Exploration on Effect of Yinhuotang on Depression-like Behavior in Mice with Bilateral Ovariectomy Induced by Chronic Stress Based on E2/ERβ Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effect of Yinhuotang （YHT） on the depression-like behavior of mice with bilateral ovariectomy （OVX） induced by chronic stress and explore its action mechanism based on estradiol （E₂）-estrogen receptor <italic>β </italic>（ER<italic>β</italic>） pathway. Method:The experiment was divided into two parts. In the first part， mice were randomly divided into the sham operation （Sham） group， model （OVX） group， positive drug （E₂， 0.13 mg·kg^-1） group， and YHT （23.4 g·kg^-1） group. The OVX model was reproduced by OVX combined with chronic unpredictable mild stress （CUMS）. On the 8th day after OVX， the mice in each group were exposed to CUMS and treated with drugs. The changes in immobility time， horizontal movement score， and vertical movement score of mice in each group were observed in forced swimming test （FST）， tail suspension test （TST） and open-field test （OFT）， respectively. Serum and brain E₂ levels were detected by enzyme-linked immunosorbent assay （ELISA）， the aromatizing enzyme （Cyp19） mRNA expression by real-time fluorescence polymerase chain reaction （Real-time PCR）， the expression of ER<italic>α</italic> and ER<italic>β</italic> in dentate gyrus of hippocampus by immunohistochemistry （IHC）， and the total ER<italic>α</italic> and ER<italic>β</italic> levels in the brain by Western blotting. In the second part， the mice were divided into the Sham group， OVX group， YHT group， and blocker （Y+B， 23.4 g·kg^-1+100 μg·kg^-1） group. Mice in the Y+B group were first treated with intragastric administration of YHT and then with intraperitoneal injection of ER<italic>β</italic> blocker （PHTPP） on the next day. The changes in immobility time， horizontal motor score， and vertical motor score were observed in the three behavioral tests. Result:Compared with the Sham group， the OVX group displayed significantly increased immobility time， decreased horizontal and vertical movement scores （<italic>P</italic><0.01）， down-regulated serum and brain E₂levels （<italic>P</italic><0.01） and Cyp19 mRNA expression in the brain （<italic>P</italic><0.01）， and up-regulated total ER<italic>β</italic> in dentate gyrus and brain （<italic>P</italic><0.01）. However， there was no significant change in total ER<italic>α</italic> expression in the dentate gyrus or brain. As revealed by comparison with the OVX group， the immobility time of the E₂ group was decreased significantly， while the horizontal and vertical movement scores were increased significantly （<italic>P</italic><0.01）. The E₂ levels in the serum was significantly elevated （<italic>P</italic><0.01）. The Cyp19 mRNA expression in the brain and the total ER<italic>α</italic> expression in the dentate gyrus and brain were not significantly changed， while the expression levels of total ER<italic>β</italic> in dentate gyrus and brain were significantly increased （<italic>P</italic><0.01）. In the YHT group， the immobility time declined significantly， and the horizontal and vertical movement scores rose significantly （<italic>P</italic><0.01）. The serum E₂ did not increase， whereas the brain E₂ increased significantly （<italic>P</italic><0.01）. The expression of Cyp19 gene in the brain was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. There was no significant change in the total ER<italic>α</italic> of dentate gyrus and brain， but the expression levels of total ER<italic>β</italic> in dentate gyrus and brain were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. PHTPP reversed the effects of YHT on OVX mice in FST， TST and OFT. Conclusion:YHT promotes the synthesis and release of endogenous estrogen in brain and improves the depression-like behavior of OVX mice induced by chronic stress， which is possibly related to the activation of E₂/ER<italic>β</italic> pathway.

