17β-雌二醇对人成骨样MG-63细胞CTGF和PAIP-1基因表达的影响

目的 观察结缔组织生长因子（CTGF）和多聚腺苷酸结合蛋白相互作用蛋白1（PAIP-1）mRNA在MG-63细胞培养不同阶段的表达，研究17β-雌二醇（E2）对MG-63细胞CTGF和PAIP-1 mRNA表达的影响。方法 半定量RT—PCR检测MG-63细胞碱性磷酸酶（ALP）、骨钙素（OC）和Ⅰ型胶原mRNA的表达，半定量RT-PCR和Northern印迹方法检测MG-63细胞CTGF和PAIP-1 mRNA表达，改良的钙-钴法ALP染色、vail Gieson法Ⅰ型胶原染色和0．1％茜素红矿化结节染色。结果 （1）半定量RT-PCR和组织化学染色结果均证实，MG-63细胞接种培养后0～9d为细胞增殖阶段，7～18d为基质成熟阶段，17～18d后为基质矿化阶段；（2）在MG-63细胞培养的不同阶段，均检测到CTGF和PAIP-1 mRNA的表达，E2可显著下调MG-63细胞CTGF mRNA的表达，并呈剂量依赖关系（P〈0．01），但时间依赖性关系不明显。17β-E2对MG-63细胞PAIP-1 mRNA表达无明显作用（P〉0．05）。结论 E2可剂量依赖性下调MG-63细胞CTGF mRNA的表达，但对PAIP-1 mRNA表达无明显作用。     

Effects of 17β-estradiol on the expression of matrix metalloproteinase-1, -2 and tissue inhibitor of metalloproteinase-1 in human osteoblast-like cell cultures

Estrogen can effectively prevent estrogen deficiency-induced bone loss in animals and humans. However, its mechanism remains unknown. Osteoblast-derived Matrix metalloproteinse-1 (MMP-1), MMP-2, and tissue inhibitor of metalloproteinase-1 (TIMP-1) recently were implicated as playing important roles in initiating bone resorption. Therefore, we tested the effects of 17β-estradiol (E₂) on MMP-1, MMP-2, and TIMP-1 production in cultures of human osteoblastic MG-63 cells and normal human osteoblasts (hOB). MMP-1, MMP-2 and TIMP-1 concentrations in the culture medium were determined by ELISA, and activity of MMP-2 was assessed by ELISA. After 12–48 h of treatment, E₂ at 10⁻⁸M decreased MMP-1 level in cultures of MG-63 cells or hOB. Treatment with increasing dose of E₂ in MG-63 cells or hOB caused a dose-dependent decrease in MMP-1 synthesis. E₂ had no influence on MMP-2 and TIMP-1 production in MG-63 cells or hOB cultures, as well as activation of latent MMP-2. In conclusion, E₂ represses MMP-1 synthesis, and this effect may contribute to its action on the inhibition of bone resorption, followed by prevention of bone loss. Increasing MMP-1 production followed by estrogen deficiency may contribute to the mechanisms involved in postmenopausal osteoporosis.     

Effects of three MAPK inhibitors on the expressions of TGF-β1 and α-SMA mRNA and protein in LX-2 cells induced by sodium arsenite

<正>Objective To investigate the effects of three mitogen-activated protein kinase (MAPK) inhibitors on the expressions of transforming growth factor-β1 (TGF-β1),α-smooth actin (α-SMA) mRNA and protein in human liver stellate cells (LX-2 cells) activated by sodium arsenite. Methods LX-2 cells cultured in vitro in the     

Combined effects of rat Schwann cells and 17β-estradiol in a spinal cord injury model

