful as an auxiliary method for predicting bioequivalence of generic drugs and analyze the factors responsible for bioequivalence risk.

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 of zedoary oil for cat D and cat K expression in A549 cell line].

OBJECTIVE: To explore the Zedoary oil on A549 cell line of collagen deposition cat D and cat K expression. METHOD: The A549 cell line were treat by Zedoary oil on four different concentrations (0, 40, 80, 120 mg x L(-1)) in different time. Dynamic changes of collagen in A549 cell using Picric-sirius red method. Cat D and Cat K expression of level were detected by using western blot. RESULT: The collagen content showed that Zedoary oil had an inhibitory effect on the deposition of A549 cells. The results of western blot showed that the expression of cat D and cat K were up-regulated significangly in A549 cells of Zedoary oil groups compared with that in controls. CONCLUSION: A549 cell of collagen deposition were reduced by Zedoary oil. The effects may due to the up-regulation of cat D and cat K.

[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.

Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts.

BACKGROUND: Lung fibrosis is characterized by fibroblast proliferation and the deposition of collagens. Curcumin, a polyphenol antioxidant from the spice tumeric, has been shown to effectively counteract fibroblast proliferation and reducing inflammation and fibrotic progression in animal models of bleomycin-induced lung injury. However, there is little mechanistic insight in the biological activity of curcumin. Here, we study the effects of curcumin on the expression and activity of cathepsins which have been implicated in the development of fibrotic lung diseases. METHODS: We investigated the effects of curcumin administration to bleomycin stimulated C57BL/6 mice and human fetal lung fibroblasts (HFL-1) on the expression of cathepsins K and L which have been implicated in matrix degradation, TGF-ß1 modulation, and apoptosis. Lung tissues were evaluated for their contents of cathepsins K and L, collagen, and TGF-ß1. HFL-1 cells were used to investigate the effects of curcumin and cathepsin inhibition on cell proliferation, migration, apoptosis, and the expression of cathepsins K and L and TGF-ß1. RESULTS: Collagen deposition in lungs was decreased by 17-28% after curcumin treatment which was accompanied by increased expression levels of cathepsins L (25%-39%) and K (41%-76%) and a 30% decrease in TGF-ß1 expression. Moreover, Tunel staining of lung tissue revealed a 33-41% increase in apoptotic cells after curcumin treatment. These in vivo data correlated well with data obtained from the human fibroblast line, HFL-1. Here, cathepsin K and L expression increased 190% and 240%, respectively, in the presence of curcumin and the expression of TGF-ß1 decreased by 34%. Furthermore, curcumin significantly decreased cell proliferation and migration and increased the expression of surrogate markers of apoptosis. In contrast, these curcumin effects were partly reversed by a potent cathepsin inhibitor. CONCLUSION: This study demonstrates that curcumin increases the expression of cathepsins K and L in lung which an effect on lung fibroblast cell behavior such as proliferation, migration and apoptosis rates and on the expression of TGF-ß1 in mouse lung and HFL-1 cells. These results suggest that cathepsin-inducing drugs such as curcumin may be beneficial in the treatment of lung fibrosis.

[Study total glucosides of Radix Paeoniae Rubra induced K562 tumor cell apoptosis of signaling pathways and related gene changes].

OBJECTIVE: To study the total glucosides of Radix Paeoniae Rubra induced K562 tumor cell apoptosis of signaling pathways and related gene changes. METHOD: By MTT and flow cytometric, real-time quantitatie polymerase chain reaction (PCR) and Western blot methods researched the level of genes and proteins. RESULT: The total glucosides of Radix Paeoniae Rubra could inhibit K562 cell growth by MTT method, there was the relationship of concentration-time between them, TGC prompted K562 cell translocation of phosphatidylserine, may be apoptosis through non-receptor-dependent pathways because caspase-3 mRNA, caspase-9 mRNA and cytochrome C increased, caspase-8 mRNA had no significant change. The expression of Bcl-2 protein and Bcl-X1 protein could decreased, the expression of Bax protein could increased by regulating gene expression. CONCLUSION: TGC induced K562 cell apoptosis that might be through the mitochondria pathway, when cell apoptosis occured, Bcl-2 or Bcl-XI proteins combination of Bax protein would be displacement, then the mitochondrial membrane became permeable to release a series of material, then lead to cell death.

