Role of estrogen receptor β selective agonist in ameliorating portal hypertension in rats with CCl4-induced liver cirrhosis

AIM: To investigate the role of diarylpropionitrile(DPN), a selective agonist of estrogen receptor β(ERβ), in liver cirrhosis with portal hypertension(PHT) and isolated hepatic stellate cells(HSCs).METHODS: Female Sprague-Dawley rats were ovariectomized(OVX), and liver cirrhosis with PHT was induced by CCl4 injection. DPN and PHTPP, the selective ERβ agonist and antagonist, were used as drug interventions. Liver fibrosis was assessed by hematoxylin and eosin(HE) and Masson's trichrome staining and by analyzing smooth muscle actin expression. Hemodynamic parameters were determined in vivo using colored microspheres technique. Protein expression and phosphorylation were determined by immunohistochemical staining and Western blot analysis. Messenger RNA levels were analyzed by quantitative real-time polymerase chain reaction(q RT-PCR). Collagengel contraction assay was performed using gel lattices containing HSCs treated with DPN, PHTPP, or Y-27632 prior to ET-1 addition. RESULTS: Treatment with DPN in vivo greatly lowered portal pressure and improved hemodynamic parameters without affecting mean arterial pressure, which was associated with the attenuation of liver fibrosis and intrahepatic vascular resistance(IHVR). In CCl4-treated rat livers, DPN significantly decreased the expression of Rho A and ROCK Ⅱ, and even suppressed ROCK Ⅱ activity. Moreover, DPN remarkedly increased the levels of endothelial nitric oxide synthase(e NOS) and phosphorylated e NOS, and promoted the activities of protein kinase G(PKG), which is an NO effector in the liver. Furthermore, DPN reduced the contractility of activated HSCs in the 3-dimensional stress-relaxed collagen lattices, and decreased the ROCK Ⅱ activity in activated HSCs. Finally, in vivo /in vitro experiments demonstrated that MLC activity was inhibited by DPN.CONCLUSION: For OVX rats with liver cirrhosis, DPN suppressed liver Rho A/ROCK signal, facilitated NO/PKG pathways, and decreased IHVR, giving rise to reduced portal pressure. Therefore, DPN represents a relevant treatment choice against PHT in cirrhotic patients, especially postmenopausal women.     

Rapid effects of estrogen receptor α and β selective agonists on learning and dendritic spines in female mice

Estrogen receptor (ER) agonists rapidly affect neural plasticity within 1 h, suggesting they play a functional role in learning and memory. However, behavioral learning experiments on such a rapid time scale are lacking. Therefore we investigated whether the ERα agonist propyl pyrazole triol (PPT) and ERβ agonist diarylpropionitrile (DPN) could affect social recognition, object recognition, or object placement learning within 40 min of drug administration. At the same time, we examined their effects on CA1 hippocampal dendritic spines. Ovariectomized female CD1 mice were administered a range of PPT or DPN doses (0, 30, 50, 75, or 150 μg/mouse). PPT at the middle doses improved social recognition, facilitated object recognition and placement at a dose of 75 μg, and increased dendritic spine density in the stratum radiatum and lacunosum-moleculare. In contrast, DPN impaired social recognition at higher doses, did not affect object recognition, but slightly facilitated object placement learning at the 75-μg dose. DPN did not affect spines in the stratum radiatum but decreased spine density and increased spine length in the lacunosum-moleculare. This suggests that rapid estrogen-mediated learning enhancements may predominantly be mediated through ERα, while the effects of DPN are weaker and may depend on the learning paradigm. The role of ERα and ERβ in learning and memory may vary depending on the timing of drug administration, as genomic studies often implicate ERβ in enhancing effects on learning and memory. To our knowledge, this is the first report of estrogens' effects on learning within such a short time frame.     

