Expression Of CRFR1 Research Articles

Urocortin suppresses endometrial cancer cell migration via CRFR2 and its system components are differentially modulated by estrogen

Urocortin (UCN1) peptide shares structural and functional homology with corticotropin‐releasing factor (CRF). UCN1 is significantly reduced in endometrial adenocarcinoma compared to healthy controls. However, there are no data which evaluate the effects of UCN1 in the endometrium, or how it is modulated. We used proliferation and transwell assays to determine the effect of UCN1 on the proliferation and migration of Ishikawa and HEC1A cells. We also determined the expression levels of UCN1 and its receptors produced by estrogen receptor agonists, and the effect of UCN1 on estrogen receptor expression, using quantitative polymerase chain reaction. UCN1 suppressed migration of endometrial cancer cells in vitro. This effect appears to be specific to CRF receptor 2 (CRFR2), as selective antagonism of CRFR2 but not CRFR1 completely eliminated suppression of migration. Activation of ERA reduced UCN1 expression, but only had a small effect on the expression of CRFR1. However, expression of CRFR2 was more notably reduced at both the mRNA and protein levels by activation of ERB. UCN1 in turn reduced both ERA and ERB expression, as assessed by real‐time quantitative PCR. We demonstrate that UCN1 significantly suppresses the migration of endometrial cancer cells but has no effect on their proliferation. Thus, loss of UCN1 in endometrial cancer may promote invasion and metastatic spread. There is a complex relationship between the UCN1 system and estrogen receptors, which may provide insights into endometrial carcinogenesis, a disease known to be driven by estrogen excess.

Cardiac CRFR1 Expression Is Elevated in Human Heart Failure and Modulated by Genetic Variation and Alternative Splicing.

Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P < .001) and CRFR2α lower (P = .012) in patients. In patients and donors, genetic variation within CRFR1, represented by the chromosome 17q21.31 inversion polymorphism, was associated with markedly higher CRFR1 expression (P < .001), making CRFR1 and CRFR2α expression almost equivalent in some patients. A novel, truncated splice variant of CRFR1, designated CRFR1j, was identified and shown to exert a dominant-negative effect on CRFR1 signaling in vitro. The novel variant was expressed in a greater proportion of patients (60%) than donors (3%, P < .001). In summary, cardiac expression of CRFR1, CRF, and Ucn3 genes is elevated in heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling.

Differentiation of Keratinocytes Modulates Skin HPA Analog.

It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

Corticotropin-releasing factor augments LPS-induced immune/inflammatory responses in JAWSII cells.

The effect of corticotropin-releasing factor (CRF) on gastrointestinal tract inflammation via modulation of the immune system cells such as dendritic cells (DCs) is not known. We investigated the expression of CRF and its receptors CRFR1 and CRFR2 in a colonic DC model (JAWSII cells) in a pro-inflammatory and anti-inflammatory milieu. The mRNA expression of CRF and protein expression of CRFR1 and CRFR2 was assessed, and lipopolysaccharide (LPS) was used to induce the maturation of JAWSII cells. JAWSII cells were divided into four groups: control, CRF, LPS, and LPS + CRF. The levels of secreted cytokines IL-6, IL-4, TNF-α, and MIP-1α were determined, both in JAWSII cells and in the culture supernatant, by qRT-PCR and ELISA. The expression of CRFR1 and CRFR2 in JAWSII cells was accompanied by a low CRF expression. Compared with control group, CRF group did not affect the expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and MIP-1α, but LPS treatment significantly increased the expression of these cytokines, indicating maturation of JAWSII cells. CRF further augmented the production of IL-6, TNF-α, and MIP-1α in mature JAWSII cells, with no increase in TNF-α mRNA expression. However, the expression of anti-inflammatory factor IL-4 did not change after LPS treatment. CRF treatment decreased the expression of IL-4 in both mature and immature JAWSII cells. JAWSII cells produce low level of CRF and express CRFR1 and CRFR2 surface receptors. CRF promotes immune/inflammatory responses in mature JAWSII cells when induced by LPS treatment.

Dynamic Alterations of miR-34c Expression in the Hypothalamus of Male Rats after Early Adolescent Traumatic Stress.

Several types of microRNA (miRNA) overexpression in the brain are associated with stress. One of the targets of miR-34c is the stress-related corticotrophin releasing factor receptor 1 mRNA (CRFR1 mRNA). Here we will probe into the short-term effect and long-term effect of early adolescent traumatic stress on the expression of miR-34c and CRFR1 mRNA. Traumatic stress was established by electric foot shock for six consecutive days using 28-day rats. The anxiety-like behaviors, memory damage, CRFR1 protein, CRFR1 mRNA, and miR-34c expression were detected in our study. The results of our study proved that exposure to acute traumatic stress in early adolescent can cause permanent changes in neural network, resulting in dysregulation of CRFR1 expression and CRFR1 mRNA and miR-34c expression in hypothalamus, anxiety-like behavior, and memory impairment, suggesting that the miR-34c expression in hypothalamus may be an important factor involved in susceptibility to PTSD.