Anti-depressant Mechanism of Qing' ewan in CUMS Rats Based on Estrogen Receptor Pathway / 中国实验方剂学杂志

Objective::To study the anti-depressive effect of Qing' ewan in treating chronic unpredictable mild stress (CUMS) in rats, and the regulatory effect on estrogen receptor and estrogen receptor-related signaling pathways, in order to explore its anti-depressive mechanism. Method::The CUMS model was established. The experiment was divided into normal control group, model group, escitalopram oxalate group (positive control) and Qing' ewan groups (1.71, 5.13, 15.39 g·kg-1). After 4 weeks of modeling, rats were treated with corresponding drugs for 2 weeks. Behavioral evaluation [sucrose preference test (SPT), forced swimming test (FST), open field test (OFT)] was conducted to assess if the CUMS model was successful. Western blot was used to analyze the protein expression levels of estrogen receptor α (ERα), estrogen receptor β (ERβ), brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB). Result::Compared with the normal group, the sucrose consumption rate and the score of OFT in the model group decreased(P<0.05, P<0.01), the immobility time of FST prolonged significantly(P<0.01), and the protein expression levels of ERα, ERβ, BDNF and TrkB decreased(P<0.05, P<0.01). Compared with the model group, the behavioral performance of the treated group was improved, the sucrose consumption rate and the score of OFT increased(P<0.05, P<0.01), and the immobility time decreased(P<0.05). The protein expressions of ERα, ERβ, BDNF and TrkB in the treated group were significantly up-regulated(P<0.05, P<0.01), especially the middle-dose Qing' ewan group (5.13 g·kg-1). Conclusion::Qing' ewan can improve depression-like behavior in CUMS rats. Its mechanism may be related to the neuroprotective effect by up-regulating the expressions of ERα and ERβ and activating estrogen receptor-mediated ERβ/BDNF/TrkB pathways.

Effects of estrogen receptor antagonist on the expressions of Erα, Erβ and p57kip2 in JEC cells of human endometrial carcinoma / 临床与实验病理学杂志

Purpose To explore the effects of estrogen receptor antagonist on the expression of estrogen receptor subtype (ERα, ERβ), and p57kip2 protein in human endometrioid carcinoma cells named JEC. Methods The JEC cells (moderately differentiated EC cells) cultured in vitro were treated with β-Estradiol (E2) (10～6 mol/L) and two types of estrogen receptor antagonists, tamoxifen (TAM) and fulvestrant (ICI182780) (10-6 mol/L). After 24, 48, 72 h, MTT was used to detect the growth condition of JEC cells, and the light microscopy and electron microscopy were used to observe the growth condition and morphological changes of cells, Western blot was used to detect the expression of ERα, ERβ, PR-A, PR-B and P57kip2 protein in JEC cells. Results MTT results: Compared with the control group, E2 could promote the proliferation of JEC cells significantly (P＜0.05), and ICI182780 could inhibit the proliferation of JEC cells obviously (P＜0.05). Compared with the E2 group, the proliferation ability of JEC cells in E2 + ICI182780 group were lower(P＜0.05). Morphological change: Compared with the control group, the cells density of E2 group increased obviously, and the pathologic mitosis was easy to seen in some cells. The cells density decreased obviously in ICI182780 group. Compared with E2 group, the cells density of E2 + TAM group and E2 + ICI182780 group were decreased, and pathological mitotic figures were difficult to seen. Western blot results: Compared with the control group, the expression of ERβ protein increased, and the expression of p57kip2 protein decreased in E2 group (P＜0.05). The expression of ERβ protein decreased, and the expression of p57kip2 protein increased in ICI182780 group and TAM group, and the difference was statistically significant between ICI182780 group and control group (P＜0.05). Compared with the E2 group, the expression of ERβ protein decreased, and the expression of p57kip2 protein increased in E2 + ICI182780 group and E2 + TAM group, and the difference was statistically significant between E2 + ICI182780 group and E2 group (P＜0.05). ERa protein of JEC cells did not expressed in experimental group or control group. Conclusion ERa protein are not expressed in JEC cells. ICI182780 have a stronger role in antagonizing estrogen, and may induce the expression of p57kip2 protein by down-regulating the expression of ERβ protein in JEC cells, block the cell cycle progression and inhibit the growth of tumor cells. TAM has a weaker estrogen like effect on the growth of JEC cells. It is possible that combined detection of the expression of ERa and p57kip2 protein in EC has an important reference value for individualized selection of endocrine therapy for EC patients.