Spinal cord injury (SCI) is a devastating traumatic event which burdens the affected individuals and the health system. Schwann cell (SC) transplantation is a promising repair strategy after SCI. However, a large number of SCs do not survive following transplantation. Previous studies demonstrated that 17β-estradiol (E2) protects different cell types and reduces tissue damage in SCI experimental animal model. In the current study, we evaluated the protective potential of E2 on SCs in vitro and investigated whether the combination of hormonal and SC therapeutic strategy has a better effect on the outcome after SCI. Primary SC cultures were incubated with E2 for 72 h. In a subsequent experiment, thoracic contusion SCI was induced in male rats followed by sustained administration of E2 or vehicle. Eight days after SCI, DiI-labeled SCs were transplanted into the injury epicenter in vehicle and E2-treated animals. The combinatory regimen decreased neurological and behavioral deficits and protected neurons and oligodendrocytes in comparison to vehicle rats. Moreover, E2 and SC significantly decreased the number of Iba-1+ (microglia) and GFAP⁺ cells (astrocyte) in the SCI group. In addition, we found a significant reduction of mitochondrial fission-markers (Fis1) and an increase of fusion-markers (Mfn1 and Mfn2) in the injured spinal cord after E2 and SC treatment. These data demonstrated that E2 protects SCs against hypoxia-induced SCI and improves the survival of transplanted SCs.

     

Insulinotropic and antidiabetic effects of 17β-estradiol and the GPR30 agonist G-1 on human pancreatic islets

We have recently shown that 17β-estradiol (E2) and the synthetic G protein-coupled receptor 30 (GPR30) ligand G-1 have antiapoptotic actions in mouse pancreatic islets, raising the prospect that they might exert beneficial effects also in human islets. The objective of the present study was to identify the expression of GPR30 in human islets and clarify the role of GPR30 in islet hormone secretion and β-cell survival. GPR30 expression was analyzed by confocal microscopy, Western blot, and quantitative PCR in islets from female and male donors. Hormone secretion, phosphatidylinositol hydrolysis, cAMP content, and caspase-3 activity in female islets were determined with conventional methods and apoptosis with the annexin-V method. Confocal microscopy revealed GPR30 expression in islet insulin, glucagon, and somatostatin cells. GPR30 mRNA and protein expression was markedly higher in female vs. male islets. An amplifying effect of G-1 or E2 on cAMP content and insulin secretion from isolated female islets was not influenced by the E2 genomic receptor (ERα and ERβ) antagonists ICI 182,780 and EM-652. Cytokine-induced (IL-1β plus TNFα plus interferon-γ) apoptosis in islets cultured for 24 h at 5 mmol/liter glucose was almost abolished by G-1 or E2 treatment and was not affected by the nuclear estrogen receptor antagonists. Concentration-response studies on female islets from healthy controls and type 2 diabetic subjects showed that both E2 and G-1 displayed important antidiabetic actions by improving glucose-stimulated insulin release while suppressing glucagon and somatostatin secretion. In view of these findings, we propose that small molecules activating GPR30 could be promising in the therapy of diabetes mellitus.     

Differential expression of CTGF in pre- and post-ovulatory granulosa cells in the hen ovary is regulated by TGFβ1 and gonadotrophins

Connective tissue growth factor (CTGF) is a cysteine-rich, matrix-associated heparin-binding protein that is important in many cell types as a regulator of cell proliferation, angiogenesis, cell remodelling and other cellular processes. CTGF is necessary for normal follicle growth and luteinisation in mammals. The avian follicular hierarchy provides an excellent experimental model to study developmental events, particularly the role of cellular remodelling factors in the process of folliculogenesis. In this study, we examined CTGF expression and regulation in the hen ovary. CTGF expression was increased considerably as follicular development proceeds in pre-ovulatory follicles, peaking in expression at the time of ovulation. Immunohistochemistry revealed that CTGF protein was concentrated in the cytoplasm of follicular granulosa cells throughout the ovulation cycle. We isolated granulosa cells from the follicles at two key stages of the ovulation cycle (in terms of cellular alteration): during pre-ovulatory growth and during post-ovulatory regression. Follicle-stimulating hormone (FSH) and luteinising hormone (LH) inhibited CTGF expression in pre-ovulatory granulosa cells but stimulated CTGF expression in post-ovulatory granulosa cells. Moreover, TGFβ1 stimulated CTGF expression in both pre- and post-ovulatory granulosa cells. Nevertheless, TGFβ1 could rescue the inhibition of gonadotrophins on pre-ovulatory granulosa CTGF expression but could not further stimulate CTGF expression in gonadotrophin-treated post-ovulatory granulosa cells. The results of this study indicate that CTGF expression in avian granulosa cells is modulated by a combination of gonadotrophins and TGFβ1 according to the different stages of follicle maturation and degradation. The results also suggest that the gonadotrophic action on post-ovulatory follicles in the avian ovary differs from the gonadotrophin-induced luteinisation in mammals.     

Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo marmorata exposed to 17β-estradiol

In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo marmorata exposed to 17β-estradiol (E₂). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E₂, synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E₂ treatment are discussed.     

Rapid and widespread effects of 17β-estradiol on intracellular signaling in the male songbird brain: a seasonal comparison

Across vertebrate species, 17β-estradiol (E(2)) acts on the brain via both genomic and nongenomic mechanisms to influence neuronal physiology and behavior. Nongenomic E(2) signaling is typically initiated by membrane-associated estrogen receptors that modulate intracellular signaling cascades, including rapid phosphorylation of ERK. Phosphorylated ERK (pERK) can, in turn, rapidly phosphorylate tyrosine hydroxylase (TH) and cAMP response element-binding protein (CREB). Recent data suggest that the rapid effects of E(2) on mouse aggressive behavior are more prominent during short photoperiods (winter) and that acute aromatase inhibition reduces songbird aggression in winter only. To date, seasonal plasticity in the rapid effects of E(2) on intracellular signaling has not been investigated. Here, we compared the effects of acute (15 min) E(2) treatment on pERK, pTH, and pCREB immunoreactivity in male song sparrows (Melospiza melodia) pretreated with the aromatase inhibitor fadrozole during the breeding and nonbreeding seasons. We examined immunoreactivity in 14 brain regions including portions of the song control system, social behavior network, and the hippocampus (Hp). In both seasons, E(2) significantly decreased pERK in nucleus taeniae of the amygdala, pTH in ventromedial hypothalamus, and pCREB in mesencephalic central gray, robust nucleus of the arcopallium, and caudomedial nidopallium. However, several effects were critically dependent upon season. E(2) decreased pERK in caudomedial nidopallium in the breeding season only and decreased pCREB in the medial preoptic nucleus in the nonbreeding season only. Remarkably, E(2) decreased pERK in Hp in the breeding season but increased pERK in Hp in the nonbreeding season. Together, these data demonstrate that E(2) has rapid effects on intracellular signaling in multiple regions of the male brain and also demonstrate that rapid effects of E(2) can be profoundly different across the seasons.     

Effects of bile acid conjugates on biliary excretion of estradiol-17β-glucuronide in the rat

Estradiol-17β-glucuronide is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. We previously reported that biliary estradiol-17β-glucuronide excretion was delayed in Eisai hyperbililubinemic rats (EHBR) and inhibited by sulfobromophthalein whereas dibromosulfophthalein had not effect on biliary estradiol-17β-glucuronide excretion. In the present study, we examined the effects of bile acid conjugates on biliary excretion of tracer doses of [³H]estradiol-17β-glucuronide and [¹⁴C]estradiol intravenously injected to rats. Biliary excretion of estradiol-17β-glucuronide and estradiol metabolites was significantly delayed by the infusion of taurolithocholate-3-sulfate or ursodeoxycholate-3-O-glucuronide, but not by ursodeoxycholate-3,7-disulfate. These findings indicate that estradiol-17β-glucuronide is excreted into bile at least partially by the canalicular multispecific organic anion transporter which is common for sulfobromophthaleinglutathione and some bile acid sulfates and glucuronides.     

