The ancient Chinese formula Longdan Xiegan Tang improves antipsychotic-induced hyperprolactinemia by repairing the hypothalamic and pituitary TGF-ß1 signaling in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Antipsychotics often induce hyperprolactinemia. The transforming growth factor (TGF)-beta1 signaling in the pituitary and hypothalamus inhibits prolactin synthesis and secretion, and its impairment is implicated in neuropsychiatric disorders. Longdan Xiegan Tang (LXT) alone or together with antipsychotics have been used to treat various neuropsychiatric diseases and hyperprolactinemia-associated disorders. AIM OF THE STUDY: To investigate the effect of LXT on hyperprolactinemia and involvement of the TGF-beta1 signaling. MATERIALS AND METHODS: Male rats were co-administered with olanzapine (5 mg/kg) and LXT extract (50 and 500 mg/kg) (p.o., × 8 weeks). Plasma concentrations of prolactin and TGF-beta1 were determined by ELISA. Protein expression was analyzed by Western blot. RESULTS: Treatment of rats with LXT extract suppressed olanzapine-induced increase in plasma prolactin concentration and overexpression of pituitary and hypothalamic prolactin protein. Importantly, LXT restored olanzapine-induced decrease in protein expression of the key components of the TGF-beta1 signaling, TGF-beta1, type II TGF-beta receptor, type I TGF-beta receptor and phosphorylated SMAD3 in the pituitary and hypothalamus. Further, it antagonized downregulation of pituitary and hypothalamic dopamine D2 receptor (D2R) protein level, and inhibited pituitary estrogen receptor (ER) alpha and ERbeta protein expression. CONCLUSIONS: The present results suggest that LXT ameliorates antipsychotic-induced hyperprolactinemia in rats by repairing the pituitary and hypothalamic TGF-beta1 signaling possibly via D2R, ERs or/and other pathways. Our findings may also provide scientific elucidation for use of the ancient Chinese formula to treat the impaired TGF-beta1 signaling-associated neuropsychiatric disorders.

Effects of acupuncture on the levels of serum estradiol and pituitary estrogen receptor beta in a rat model of induced super ovulation.

AIMS: Acupuncture is frequently recommended as a complementary therapy for infertility. However, whether acupuncture can prevent early ovarian hyperstimulation syndrome has not been examined and its potential mechanisms are not well understood. MAIN METHODS: Forty rats were randomized into four groups: Control, Ovarian Stimulation Model, Acupuncture, and Human Chorionic Gonadotropin (HCG). Serum estradiol, progesterone, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels were measured by enzyme-linked immunosorbent assay. Pituitary ER mRNA and ERß expression were detected by real-time PCR and western blotting respectively. The pathology of rat ovaries were observed by light microscopy. KEY FINDINGS: We observed significantly lower estradiol levels in the Acupuncture group than in the Model group and increased LH levels in the HCG group than in Model and Acupuncture groups. Testosterone and FSH levels were significantly lower in the Acupuncture group than in the HCG group. Western blotting showed significantly lower pituitary ERß expression in the Model group than in the Control group and higher expression in the Acupuncture group than in the Model group. Real-time PCR showed lower pituitary ER mRNA expression in the Acupuncture group than in the Model group. Hematoxylin and eosin staining showed a lower proportion of atretic follicles in Acupuncture and HCG groups than in Model and Control groups. Instead, Acupuncture and HCG groups showed growing and mature follicles. SIGNIFICANCE: Our results demonstrate a relationship between acupuncture and the hypothalamic-pituitary-gonadal axis, and the potential mechanism underlying the preventative effects of acupuncture on the incidence of early ovarian hyperstimulation syndrome.

Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation.

BACKGROUND: Phytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 µM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts. METHODS: Thus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed. RESULTS: Exposure to the combination of 100 µM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells. CONCLUSION: From these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.

Hyperoxia-induced changes in estradiol metabolism in postnatal airway smooth muscle.

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.

Antidepressant effect of Stillen.