Multiple functional therapeutic effects of the estrogen receptor β agonist indazole-Cl in a mouse model of multiple sclerosis

Currently available immunomodulatory therapies do not stop the pathogenesis underlying multiple sclerosis (MS) and are only partially effective in preventing the onset of permanent disability in patients with MS. Identifying a drug that stimulates endogenous remyelination and/or minimizes axonal degeneration would reduce the rate and degree of disease progression. Here, the effects of the highly selective estrogen receptor (ER) β agonist indazole chloride (Ind-Cl) on functional remyelination in chronic experimental autoimmune encephalomyelitis (EAE) mice were investigated by assessing pathologic, functional, and behavioral consequences of both prophylactic and therapeutic (peak EAE) treatment with Ind-Cl. Peripheral cytokines from autoantigen-stimulated splenocytes were measured, and central nervous system infiltration by immune cells, axon health, and myelination were assessed by immunohistochemistry and electron microscopy. Therapeutic Ind-Cl improved clinical disease and rotorod performance and also decreased peripheral Th1 cytokines and reactive astrocytes, activated microglia, and T cells in brains of EAE mice. Increased callosal myelination and mature oligodendrocytes correlated with improved callosal conduction and refractoriness. Therapeutic Ind-Cl-induced remyelination was independent of its effects on the immune system, as Ind-Cl increased remyelination within the cuprizone diet-induced demyelinating model. We conclude that Ind-Cl is a refined pharmacologic agent capable of stimulating functionally relevant endogenous myelination, with important implications for progressive MS treatment.Multiple sclerosis (MS) is an autoimmune, demyelinating, and neurodegenerative disease of the central nervous system (CNS) that affects 2–2.5 million people worldwide. Currently approved MS drugs reduce relapse rates but fail to reverse or prevent neurodegeneration and disability progression. Disease-modifying drugs capable of restoring neuronal function via axon remyelination (RM) represent a major unmet goal for MS therapeutics.Oligodendrocyte (OL) progenitor cells (OPCs) are responsible for remyelinating axons, make up at least 3% of all white matter cells, and are present in and around MS lesions; however, they remain largely quiescent in the adult CNS (1). Although endogenous RM can occur in patients with MS, as evidenced by shadow plaques, it is short-lived, incomplete, and relatively ineffective (2). Transition to progressive MS is characterized by increased axon loss, which correlates with RM failure (3). Hence, a treatment that stimulates endogenous OPCs to differentiate and remyelinate axons would reduce axon degeneration and restore neuronal function.Experimental autoimmune encephalomyelitis (EAE) affords researchers an in-depth, mechanistic understanding of immune-mediated, demyelinating neurodegeneration and anti-inflammatory effects of currently approved MS drugs. Our recent work has demonstrated promising neuroprotective effects of the estrogen receptor (ER) β agonist 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) (4, 5). Although DPN, acting through ERβ, has a desirable palliative effect in EAE, it possesses only 70-fold binding selectivity for ERβ over ERα and lacks anti-inflammatory effects (6, 7). A more selective ERβ agonist capable of immunomodulation would be more efficacious in treating inflammatory demyelinating neurodegeneration.The structurally unique ERβ ligand indazole chloride (Ind-Cl), based on a halogen-substituted phenyl-2H-indazole core, is a preclinical development candidate with a strong dossier, including in vitro pharmacology using rodent and human cells, selectivity and potency data, promising absorption-distribution-metabolism-excretion findings, and pharmacokinetic profiling that includes confirmation of brain penetrability (mouse brain/plasma: ∼1.0) (7, 8). It is a highly ERβ-selective (>100-fold) small molecule agonist that is administered s.c. and can be developed for oral administration (7).Here, we explored pathologic, functional, and behavioral consequences of prophylactic and therapeutic (after onset of peak EAE) Ind-Cl in chronic EAE mice. Importantly, our recent finding of Ind-Cl-induced RM was confirmed, using the chronic cuprizone (CPZ)-induced demyelinating model (9), supporting Ind-Cl’s remyelinating capabilities independent of its effects on primary inflammation. Our results demonstrate that prophylactic and therapeutic Ind-Cl have significant beneficial effects in a murine model of progressive MS. Specifically, Ind-Cl attenuates clinical disease, and its functional immunomodulatory, remyelinating, and neuroprotective effects manifest in axon conduction and myelination improvements. Importantly, these effects correlate with improved motor function. Thus, Ind-Cl could impart much-needed, unique therapeutic benefits in progressive MS and other demyelinating disorders.     

