Estrogen altered visceromotor reflex and P2X3 mRNA expression in a rat model of colitis

P2X₃ and P2X_2/3 receptors are expressed in peripheral tissues and dorsal root ganglia (DRG) and participate in peripheral pain. However, the mechanisms underlying P2X receptor-mediated nociception at different ovarial hormone levels has not been examined. In this study, 24 female rats were randomly divided into sham-operated (sham), ovariectomized (OVX), estrogen-treated, and estrogen–progesterone-treated groups with colitis. In each group, the visceromotor reflex (VMR) to colorectal distension was tested and the DRG were harvested for a real-time PCR analysis of P2X₃ and P2X₂ receptor mRNA. In OVX rats with colitis we found that the VMR to colorectal distension and P2X₃ receptor mRNA in DRG were both significantly decreased. Estrogen replacement reversed the decrease. However, neither the VMR nor the P2X₃ mRNA level in DRG from OVX colitis rats was reversed by the complex of estrogen and progesterone. Patch-clamp recording showed that in colitis rats, estradiol rapidly potentiated the sustained and transient currents evoked by ATP to 336 ± 49% and 122 ± 12% of controls, respectively, in a subpopulation of DRG neurons, which were blocked by ICI 182, 780, an antagonist of the estrogen receptor. Whereas progesterone rapidly inhibited the transient currents induced by ATP to 67 ± 10% of control and had no effect on the sustained currents evoked by the same agonist. These results indicate that P2X₃ receptors are likely to be an important contributor to the altered colonic functions in colitis rats, where the underlying mechanisms are closely related to endogenous estrogen modulation.     

Sexual responses of the male rat medial preoptic area and medial amygdala to estrogen II: site specific effects of selective estrogenic drugs

In the medial preoptic area (MPO) and medial amygdala (MEA), estradiol (E(2)) aromatized from testosterone (T) may act via either estrogen receptor (ER) α or ERβ to mediate mating in male rats. We tested the hypothesis that, in the MPO, ERα exclusively mediates sexual responses to E(2) by monitoring mating in four groups of castrated male rats administered dihydrotestosterone (DHT) subcutaneously and MPO implants delivering either: cholesterol, E(2), propyl pyrazole triol (PPT, ERα-agonist) or diarylpropionitrile (DPN, ER β-agonist); a fifth group of intact males served as DPN toxicity control, receiving DPN MPO implants. In a follow-up study, either 1-methyl-4-phenyl pyridinium (MPP, ERα-antagonist) or blank MPO cannulae were implanted in castrated male rats receiving T subcutaneously, whereas intact MPP toxicity controls received MPP MEA implants. PPT or E(2) MPO implants maintained mating, but cholesterol or DPN MPO implants did not. Moreover, MPP MPO implants interfered with T reinstatement of mating suggesting that, in the MPO, ERα is necessary and sufficient for mating in androgen-maintained male rats and ERβ is not sufficient. Because it is unknown which ER subtype(s) mediate sexual responses of the MEA to E(2), we examined mating following MEA implants of cholesterol, E(2), PPT or DPN in four groups of castrated male rats administered DHT subcutaneously. E(2) MEA implants maintained mounting but mating was significantly decreased in groups receiving PPT, DPN or cholesterol MEA implants suggesting that, unlike the MPO where ERα alone is essential, sexual responses of the MEA to E(2) require simultaneous interactions among multiple ER subtypes.     

Estrogen receptor-β activation in combination with letrozole blocks the growth of breast cancer tumors resistant to letrozole therapy

Treatment with anti-estrogens or aromatase inhibitors (AI) is the main therapeutic strategy used against estrogen receptor ERα-positive breast cancer. Resistance to these therapies presents a major challenge in the management of breast cancer. Little is known about ERβ in breast carcinogenesis. Our aim in this study is to examine potential novel strategies utilizing ERβ activity to overcome AI resistance. We provide evidence that ERβ agonist can reduce the growth of AI-resistant breast cancer cells. Our data further confirm that therapeutic activation of ERβ by DPN, an ERβ agonist, blocks letrozole-resistant tumor growth in a xenograft model. Interestingly, DPN exerted tumor growth inhibition only in the presence of the AI letrozole, suggesting that combination therapy including ERβ activators and AI may be used in the clinical setting treating AI resistant breast cancer. An increase in ERβ levels, with diminished ERα/ERβ ratio, was observed in the tumors from mice treated with DPN/letrozole combination compared to single agents and control. Decreased Cyclin D1 and increased CyclinD1/CDK inhibitors p21 and p27 levels in DPN/letrozole treated tumors were observed, suggesting that the combination treatment may inhibit tumor growth by blocking G1/S phase cell cycle progression. Our data show a decrease in MAPK phosphorylation levels without affecting total levels. In addition to providing evidence suggesting the potential use of ERβ agonists in combination with letrozole in treating AI resistant breast cancer and prolonging sensitivity to AI, we also provide mechanistic evidence supporting the role of ERβ in altering the expression profile associated with resistance.     