[Protective effects of Siwuheji on cisplatin induced ovarian dysfunction in mice].

OBJECTIVE: To evaluate the protective effects of siwuheji on cisplatin-induced ovarian impairing and explore its possible mechanisms in mice. METHOD: The mice received cisplatin ip (7 mg kg(-1)) for 7 days, while fed with siwuheji for 14 days. The changes of estrous cycle, ovary and uterus coefficient, levels of estrogen in serum and morphology of ovary were recorded. RESULT: From the third day of cisplatin injection, the estrous cycle of the model group stagnated in interphase. On the thirteenth day, part of the mice fed with siquheji started with change of estrous cycle. The ovary coefficient of the model group was less than the control group, the experimental groups were no differences compared with the control group and had significant differences with the model group. The levels of E2 and P of model group were lower than the control group, but no statistical significance. The level of E2 with high-dose Siwuheji group was higher than model group, and the level of P of the low-dose group was higher than model group, but both no statistical significances. The FSH/LH ratio of model group was significantly higher than the control group, low-dose group and medium-dose group of siwuheji resumed to normal levels, and had a significantly change compared with the model group. Ovarian biopsy of model group showed the decrease in the number of developing follicle at all levels, and increase in the number of Atresia of ovarian. There were more growing follicte in the large, medium-dose group. CONCLUSION: Cisplatin can induce mice ovarian injury and functional decline, on the other hand, siwuheji can improve cisplatin-induced ovarian dysfunction.

[Effect of ferulic acid on proliferation and mechanism in human breast cancer cells].

OBJECTIVE: To investigate phytoestrogenic effects of ferulic acid in ER-positive T47D and ER-negative MDA-MB231 cells in culture. METHOD: T47D and MDA-MB231 human breast cancer cells were treated with ferulic acid and examined cell proliferation by means of MTT assay. Cell cycle distribution, ERalpha and ERbeta expression were treated by flow cytometer. The pS2 mRNA expressions were detected by real-time fluorescence quantitative PCR. RESULT: The proliferations were enhanced significantly by treatment with ferulic acid on T47D cells and the proliferation effects were inhibited by adding Faslodex (1 x 10(-8) mol x L(-1)). However, there was no significant difference on the proliferation in MDA-MB-231 cells compared with solvent control group by both treatment with ferulic acid and co-treatment with Faslodex (1 x 10(-8) mol x L(-1)). Ferulic acid stimulated the amount of T47D cells in phase S and proliferation index increased significantly. The effects were inhibited by treatment with Faslodex (1 x 10(-8) mol x L(-1)), and the amount of cells in phase S and proliferation index decreased, the amount of cells in G0/G1 phase increased, cell cycle of T47D was arrested in G0/G1 phase. Ferulic acid up-regulated pS2 mRNA expressions and increased the level of ERalpha protein expression in T47D cells. Ferulic acid did not show remarkable effect to the level of ERbeta protein expression in T47D cells. CONCLUSION: Ferulic acid possessed phytoestrogenic effect by up-regulating pS2 gene expression and the receptor subtype of ERalpha.

[Research on phytoestrogenic effect of formononetin].

OBJECTIVE: Research on the phytoestrogenic effect and its possible mechanism of formononetin. METHOD: To evaluate the estrogenic effect and mechanisms of formononetin through the test of its influence on proliferation and ER subtype expression of T47D cells. RESULT: The proliferation rates of T47D cells treated with 1 x 10(-7) -1 x 10(-6) mol x L(-1) formononetin were not increased. On the influence of ICI182, 780, the proliferation rates of T47D cells treated with 1 x 10(-7) 1 x 10(-6) mol x L(-1) formononetin were decreased. Formonenetin could induce the augment of ERalpha expression significantly of T47D. CONCLUSION: Formonenetin has phytoestrogenic effect Formonenetin can not accelerate ER(+) T47D cell proliferation. But the expression level of ERalpha subtype in T47D cells change significantly with certain concentrations of formonenetin.

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.