Effects of estrogen receptor α and β gene deletion on estrogenic induction of progesterone receptors in the locus coeruleus in female mice

Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ERα (αERKO) or ERβ (βERKO) knockout mice, and their wild-type (αWT and βWT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ERβ was more abundant. Estradiol benzoate (EB) decreased ERα-positive cells in WT and βERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ERβ expression. ERβ deletion increased ERα while ERα deletion did not alter ERβ expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in αERKO animals but to a lesser extent, suggesting that ERα is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in βERKO mice were similar to those in the αERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses αER, βER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.     

Liver X receptor β and thyroid hormone receptor α in brain cortical layering

In the past year, two members of the nuclear receptor family, liver X receptor β (LXRβ) and thyroid hormone receptor α (TRα), have been found to be essential for correct migration of neurons in the developing cortex in mouse embryos. TRα and LXRβ bind to identical response elements on DNA and sometimes regulate the same genes. The reason for the migration defect in the LXRβ^−/− mouse and the possibility that TRα may be involved are the subjects of the present study. At E15.5, expression of reelin and VLDLR was similar but expression of apolipoprotein E receptor 2 (ApoER2) (the reelin receptor) was much lower in LXRβ^−/− than in WT mice. Knockout of ApoER2 is known to lead to abnormal cortical lamination. Surprisingly, by postnatal day 14 (P14), no morphological abnormalities were detectable in the cortex of LXRβ^−/− mice and ApoER2 expression was much stronger than in WT controls. Thus, a postnatal mechanism leads to increase in ApoER2 expression by P14. TRα also regulates ApoER2. In both WT and LXRβ^−/− mice, expression of TRα was high at postnatal day 2. By P14 it was reduced to low levels in WT mice but was still abundantly expressed in the cortex of LXRβ^−/− mice. Based on the present data we hypothesize that reduction in the level of ApoER2 is the reason for the retarded migration of later-born neurons in LXRβ^−/− mice but that as thyroid hormone (TH) increases after birth the neurons do find their correct place in the cortex.     

Role of peroxisome proliferator-activated receptor β agonist on angiogenesis in hindlimb ischemic diabetic rats

IntroductionStudies indicated that PPARβ agonists play a role in modulation of angiogenesis. In this study, we evaluated the effect of specific PPARβ agonist, GW0742, on angiogenesis and serum vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and nitrite concentrations in hindlimb ischemia in normal and diabetic rats.MethodsHindlimb ischemic rats were divided into four groups: control, diabetic, control, and diabetic treated with GW0742 (n = 7 each). Diabetes was induced by injection of streptozotocin (55 mg/kg, ip). GW0742 was injected 1 day after surgery (1 mg/kg, sc). After 21 days, blood samples were taken, and gastrocnemius muscles were harvested for immunohistochemistry.ResultsGW0742 significantly increased serum nitrite and VEGFR-2 concentrations and VEGF-to-VEGFR-2 ratio in control and diabetic rats. Capillary density was lower in diabetic animals compared to the control, and GW0742 significantly restored the capillary density in the control and diabetic hindlimb ischemic rats.ConclusionPPARβ agonists restore skeletal muscle angiogenesis and can be considered for prevention and/or treatment of peripheral vascular complications in diabetic subjects.     

Association between the polymorphism of estrogen receptor α and coronary artery disease in a Chinese population