Estrogen receptor-β signaling modulates epithelial barrier function

Impaired epithelial barrier function and estrogens are recognized as factors influencing inflammatory bowel disease (IBD) pathology and disease course. Estrogen receptor-β (ERβ) is the most abundant estrogen receptor in the colon and a complete absence of ERβ expression is associated with disrupted tight-junction formation and abnormal colonic architecture. The aim of this study was to determine whether ERβ signaling has a role in the maintenance of epithelial permeability in the colon. ERβ mRNA levels and colonic permeability were assessed in IL-10-deficient mice and HLA-B27 rats by RT-PCR and Ussing chambers. ERβ expression and monolayer resistance were measured in HT-29 and T84 colonic epithelial monolayers by RT-PCR and electric cell-substrate impedance sensing. The effect of 17β-estradiol and an estrogen agonist [diarylpropionitrile (DPN)] and antagonist (ICI 182780) on epithelial resistance in T84 cells was measured. Expression of ERβ and proinflammatory cytokines was investigated in colonic biopsies from IBD patients. Levels of ERβ mRNA were decreased, whereas colonic permeability was increased, in IL-10-deficient mice and HLA-B27 transgenic rats prior to the onset of colitis. T84 cells demonstrated higher resistance and increased levels of ERβ mRNA compared with HT-29 cells. 17β-estradiol and DPN induced increased epithelial resistance in T84 cells, whereas an ERβ blocker prevented the increased resistance. Decreased ERβ mRNA levels were observed in colonic biopsies from IBD patients. This study suggests a potential role for ERβ signaling in the modulation of epithelial permeability and demonstrates reduced ERβ mRNA in animal models of colitis and colon of patients with inflammatory bowel disease.     

17β-雌二醇和跑台运动对去卵巢大鼠骨组织及子宫ERα蛋白表达影响的对比研究

目的：雌激素受体α（ERα）作为雌激素调节骨效应的主要受体在雌激素对骨量和骨代谢的调节中发挥重要的作用,为此本研究比较了17β-雌二醇（E2）和跑台运动对去卵巢大鼠骨组织和子宫ERα蛋白表达影响的异同。方法：将40只健康3月龄雌性SD大鼠,按体重分层后随机分为假手术、去卵巢、雌激素和运动四个组。手术1周后,雌激素组大鼠每周按体重颈部皮下注射三次17β-雌二醇,每次25μg／kg体重。运动组每周进行4次45min、速度18m／min、坡度5°的跑台训练。连续给药或运动处理14周后。采用放射免疫法和免疫组织化学法分别检测血清E2水平以及胫骨和子宫ERα蛋白表达的变化。结果：大鼠去卵巢后,子宫重量、子宫重量指数和血清E2水平显著下降,补充外源性17β-雌二醇后,三项指标均显著增加;但运动处理只能增加血清E2水平,对子宫重量和子宫重量指数均无显著影响。子宫EIh蛋白免疫组化结果显示：ERα在去卵巢组子宫内膜腔上皮、腺上皮及基质中的表达显著低于假手术组,而在雌激素组中的表达高于去卵巢组,运动组各部位ERα表达虽有所增加,但是增加的程度远低于雌激素组。胫骨近端ERα蛋白免疫组化结果显示：去卵巢后,胫骨近端骨骺端软骨细胞的细胞核内ERα表达减少,运动和雌激素干预后,胫骨近端ERα表达增加。结论：运动和雌激素处理均能刺激去卵巢大鼠子宫和骨组织ERα蛋白的表达,但运动处理无雌激素处理增加子宫重量的副作用。可见在绝经后骨质疏松的预防中,运动处理可能要优于雌激素处理。     

Systemic administration of diarylpropionitrile (DPN) or phytoestrogens does not affect anxiety-related behaviors in gonadally intact male rats