Bidirectional regulation of angiogenesis by phytoestrogens through estrogen receptor-mediated signaling networks / 中国天然药物

Sex hormone estrogen is one of the most active intrinsic angiogenesis regulators; its therapeutic use has been limited due to its carcinogenic potential. Plant-derived phytoestrogens are attractive alternatives, but reports on their angiogenic activities often lack in-depth analysis and sometimes are controversial. Herein, we report a data-mining study with the existing literature, using IPA system to classify and characterize phytoestrogens based on their angiogenic properties and pharmacological consequences. We found that pro-angiogenic phytoestrogens functioned predominantly as cardiovascular protectors whereas anti-angiogenic phytoestrogens played a role in cancer prevention and therapy. This bidirectional regulation were shown to be target-selective and, for the most part, estrogen-receptor-dependent. The transactivation properties of ERα and ERβ by phytoestrogens were examined in the context of angiogenesis-related gene transcription. ERα and ERβ were shown to signal in opposite ways when complexed with the phytoestrogen for bidirectional regulation of angiogenesis. With ERα, phytoestrogen activated or inhibited transcription of some angiogenesis-related genes, resulting in the promotion of angiogenesis, whereas, with ERβ, phytoestrogen regulated transcription of angiogenesis-related genes, resulting in inhibition of angiogenesis. Therefore, the selectivity of phytoestrogen to ERα and ERβ may be critical in the balance of pro- or anti-angiogenesis process.

Molecular mechanism for inhibitory effect of puerarin on IL-6 secretion in human osteoblast-like MG-63 cells / 中国药理学通报

Aim To investigate the molecular mecha-nism for the inhibitory effect of puerarin on IL-6 secre-tion in human osteoblast-likeMG-63 cells. Methods According to our previous studies, human osteoblast-like MG-63 cells containing two estrogen receptor ( ER) isoforms are a suitable model for this study. En-zyme-linked immnosorbent assay (ELISA), RT-PCR, luciferase reporter assay and small interfering RNA were performed to investigate the effect of puerarin on IL-6 expression in osteoblast-derived cells and underly-ing molecular mechanism. Results Puerarin could obviously inhibit IL-6 expression and IL-6 promoter ac-tivity by human osteoblastic MG-63 cells. Treatment with the ER antagonist ICI 182,780 abrogates the a-bove actions of puerarin on osteoblast-derived cells. U-sing siRNA technology, we further demonstrated that the effects of puerarin on IL-6 production were media-ted by ERα. Conclusion The effect of puerarin on IL-6 production in osteoblast is mediated by ERα.

Construction and identification of ERβ419 gene silencing recombined vectors of beagle ERβisoforms / 中国比较医学杂志

Objective To construct and identify retroviral-mediated short hairpin RNA ( shRNA ) expression vectors of ERβ419, and explore ERβ419 unknown biological function in beagles in future.Methods To screen out the most effective gene silencing sequence of beagle ERβ419 mRNA using qRT-PCR and Western Blot assays, imitate beagle estrogen target cells.Results qRT-PCR results showed, ERβ419-shRNA1 ( P <0.01 ) and ERβ419-shRNA3 ( P <0.01)differed significantly, Western Blot result as same as qRT-PCR,ERβ419-shRNA3 is the best choice.Conclusion Beagles ERβ419-shRNA3 retrain most effectively target gene repression. It is applied to explore ERβ419 unknown biological function in beagles reproductive system, and to prevent and treat beagles reproductive function diseases.