Stillen has been used to treat patients with gastric mucosal ulcers and has an anti-inflammatory effect. It is well-known that neuro-inflammatory reactions are related to depression. Here we evaluated the antidepressant-like effect of Stillen on mice subjected to the forced swimming test (FST). Stillen and eupatilin (a major component of Stillen) significantly decreased immobility times compared with the FST control group. In the Stillen-administered group, increased levels of 5-hydroxytryptamine (serotonin) and brain-derived neurotrophic factor protein were observed in the hippocampus. Nissl bodies also increased in the hippocampus neuronal cytoplasm of the Stillen-administered group. Stillen decreased levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (at the mRNA and protein levels) in the hippocampus and serum, compared with the control group. In addition, the mRNA expression of estrogen receptor-ß increased after Stillen administration in the hippocampus. These findings suggest that Stillen should be viewed as a candidate antidepressant.

Effects of the phytoestrogen, genistein, and protein tyrosine kinase inhibitor-dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles.

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P(4)) and estradiol (E(2)) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and ß (ERα and ERß) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P(4) secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P(4) secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E(2). In contrast, lavendustin C did not alter basal E(2) secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERß (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERß. However, the genistein action on P(4) and E(2) production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.

Soy isoflavones administered pre- and postnatally may affect the ERα and ERß expression and elements' content in bones of mature male rats.

The aim of this study was to assess the influence of soy isoflavones, administered pre- and later postnatally, on the estrogen receptor α (ERα) and ß (ERß) expression in bones and to examine the mineral metabolism of the skeletal system in male rats. In bones, ERs were examined with an immunohistochemical method; in blood, estradiol with chemiluminescence immunoassay and in blood and bones, calcium and magnesium with atomic absorption spectrometry and fluorides with a potentiometric method were examined. Decreased immunoexpression of ERα and the increased intensity of immunofluorescence of ERß in osteocytes in the femur of experimental rats were observed. In the serum of treated rats, a significantly higher concentration of estradiol and lower calcium were observed. The content of magnesium and fluoride were significantly higher in the bones of the examined animals. The data presented show that pre- and postnatal supplementation of male rats with soy isoflavones may considerably increase the concentration of estrogens in serum, with a concurrent effect on the mineral composition of bones.

Differential expression of androgen and estrogen receptors in PCB (Aroclor 1254)-exposed rat ventral prostate: impact of alpha-tocopherol.

Polychlorinated biphenyls (PCBs) are environmental toxicants, which affect male fertility by altering the androgen and estrogen levels. PCB-induced toxic manifestations are associated with the production of reactive oxygen species. Vitamin E (α-tocopherol) is a major lipophilic chain breaking antioxidant, which protects polyunsaturated fatty acids in tissues against peroxidation, a property that could be beneficial in the male reproductive biology. The purpose of this study was to determine the impact of α-tocopherol on PCB (Aroclor 1254)-induced changes in androgen receptor (AR) and estrogen receptors (ERs) expression in Wistar rat ventral prostate. Rats were divided into 3 groups of 6 animals each. Group I rats were administered corn oil (vehicle) intraperitoneally (i.p.); Group II rats were treated with 2 mg kg(-1)day(-1) of PCB (i.p.); Group III rats were treated with 2 mg kg(-1)day(-1) of PCB (i.p.) along with simultaneous oral supplementation of 50 mg kg(-1)day(-1) of α-tocopherol. Serum testosterone and estradiol titers were assayed. Prostatic acid phosphatase activity (PAcP), citric acid concentration, generation of hydrogen peroxide (H(2)O(2)) and lipid peroxides (LPO) were estimated. mRNA and protein expression of AR, ER-α and ER-ß in ventral prostate were quantified. Serum testosterone, estradiol, PAcP, citric acid levels, AR and ER-α expressions were significantly decreased while H(2)O(2) generation, LPO, ER-ß were increased in PCB-exposed animals. Simultaneous supplementation of α-tocopherol in PCB-exposed rats resulted in significant restoration of all the parameters to the control. The results suggest that α-tocopherol has definite protective effect against PCB-induced toxicity in ventral prostatic dysfunction.