The development of highly selective agonists for the two major subforms of the estrogen receptor (ERα and ERβ) has produced new experimental methodologies for delineating the distinct functional role each plays in neurobehavioral biology. It has also been suggested that these compounds might have the potential to treat estrogen influenced behavioral disorders, such as anxiety and depression. Prior work has established that the ERβ agonist, diarylpropionitrile (DPN) is anxiolytic in gonadectomized animals of both sexes, but whether or not this effect persists in gonadally intact individuals is unknown. Isoflavone phytoestrogens, also potent but less selective ERβ agonists, have also been shown to influence anxiety in multiple species and are becoming more readily available to humans as health supplements. Here we determined the effects of 0.5, 1 or 2 mg/kg DPN, 1 mg/kg of the ERα agonist propyl-pyrazole-triol (PPT), 3 or 20 mg/kg of the isoflavone equol (EQ) and 3 or 20 mg/kg of the isoflavone polyphenol resveratrol (RES) on anxiety behavior in the gonadally intact male rat using the light/dark box and the elevated plus maze. We first determined that DPN can be successfully administered either orally or by subcutaneous injection, although plasma DPN levels are significantly lower if given orally. Once injected, plasma levels peak rapidly and then decline to baseline levels within 3 h of administration. For the behavioral studies, all compounds were injected and the animals were tested within 3 h of treatment. None of the compounds, at any of the doses, significantly altered anxiety-related behavior. Plasma testosterone levels were also not significantly altered suggesting that these compounds do not interfere with endogenous androgen levels. The results suggest that the efficacy of ERβ agonists may depend on gonadal status. Therefore the therapeutic potential of ERβ selective agonists to treat mood disorders may be limited.     

Influence of ERβ selective agonism during the neonatal period on the sexual differentiation of the rat hypothalamic-pituitary-gonadal (HPG) axis

Background

It is well established that sexual differentiation of the rodent hypothalamic-pituitary-gonadal (HPG) axis is principally orchestrated by estrogen during the perinatal period. Here we sought to better characterize the mechanistic role the beta form of the estrogen receptor (ERβ) plays in this process.

Methods

To achieve this, we exposed neonatal female rats to three doses (0.5, 1 and 2 mg/kg) of the ERβ selective agonist diarylpropionitrile (DPN) using estradiol benzoate (EB) as a positive control. Measures included day of vaginal opening, estrous cycle quality, GnRH and Fos co-localization following ovariectomy and hormone priming, circulating luteinizing hormone (LH) levels and quantification of hypothalamic kisspeptin immunoreactivity. A second set of females was then neonatally exposed to DPN, the ERα agonist propyl-pyrazole-triol (PPT), DPN+PPT, or EB to compare the impact of ERα and ERβ selective agonism on kisspeptin gene expression in pre- and post-pubescent females.

Results

All three DPN doses significantly advanced the day of vaginal opening and induced premature anestrus. GnRH and Fos co-labeling, a marker of GnRH activation, following ovariectomy and hormone priming was reduced by approximately half at all doses; the magnitude of which was not as large as with EB or what we have previously observed with the ERα agonist PPT. LH levels were also correspondingly lower, compared to control females. No impact of DPN was observed on the density of kisspeptin immunoreactive (-ir) fibers or cell bodies in the arcuate (ARC) nucleus, and kisspeptin-ir was only significantly reduced by the middle (1 mg/kg) DPN dose in the preoptic region. The second experiment revealed that EB, PPT and the combination of DPN+PPT significantly abrogated preoptic Kiss1 expression at both ages but ARC expression was only reduced by EB.

Conclusion

Our results indicate that selective agonism of ERβ is not sufficient to completely achieve male-typical HPG organization observed with EB or an ERα agonist.     

Estrogen rapidly modulates mustard oil-induced visceral hypersensitivity in conscious female rats: A role of CREB phosphorylation in spinal dorsal horn neurons

This study investigated the effect of sex hormones on mustard oil (MO)-induced visceral hypersensitivity in female rats and analyzed possible involved signaling pathways. Female rats, either intact or ovariectomized (OVX), were prepared for abdominal muscle electromyography in response to colorectal distension after intracolonic instillation of MO. The effect of MO intracolonic sensitization was evaluated in intact rats, OVX rats, and OVX rats pretreated with a single injection of 17beta-estradiol (E), progesterone (P), E+P, or vehicle. cAMP-responsive element-binding protein (CREB) and phosphorylated CREB (pCREB) were detected in the superficial dorsal horn of L6 and S1 in MO or mineral oil-treated OVX rats with/without colorectal distension and estrogen replacement. The distal colorectum was removed for histological evaluation of inflammatory severity in MO-treated intact or OVX rats. The MO-treated rats had significantly higher visceromotor reflex than controls (enhanced visceral hypersensitivity), whereas OVX eliminated this hypersensitivity. After a single injection of E or E+P, the rats rapidly restored MO-induced visceral hypersensitivity within 2 h. Estrogen also rapidly induced a dose-dependent increase in pCREB expression in the superficial dorsal horn neurons in MO-treated, but not mineral oil-treated, OVX rats. The present study suggests that estrogen can rapidly modulate visceral hypersensitivity induced by MO intracolonic instillation in conscious female rats, which may involve spinal activation of the cAMP response element-mediated gene induction pathway.     