BackgroundThe role of estrogen receptor α (ERα) polymorphism in coronary artery disease (CAD) was investigated previously in several populations. There are few data on relation between ERa polymorphism and CAD in Chinese population. Our study was to investigate the possible association between ERα polymorphism and CAD in Chinese population.MethodsA total of 539 patients with CAD and 539 age and sex matched controls were examined for ERa polymorphism. DNA was obtained and ERa polymorphism was analyzed by the polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP).ResultsThe frequencies of the PvuII C allele were significantly higher in CAD patients than in control individuals (P < 0.05). Using T allele as a reference, the odds ratio for CAD patients with C allele was 1.24 (95%CI = 1.03-1.48). Using TT genotype as a reference, the odds ratio for TC genotype was 1.17 (95%CI = 0.90-1.50), and for CC genotype was 1.58 (95%CI = 1.05-2.38). The odds ratio for CC genotype was 1.42 (95%CI = 0.94-2.15) in women and 1.72 (95%CI = 1.41-2.10) in men. There were no significant differences in XbaI allele and genotype between CAD patients and control individuals.ConclusionsThe ERa PvuII polymorphism is associated with the increased risk of CAD in men of a Chinese population. Further research is needed to investigate the mechanism underlying the association between ERα polymorphism and CAD.     

Anxiolytic effects and neuroanatomical targets of estrogen receptor-β (ERβ) activation by a selective ERβ agonist in female mice

The dichotomous anxiogenic and anxiolytic properties of estrogens have been reported to be mediated by two distinct neural estrogen receptors (ER), ERα and ERβ, respectively. Using a combination of pharmacological and genetic approaches, we confirmed that the anxiolytic actions of estradiol are mediated by ERβ and extended and these observations to demonstrate the neuroanatomical targets involved in ERβ activation in these behavioral responses. We examined the effects of the biologically active S-enantiomer of diarylpropionitrile (S-DPN) on anxiety-related behavioral measures, the corresponding stress hormonal response to hypothalamo-pituitary-adrenal axis reactivity, and potential sites of neuronal activation in mutant female mice carrying a null mutation for ERβ gene (βERKO). S-DPN administration significantly reduced anxiety-like behaviors in the open field, light-dark exploration, and the elevated plus maze (EPM) in ovariectomized wild-type (WT) mice, but not in their βERKO littermates. Stress-induced corticosterone (CORT) and ACTH were also attenuated by S-DPN in the WT mice but not in the βERKO mice. Using c-fos induction after elevated plus maze, as a marker of stress-induced neuronal activation, we identified the anterodorsal medial amygdala and bed nucleus of the stria terminalis as the neuronal targets of S-DPN action. Both areas showed elevated c-fos mRNA expression with S-DPN treatment in the WT but not βERKO females. These studies provide compelling evidence for anxiolytic effects mediated by ERβ, and its neuroanatomical targets, that send or receive projections to/from the paraventricular nucleus, providing potential indirect mode of action for the control of hypothalamo-pituitary-adrenal axis function and behaviors.     

Peroxisome proliferator-activated receptor γ agonist reduces the severity of post-ERCP pancreatitis in rats

INTRODUCTIONAcute pancreatitis is one of the major and serious complications after diagnostic or therapeutic endoscopic retrograde cholangiopancreatography(ERCP).Despite the technical improvements of recent years and the experience of endoscopists,the inc…     

Peroxisome proliferator-activated receptor γ agonist reduces the severity of post-ERCP pancreatitis in rats

AIM: To determine the effects of prophylactic peroxi-some proliferator-activated receptor (PPARgamma) agonist administration in an experimental model of post-endoscopic retrograde cholangiopancreatography (post-ERCP) acute pancreatitis. METHODS: Post-ERCP pancreatitis was induced in male Wistar rats by infusion of contrast medium into the pancreatic duct. In additional group, rosiglitazone, a PPARgamma agonist, was administered 1 h before infusion of contrast medium. Plasma and pancreas samples were obtained 6 h after the infusion. RESULTS: Infusion of contrast medium into the pan-creatic duct resulted in an inflammatory process characterized by increased lipase levels in plasma, and edema and myeloperoxidase activity (MPO) in pancreas. This result correlated with the activation of nuclear factor kappaB (NFkappaB) and the inducible NO synthase (iNOS) expression in pancreatic cells. Rosiglitazone reduced the increase in lipase and the level of edema and the increase in myeloperoxidase as well as the activation of NFkappaB and iNOS expression. CONCLUSION: A single oral dose of rosiglitazone, given 1 h before post-ERCP pancreatitis induction is effective in reducing the severity of the subsequent inflammatory process. The protective effect of rosiglitazone was associated with NFkappaB inhibition and the blockage of leukocyte infiltration in pancreas.     