Differential effects of 17β-estradiol and 11-ketotestosterone on the endocrine stress response in zebrafish (Danio rerio)

Sexually dimorphic stress responses are present in species across all vertebrate taxa and it has been suggested that these effects are mediated by circulating sex steroids. While a few species of fish have been identified as having a sexually dimorphic stress response, there is conflicting evidence as to the effects of sex steroids on the stress axis. In this study, we tested whether zebrafish exhibit a sexually dimorphic cortisol stress response and whether 17β-estradiol (E2) or 11-ketotestosterone (11KT) modulate the activity of the hypothalamic-pituitary-interrenal (HPI) axis. To accomplish this, we quantified the whole body cortisol response to a physical stressor, cortisol release in vitro, and the expression of key HPI axis regulating genes of control and E2- or 11KT-exposed zebrafish. Under control conditions no dimorphisms in the HPI axis were apparent at rest or in response to a standardized stressor. In contrast, E2-exposure blunted the cortisol response of male fish in vivo and in vitro and as well as corticotropin-releasing factor (crf) expression in the pre-optic area (POA) of the brain. While the expression of some interrenal genes was suppressed by E2-exposure, these changes occurred in both male and female zebrafish. 11KT-exposure increased whole-body cortisol of males at rest and vortex-exposed females, but had no impact on the rate of cortisol synthesis in vitro or on POA crf expression. Therefore, while we found no evidence that zebrafish exhibit a sexually dimorphic cortisol stress response, both E2 and 11KT can modulate the activity of the HPI axis in this species and do so via different mechanisms.     

Acute effects of 17β-estradiol and genistein on insulin sensitivity and spatial memory in aged ovariectomized female rats

Aging is characterized by decline in metabolic function and insulin resistance, and both seem to be in the basis of neurodegenerative diseases and cognitive dysfunction. Estrogens prevent age-related changes, and phytoestrogens influence learning and memory. Our hypothesis was that estradiol and genistein, using rapid-action mechanisms, are able to modify insulin sensitivity, process of learning, and spatial memory. Young and aged ovariectomized rats received acute treatment with estradiol or genistein. Aged animals were more insulin-resistant than young. In each age, estradiol and genistein-treated animals were less insulin-resistant than the others, except in the case of young animals treated with high doses of genistein. In aged rats, no differences between groups were found in spatial memory test, showing a poor performance in the water maze task. However, young females treated with estradiol or high doses of genistein performed well in spatial memory task like the control group. Only rats treated with high doses of genistein showed an optimal spatial memory similar to the control group. Conversely, acute treatment with high doses of phytoestrogens improved spatial memory consolidation only in young rats, supporting the critical period hypothesis for the beneficial effects of estrogens on memory. Therefore, genistein treatment seems to be suitable treatment in aged rats in order to prevent insulin resistance but not memory decline associated with aging. Acute genistein treatment is not effective to restore insulin resistance associated to the early loss of ovarian function, although it can be useful to improve memory deficits in this condition.     

Effects of c-myb antisense RNA on TGF-β1 and α1-I collagen expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA oncell proliferation and the expression of c-myb,TGF-131 andα1-I collagen in cultured hepatic stellate cells (HSC) from rats.METHODS:Recombinant retroviral vector of c-myb antisensegene (pDOR-myb) was constructed,and then transfectedinto retroviral package cell line PA317 by means of DOTARThe pseudoviruses produced from the resistant PA317 cellswere selected with G418 to infect HSCs isolated from ratlivers.The cell proliferation was measured by 3-[4,5-Dimethylthiazolzyl]-2,5-diphenyl tetrazo-dium bromide(MIT) method.The expression of c-myb,α_1-I collagen andTGF-β1 mRNA,and c-myb protein in HSCs was detectedwith semi-quantitive reverse transeription-polymerase chainreaction (RT-PCR) and Western-blot respectively.RESULTS:HSCs from rats were isolated successfully withthe viability>98%.In the pDOR-myb infected HSCs,the c-myb protein expression,cell proliferation,and α_1-I collagenand TGF-β1 mRNA expression were repressed significantlycompared with their corresponding control groups (P<0.01).CONCLUSION:c-myb plays a key role in activation andproliferation of HSC.c-myb antisense RNA can inhibit cellproliferation,α_1-I collagen and TGF-β1 mRNA expression,suggesting that inhibition of c-myb gene expression mightbe a potential way for the treatment of liver fibrosis.     