Neonatal agonism of ERβ impairs male reproductive behavior and attractiveness

The organization of the developing male rodent brain is profoundly influenced by endogenous steroids, most notably estrogen. This process may be disrupted by estrogenic endocrine disrupting compounds (EDCs) resulting in altered sex behavior and the capacity to attract a mate in adulthood. To better understand the relative role each estrogen receptor (ER) subtype (ERα and ERβ) plays in mediating these effects, we exposed male Long Evans rats to estradiol benzoate (EB, 10 μg), vehicle, or agonists specific for ERβ (DPN, 1 mg/kg) or ERα (PPT, 1 mg/kg) daily for the first four days of life, and then assessed adult male reproductive behavior and attractiveness via a partner preference paradigm. DPN had a greater adverse impact than PPT on reproductive behavior, suggesting a functional role for ERβ in the organization of these male-specific behaviors. Therefore the impact of neonatal ERβ agonism was further investigated by repeating the experiment using vehicle, EB and additional DPN doses (0.5 mg/kg, 1 mg/kg, and 2 mg/kg bw). Exposure to DPN suppressed male reproductive behavior and attractiveness in a dose dependent manner. Finally, males were exposed to EB or an environmentally relevant dose of genistein (GEN, 10 mg/kg), a naturally occurring xenoestrogen, which has a higher relative binding affinity for ERβ than ERα. Sexual performance was impaired by GEN but not attractiveness. In addition to suppressing reproductive behavior and attractiveness, EB exposure significantly lowered the testis to body weight ratio, and circulating testosterone levels. DPN and GEN exposure only impaired behavior, suggesting that disrupted androgen secretion does not underlie the impairment.     

NGF Attenuates High Glucose-Induced ER Stress,Preventing Schwann Cell Apoptosis by Activating the PI3K/Akt/GSK3β and ERK1/2 Pathways

Diabetic peripheral neuropathy (DPN) is one of the most common and troublesome complications of diabetes mellitus. It has been demonstrated that nerve growth factor (NGF) exerts a pivotal role in the regulation of neuronal growth and the promotion of DPN recovery. However, the exact molecular mechanisms are not well understood. Recent studies have indicated that as a novel therapeutic target, endoplasmic reticulum (ER) stress participates in the onset and progression of DPN. In the present study, it has been demonstrated that NGF prevents the sciatic nerve from degeneration and demyelination in DPN rats. Thus, RSC 96 cells, which retain the characteristic features of Schwann cells (SCs), were cultured in medium containing 30 mM glucose (high glucose, HG) to mimic SCs in DPN mice. The 50-ng/ml dose of NGF was identified to be the optimal concentration for treating an excessive ER stress level under HG conditions for 24 h. We found that NGF treatment significantly inhibits HG-induced ER stress and subsequently suppresses ER-related apoptosis. Further, NGF administration also activates the upstream signaling pathway of ER stress, PI3K/Akt/GSK3β signaling and ERK1/2 signaling. Co-treatment with the PI3K inhibitor LY294002 or ERK1/2 inhibitor U0126 significantly reverses the protective role of NGF on HG-induced excessive ER stress and subsequent apoptosis. These observations suggest that the neuroprotective role of NGF in DPN is mediated by the inhibition of excessive ER stress via the activation of the PI3K/Akt/GSK3β and ERK1/2 signaling pathways.     