Seladin-1 as a target of estrogen receptor activation in the brain: a new gene for a rather old story?

Experimental evidence indicates that estrogen exerts neuroprotective effects. According to the fact that Alzheimer's disease (AD) is more common in post-menopausal women, estrogen treatment has been proposed. However, the beneficial effect of estrogen or selective estrogen receptor modulators (SERMs) in preventing or treating AD is a controversial issue, which will be summarized in this review. Recently, a novel gene, named selective AD indicator-1 (seladin-1), has been isolated and found to be down-regulated in brain regions affected by AD. Seladin-1, which is considered the human homolog of the plant protein DIMINUTO/DWARF1, confers protection against beta-amyloid-mediated toxicity and from oxidative stress and is an effective inhibitor of caspase 3 activity, a key mediator of apoptosis. This review will present the up-to-date findings regarding seladin-1 and DIMINUTO/DWARF1. In addition, the possibility that seladin-1 may be a downstream effector of estrogen receptor activation in the brain, based on our recent experimental findings using a human fetal neuronal model, will be addressed.     

The TLR-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor α signaling

Plasmacytoid dendritic cells (pDCs) produce large amounts of type I interferons (IFN-α/β) in response to viral or endogenous nucleic acids through activation of their endosomal Toll-like receptors (TLR-7 and TLR-9). Enhanced TLR-7-mediated IFN-α production by pDCs in women, compared with men, has been reported, but whether sex hormones, such as estrogens, are involved in this sex-based difference is unknown. Here we show, in humanized mice, that the TLR-7-mediated response of human pDCs is increased in female host mice relative to male. In a clinical trial, we establish that treatment of postmenopausal women with 17β-estradiol markedly enhances TLR-7- and TLR-9-dependent production of IFN-α by pDCs stimulated by synthetic ligands or by nucleic acid-containing immune complexes. In mice, we found exogenous and endogenous estrogens to promote the TLR-mediated cytokine secretion by pDCs through hematopoietic expression of estrogen receptor (ER) α. Genetic ablation of ERα gene in the DC lineage abrogated the enhancing effect of 17β-estradiol on their TLR-mediated production of IFN-α, showing that estrogens directly target pDCs in vivo. Our results uncover a previously unappreciated role for estrogens in regulating the innate functions of pDCs, which may account for sex-based differences in autoimmune and infectious diseases.     

PPARγ agonist improves neuronal insulin receptor function in hippocampus and brain mitochondria function in rats with insulin resistance induced by long term high-fat diets

We previously demonstrated that a high-fat diet (HFD) consumption can cause not only peripheral insulin resistance, but also neuronal insulin resistance. Moreover, the consumption of an HFD has been shown to cause mitochondrial dysfunction in both the skeletal muscle and liver. Rosiglitazone, a peroxizome proliferator-activated receptor-γ ligand, is a drug used to treat type 2 diabetes mellitus. Recent studies suggested that rosiglitazone can improve learning and memory in both human and animal models. However, the effects of rosiglitazone on neuronal insulin resistance and brain mitochondria after the HFD consumption have not yet been investigated. Therefore, we tested the hypothesis that rosiglitazone improves neuronal insulin resistance caused by a HFD via attenuating the dysfunction of neuronal insulin receptors and brain mitochondria. Rosiglitazone (5 mg/kg · d) was given for 14 d to rats that were fed with either a HFD or normal diet for 12 wk. After the 14(th) week, all animals were euthanized, and their brains were removed and examined for insulin-induced long-term depression, neuronal insulin signaling, and brain mitochondrial function. We found that rosiglitazone significantly improved peripheral insulin resistance and insulin-induced long-term depression and increased neuronal Akt/PKB-ser phosphorylation in response to insulin. Furthermore, rosiglitazone prevented brain mitochondrial conformational changes and attenuated brain mitochondrial swelling, brain mitochondrial membrane potential changes, and brain mitochondrial ROS production. Our data suggest that neuronal insulin resistance and the impairment of brain mitochondria caused by a 12-wk HFD consumption can be reversed by rosiglitazone.