Estrogen receptor α and ß expressions in hypothalamus-pituitary-ovary axis in rats exposed lactationally to soy isoflavones and bisphenol A.

OBJECTIVES: This paper aims to investigate the uterotrophic activities of lactational exposure to combination of soy isoflavones (SIF) and bisphenol A (BPA) and to examine estrogen receptor α (ERα) and estrogen receptor ß (ERß) expressions in hypothalamus-pituitary-ovary axis and uterus. METHODS: Maternal rats that were breeding about 8 litters were randomly divided into four groups with seven dams in each group. Dams in different treatment groups received corn oil (control), 150 mg/kg BW of SIF, 150 mg/kg BW of BPA or combination of 150 mg/kg BW of SIF and 150 mg/kg BW of BPA, respectively, from postnatal day 5 to 11 (PND5-11) by gavage. On PND12 and PND70, 10 female litters were killed and hypothalamus, pituitary, ovary and uterus were collected. ERα and ERß expressions in these organs were detected with Western blotting assay. And vaginal opening time and estrus cycle were examined in animals fed for PND70. RESULTS: On PND12, the relative uterine weight of rats treated with ISF or BPA or their combination was significantly higher than that of untreated rats (P<0.05). But the relative uterine weight of rats in the co-exposure group was slightly lower than that in the group only exposed to SIF or BPA. On PND 70, however, the relative uterine weight in each treatment group was not statistically different from that in the control group (P>0.05). Vaginal opening time and estrus cycle in groups treated with SIF or BPA or their combination were similar to those in the control group (P>0.05). Exposure to SIF or BPA or their combination could up-regulate or down-regulate ERα and ERß expressions in hypothalamus, pituitary, ovary and uterus on PND12 and PND70. These regulation patterns for ERα and ERß were different in different organs at different time points. CONCLUSION: Lactational exposure to ISF or BPA or their combination could induce uterotrophic responses in neonate rats, which disappeared in later life. But these data fail to suggest a possibility for synergic actions between SIF and BPA. It was also demonstrated that the uterotrophic effects of SIF and BPA exposure might, at least, involve modification of ERα or ERß expressions in the hypothalamus-pituitary-ovary axis.

Effects of phytoestrogen daidzein and estradiol on steroidogenesis and expression of estrogen receptors in porcine luteinized granulosa cells from large follicles.

The aims of the study were to compare the in vitro effects of daidzein or 17beta-estradiol (E(2)) on: 1) progesterone (P(4)) secretion by luteinized granulosa cells harvested from large porcine follicles, as well as 2) estrogen receptor alpha and beta (ERalpha and ERbeta) mRNA and protein expression in the cells. In addition, the effect of daidzein on E(2) secretion and viability of the granulosa cells was examined. We found that basal and gonadotropin-stimulated P(4) secretion were inhibited in granulosa cells cultured in the presence of daidzein either for 24 or 48 hours. In contrast to daidzein, E(2) reduced P(4) secretion only during 24-hour cell cultures increasing it during longer cultures. Daidzein did not affect E(2) secretion by granulosa cells. The expression of ERalpha and ERbeta mRNA, as well as ERbeta protein, was up-regulated by daidzein but unaffected by E(2). To conclude, the soy estrogen daidzein acts directly on the porcine ovary to decrease progesterone production and to increase expression of ERbeta mRNA and protein. Daidzein actions in porcine luteinized granulosa cells differ from those of estradiol and it may suggest disadvantageous effects of the phytoestrogen on reproductive processes in females.

Photoperiod affects estrogen receptor alpha, estrogen receptor beta and aggressive behavior.