Electroacupuncture alleviates diabetic neuropathic pain in rats by suppressing P2X3 receptor expression in dorsal root ganglia

Diabetic neuropathic pain (DNP) is a troublesome diabetes complication all over the world. P2X3 receptor (P2X3R), a purinergic receptor from dorsal root ganglion (DRG), has important roles in neuropathic pain pathology and nociceptive sensations. Here, we investigated the involvement of DRG P2X3R and the effect of 2 Hz electroacupuncture (EA) on DNP. We monitored the rats’ body weight, fasting blood glucose level, paw withdrawal thresholds, and paw withdrawal latency, and evaluated P2X3R expression in DRG. We found that P2X3R expression is upregulated on DNP, while 2 Hz EA is analgesic against DNP and suppresses P2X3R expression in DRG. To evaluate P2X3R involvement in pain modulation, we then treated the animals with A317491, a P2X3R specific antagonist, or α β-me ATP, a P2X3R agonist. We found that A317491 alleviates hyperalgesia, while α β-me ATP blocks EA’s analgesic effects. Our findings indicated that 2 Hz EA alleviates DNP, possibly by suppressing P2X3R upregulation in DRG.

     

Nanoparticle-encapsulated emodin decreases diabetic neuropathic pain probably via a mechanism involving P2X3 receptor in the dorsal root ganglia

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM). More than 90% of all cases of DM belong to type 2 diabetes mellitus (T2DM). Emodin is the main active component of Radix et rhizoma rhei and has anti-bacterial, anti-viral, anti-ulcerogenic, anti-inflammatory, and anti-cancer effects. Nanoparticle encapsulation of drugs is beneficial for drug targeting and bioavailability as well as for lowering drug toxicity side effects. The aim of this study was to investigate the effects of nanoparticle-encapsulated emodin (nano emodin) on diabetic neuropathic pain (DNP) mediated by the Purin 2X3 (P2X3) receptor in the dorsal root ganglia (DRG). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) values in T2DM rats were lower than those of control rats. MWT and TWL in T2DM rats treated with nano emodin were higher compared with those in T2DM rats. Expression levels of P2X3 protein and messenger RNA (mRNA) in the DRG of T2DM rats were higher than those of controls, while levels in T2DM rats treated with nano emodin were significantly lower than those of the T2DM rats. Phosphorylation and activation of ERK1/2 in the T2DM DRG were decreased by nano emodin treatment. Nano emodin significantly inhibited currents activated by the P2X3 agonist α,β-meATP in HEK293 cells transfected with the P2X3 receptor. Therefore, nano emodin treatment may relieve DNP by decreasing excitatory transmission mediated by the DRG P2X3 receptor in T2DM rats.     

Sex specific response of cultured human bone cells to ERα and ERβ specific agonists by modulation of cell proliferation and creatine kinase specific activity

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism.     

Effects of diet-induced hypercholesterolemia on amyloid accumulation in ovariectomized mice

A central hypothesis in the study of Alzheimer’s disease (AD) is the accumulation and aggregation of β-amyloid peptide (Aβ). Recent epidemiological studies suggest that patients with elevated cholesterol and decreased estrogen levels are more susceptible to AD through Aβ accumulation. To test the above hypothesis, we used ovariectomized with diet-induced hypercholesterolemia (OVX) and hypercholesterolemia (HCL) diet alone mouse models. HPLC analysis reveals the presence of beta amyloid in the OVX and HCL mice brain. Congo red staining analysis revealed the extent of amyloid deposition in OVX and hypercholesterolemia mice brain. Overall, Aβ levels were higher in OVX mice than in HCL. Secondly, estrogen receptors α (ERα) were assessed by immunohistochemistry and this suggested that there was a decreased expression of ER α in OVX animals when compared to hypercholesterolemic animals. Aβ was quantified by Western blot and ELISA analysis. Overall, Aβ levels were higher in OVX mice than in HCL mice. Our experimental results suggested that OVX animals were more susceptible to AD with significant increase in Aβ peptide.     

Synthesis and evaluation of aryl-substituted diarylpropionitriles,selective ligands for estrogen receptor β, as positron-emission tomographic imaging agents

We have investigated halogen-substituted non-steroidal estrogens with selective binding affinity for the estrogen receptor β (ERβ that might be used for imaging the levels of this ER-subtype in breast tumors by positron emission tomography (PET). Based on diarylpropionitrile (DPN, 1a), a compound previously reported that has a 72-fold binding selectivity for ERβ, we developed a series of DPN analogs having methyl-, hydroxyl-, and halogen substituents, including fluoroethyl and fluoropropyl groups. In competitive radiometric binding assays with [³H]estradiol, all of these DPN analogs showed high ERβ/ERα selectivity; while the selectivity varied, in some cases it reached nearly 300-fold (RBA: ERα, 0.023%; ERβ, 6.25%). The absolute ERβ binding affinities, however, were not sufficient to merit further consideration for developing these ligands as PET imaging agents.     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.