Effects of c-myb antisense RNA on TGF-β1 and α1-Ⅰ collagen expression in cultured hepatic stellate cells

AIM: To investigate the effects of c-myb antisense RNA on cell proliferation and the expression of c-myb, TGF-β1 and α1-Ⅰ collagen in cultured hepatic stellate cells (HSC) from rats.METHODS: Recombinant retroviral vector of c-myb antisense gene (pDOR-myb) was constructed, and then transfected into retroviral package cell line PA317 by means of DOTAP.The pseudoviruses produced from the resistant PA317 cells were selected with G418 to infect HSCs isolated from rat livers. The cell proliferation was measured by 3-[4,5-Dimethylthiazolzyl]-2, 5-diphenyl tetrazo-dium bromide (MTT) method.The expression of c-myb, α1-Ⅰ collagen and TGF-β1 rnRNA, and c-myb protein in HSCs was detected with semi-quantitive reverse transeription-polymerase chain reaction (RT-PCR) and Western-blot respectively.RESULTS: HSCs from rats were isolated successfully with the viability &gt;98%. In the pDOR-myb infected HSCs, the cmyb protein expression, cell proliferation,and α1-Ⅰ collagen and TGF-β1 mRNA expression were repressed significantly compared with their corresponding control groups (P&lt;0.01).CONCLUSION: c-myb plays a key role in activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation, α1-Ⅰ collagen and TGF-β1 mRNA expression,suggesting that inhibition of c-myb gene expression might be a potential way for the treatment of liver fibrosis.     

Effects of daidzein on estrogen-receptor-positive and negative pancreatic cancer cells in vitro

AIM:To study the effects of daidzein on human pancreaticcancer cells in vitro.METHODS:Human estrogen-receptor (ER)-positivepancreatic cancer cells MiaPaCa-2 and ER-negative pancreaticcancer cells PANC-1 were treated by 0.1 μmol/L,1μmol/L,10/μmol/L,25 μmol/L,50 μmol/L,75 μmol/L and 100 μmol/Lof daidzein,respectively.Its antiproliferative effect wasstudied by MTT assay.RESULTS:Daidzein inhibited the growth of MiaPaCa-2and PANC-1 cells at the concentrations from 0.1 μmol/L to100/μmol/L.A dose-and time-dependent manner was found.The IC_(50) of daidzein on MiaPaCa-2 and PANC-1 cells was45 μmol/L and 75 μmol/L,respectively.After MiaPaCa-2 cellswere treated by daidzein for 3 d and at the concentrationsmore than IC_(50),the inhibitory manner was identical and theinhibition appeared a saturation phenomenon,but theinhibitory manner of daidzein on PANC-1 cells was differentfrom that of MiaPaCa-2 cells.CONCLUSION:Daidzein has antiproliferative effects onhuman estrogen-receptor-positive and negative pancreaticcancer cells,but their mechanisms may be different.     

Suppressive effects of 17β-estradiol on hepaUc fibrosis in CCl_4-induced rat model

AIM:To investigate the pathway via which 17β-estradiol(β-Est) exerts suppressive effects on rat hepatic fibrosis.METHODS:In vivo study was done in CCl_4-induced femalehepatofibrotic rats.Fibrosis-suppressive effect of β-Est(20μg/kg·d) was evaluated in intact and ovariectomizedrat models.Six weeks after the treatment,all the ratswere sacrificed and specimens of serum or liver tissuewere collected for the studies.Serum liver enzymes,fibrosis markers and estradiol levels were determined bystandard enzymatic methods,ELISA and RIA,respectively.Degrees of fibrosis and areas of hepatic stellate cells(HSCs) positive for alpha-smooth muscle actin (α-SMA) inthe liver were determined by van Gieson (VG) stain andimmunohistochemistry.In vitro studies,HSCs were isolatedby a combination of pronase-collagenase perfusion anddensity gradient centrifugation.First-passage HSCs wererandomly divided into 10 groups,and different concentrationsof β-Est,2-hydroxyestradiol (2OHE) or 2-methoxyestradiol(2MeOE) were separately added to the cell groups.Afterincubation for 72h,the degree of cell proliferation,collagenproduction,α-SMA or estrogen receptor (ER) expression wasdetermined by MTI- assay,ELISA and immunohistochemistry,respectively.RESULTS:β-Est treatment reduced aspartate aminotransfer-ase (AST),alanine aminotransferase (ALT),hyaluronic acid(HA) and type Ⅳ collagen (C Ⅳ) in sera,suppressed hepaticcollagen content,decreased the areas of HSCs positive forα-SMA significantly in both intact and ovariectomized femalehepatofibrotic rats.There was a negative correlationbetween the percentage of fibrotic area of liver tissue andthe serum estradiol level;the calculated correlationcoefficient was -0.57 (P<0.01).β-Est and its metabolitesconcentration-dependently (10~(-9)mol/L-10~(-7)mol/L) inhibitedHSC proliferation and collagen synthesis.At the concentrationof 10~(-7)mol/L,they could inhibit α-SMA expression.Theorder of potency was 2MeOE>2OHE>β-Est.CONCLUSION:β-Est may suppress hepatic fibrosis probablyvia its biologically active metabolites.     