Estrogens have important effects on male and female social behavior. Despite growing knowledge of the anatomy and behavioral effects of the two predominant estrogen receptor subtypes in mammals (ERalpha and ERbeta), relatively little is known about how these receptors respond to salient environmental stimuli. Many seasonally breeding species respond to changing photoperiods that predict seasonal changes in resource availability. We characterized the effects of photoperiod on aggressive behavior in two species of Peromyscus that exhibit gonadal regression in short days. P. polionotus (old field mice) were more aggressive than P. maniculatus (deer mice) and both species were more aggressive in short days. We used immunocytochemistry and real-time polymerase chain reaction to characterize the effects of photoperiod on ERalpha and ERbeta expression. In both species ERalpha-immunoreactive staining in the posterior bed nucleus of the stria terminalis (BNST) was increased in short vs. long days. Both species had reduced ERbeta-immunoreactive expression in the posterior BNST in short days. In the medial amygdala ERbeta immunoreactivity was increased in long days for both species. Using real-time polymerase chain reaction on punch samples that included the BNST, we observed that ERalpha mRNA was increased and ERbeta mRNA was decreased in short days. These data suggest that the effects of photoperiod on ERalpha and ERbeta expression may thus have important behavioral consequences.

Cancer-related overexpression of the peripheral-type benzodiazepine receptor and cytostatic anticancer effects of Ginkgo biloba extract (EGb 761).

The peripheral-type benzodiazepine receptor (PBR) is an 18-kDa high affinity drug- and cholesterol-binding protein that is involved in various cell functions, including cell proliferation and apoptosis. PBR was shown to be overexpressed in certain types of malignant human tumors and cancer cell lines, correlating with enhanced tumorigenicity and cell proliferation rates. The present study was conducted in order to further define the role of PBR in cancer and to extend our recent findings regarding the possible anticancer effects of the standardized Ginkgo biloba extract EGb 761. Treatment with EGb 761 decreased PBR mRNA levels and inhibited the proliferation of breast, glioma and hepatocarcinoma cell lines, further corroborating our previous contention that its mechanism of action is through the modification of PBR expression. In vivo treatment with Ginkgo biloba extract led to dose-dependent decreases in xenograft growth of both MDA-MB-231 breast cancer and U-87 glioma cell lines in nude mice, although the effects were not maintained after 50 days of treatment in the latter. The results obtained in MDA-MB-231 xenografts indicated pronounced inhibition of tumor growth, verified by MRI imaging. These results were obtained using a modified experimental protocol where the animals were treated with the extract before cell inoculation. Although an exact role for PBR in relation to the initiation and progression of various types of cancer remains to be defined, our results indicate that PBR overexpression in certain cancer cells is related to an aggressive phenotype. Since EGb 761 treatment opposes this aggressive phenotype by decreasing PBR overexpression, it could be useful in preventing or treating cancer invasiveness and metastasis.

Comparative effect of seeds of Rhynchosia volubilis and soybean on MG-63 human osteoblastic cell proliferation and estrogenicity.

The seeds of Rhynchosia volubilis (SRV) (Leguminosae) and soybean have been used in oriental folk medicine to prevent postmenopausal osteoporosis. Their beneficial effects are caused by a high content of isoflavone, which function as partial agonists or antagonists of estrogen. To compare the estrogenic effects of SRV and soybean on the MG-63 osteoblastic cell proliferation, 70% methanol extracts of SRV or soybean were treated on MG-63 cells. Although biphasic over a concentration range of 0.001 mg/ml-0.1 mg/ml, both SRV and soybean extracts increased MG-63 cell proliferation. However SRV was more effective at increasing the cell proliferation that paralleled with the greater estrogenic effects as determined by estrogen receptor alpha (ERalpha) expression, an estrogenic response element (ERE)-luciferase activity and the selective expression of insulin-like growth factor-I (IGF-I). SRV-induced IGF-I expression resulted from increases in the mRNA levels. Despite the increased expression of ERbeta, ERE activity and IGF-I expression by soybean were lower than those by SRV. Furthermore, the comparable estrogenic effects between SRV and the combined treatment of genistein and daidzein standards at 0.5 x 10(-8) M, which is a concentration of these two isoflavones similar to that of SRV at 0.001 mg/ml, demonstrate that the greater estrogenicity of SRV for MG-63 cell proliferation is mediated by the synergism of low levels of isoflavones for the selective expression of IGF-I.