Effects of differential endothelial cells growth stage on the proliferation and migration of vascular smooth muscle cells

Endothelial injury and SMC proliferation and migration are the same common pathophysiological processes of many cardiovascular diseases such as artherosclerosis, hypertension, diabete and restenosis. So it is very important to determine their functional interaction under pathology conditions.     

Effects of smoking cessation on expressions of nuclear factor-κB, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in airway epithelial cells of smoking rats

Objective To investigate the effects of smoking and smoking cessation on airway inflammation and remodeling in chronic obstructive pulmonary diseases through detecting mRNA and protein expressions of nuclear factor-κB (NF-κB), cell matrix metalloproteinase-9 (MMP-9) and cellular tissue inhibitor of metalloproteinase-1 (TIMP-1) in airway epithelial cells of smoking and smoking cessation rats. Methods Twenty-four male Wistar rats were randomly divided into control group, smoking group and smoking cessation group,eight in each group. Hybridization in situ and immunohistochemistry were used to detect mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 in airway epithelial cells of rats. Results ① Compared with control group (0.29 ± 0.06,0.29±0.06), mRNA and protein expressions of NF-κB in smoking group (0.45±0.04,0.41±0.03) and smoking cessation group (0.40±0.05,0.37±0.03) were higher (all P＜0.05). The mRNA and protein expressions of NF-κB in smoking cessation group were lower than those in smoking group (all P ＜0.05). ②Compared with control group (0.30±0.06,0.30±0.06) ,mRNA and protein expressions of MMP-9 in smoking group (0.52±0.03,0.51±0.07) and smoking cessation group (0.38±0.03,0.33±0.02) were higher (all P＜0.05). The mRNA and protein expressions of MMP-9 in smoking cessation group were lower than those in smoking group (all P＜0.05). ③Compared with control group (0.26±0.04, 0.26±0.04), mRNA and protein expressions of TIMP-1 in smoking group (0.49±0.05,0.37±0.03) and smoking cessation group (0.42±0.04,0.35±0.03) were higher (all P ＜0.05). The mRNA and protein expressions of TIMP-1 in smoking cessation group were lower than those in smoking group (all P ＜ 0.05). ④ Compared with control group (1.00±0.02,1.00±0.02), MMP-9/TIMP-1 mRNA and protein expressions were larger than one in smoking group (1.07±0.14, 1.37±0.19), and less than one in smoking cessation group (0.92±0.13,0.94±0.10) (all P ＜0.05). ⑤The mRNA and protein expressions of NF-κB and MMP-9in each group were positively correlation (r=0.87,0.66,all P ＜0.05). Conclusions In airway epithelial cells of smoking rats, mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 increase, and MMP-9/TIMP-1 is larger than one. After stoping smoking, mRNA and protein expressions of NF-κB,MMP-9 and TIMP-1 decrease, and MMP-9/TIMP-1 is less than one. This experiment explains that smoking can cause airway inflammation and remodeling, smoking cessation can reduce airway inflammation and remodeling.