Effects of the endogenous CRF on the biological phenotypes of mouse mesenteric lymph nodes den-dritic cells

Objective To investigate the expression of corticotropin-releasing factor (CRF) and its receptors including CRFR1 and CRFR2 on mouse mesenteric lymph nodes dendritic cells (MLNDC), and to analyze their effects on the biological phenotypes of intestinal dendritic cells. Methods The MLNDCs were isolated from C57BL/6 mice by using magnetic bead sorting. The purity of CD11c+ DCs was identified by flow cytometry. The double-labeling immunofluorescence and the reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to detect the expression of CRF, CRFR1 and CRFR2 on MLNDCs. The MLNDCs were exposed to CRF with or without the interference of CRFR1 and CRFR2 antagonists. Flow cytometry was used to measure the changes of surface molecules (MHCⅠ and MHCⅡ) and co-stimulatory molecules (CD80 and CD86). Results The CD11c+ DCs accounted for (80.12±6.34)% of the isolate cells with a high cell viability of more than 90%. The expression of CRF, CRFR1 and CRFR2 at mRNA level were detected in MLNDCs by RT-PCR. Results of the immunofluorescent staining assay indicated that both CRFR1 and CRFR2 were expressed on the surface of MLNDCs. The expression of CD86 on MLNDCs was inhibited by the treatment of MLNDCs with CRFR1 antagonist, but enhanced by the treatment with CRFR2 antagonist. Conclusion Both CRF and CRFRs were detected in the MLNDCs isolated from the C57BL/6 mice. The CRF could alter the biological phenotypes of MLNDCs through binding to different CRFRs (CRFR1 and CRFR2), which affected the phenotypes of MLNDCs in opposite ways. Key words: Corticotropin-releasing factor; Mesenteric lymph nodes; Dendritic cell

Involvement of mTOR in Type 2 CRF Receptor Inhibition of Insulin Signaling in Muscle Cells.

Type 2 corticotropin-releasing factor receptor (CRFR2) is expressed in skeletal muscle and stimulation of the receptor has been shown to inhibit the effect of insulin on glucose uptake in muscle cells. Currently, little is known about the mechanisms underlying this process. In this study, we first showed that both in vivo and in vitro CRFR2 expression in muscle was closely correlated with insulin sensitivity, with elevated receptor levels observed in insulin resistant muscle cells. Stimulation of CRFR2 by urocortin 2 (Ucn 2), a CRFR2-selective ligand, in C2C12 myotubes greatly attenuated insulin-induced glucose uptake. The inhibitory effect of CRFR2 signaling required cAMP production and is involved the mammalian target of rapamycine pathway, as rapamycin reversed the inhibitory effect of CRFR2 stimulation on insulin-induced glucose uptake. Moreover, stimulation of CRFR2 failed to inhibit glucose uptake in muscle cells induced by platelet-derived growth factor, which, similar to insulin, signals through Akt-mediated pathway but is independently of insulin receptor substrate (IRS) proteins to promote glucose uptake. This result argues that CRFR2 signaling modulates insulin's action likely at the levels of IRS. Consistent with this notion, Ucn 2 reduced insulin-induced tyrosine phosphorylation of IRS-1, and treatment with rapamycin reversed the inhibitory effect of Ucn 2 on IRS-1 and Akt phosphorylation. In conclusion, the inhibitory effect of CRFR2 signaling on insulin action is mediated by cAMP in a mammalian target of rapamycine-dependent manner, and IRS-1 is a key nodal point where CRFR2 signaling modulates insulin-stimulated glucose uptake in muscle cells.

Nicotine withdrawal increases stress-associated genes in the nucleus accumbens of female rats in a hormone-dependent manner.

Previous work led to our hypothesis that sex differences produced by nicotine withdrawal are modulated by stress and dopamine systems in the nucleus accumbens (NAcc). We investigated our hypothesis by studying intact females to determine whether the mechanisms that promote withdrawal are ovarian-hormone mediated. Female rats were ovariectomized (OVX) or received sham surgery (intact) on postnatal day (PND 45-46). On PND 60, they received sham surgery (controls) or were prepared with nicotine pumps. Fourteen days later, half of the rats had their pumps removed (nicotine withdrawal) and the other half received sham surgery (nicotine exposure). Twenty-four hours later, the rats were tested for anxiety-like behavior using the elevated plus maze and light/dark transfer procedures. The NAcc was then dissected for analysis of several genes related to stress (CRF, UCN, CRF-R1, CRF-R2, CRF-BP, and Arrb2) or receptors for dopamine (Drd1 and Drd2) and estradiol (Esr2). During withdrawal, intact females displayed an increase in anxiety-like behavior in both tests and CRF, UCN, and Drd1 gene expression. During nicotine exposure, intact females displayed a decrease in CRF-R1, CRF-R2, Drd3, and Esr2 gene expression and an increase in CRF-BP. This pattern of results was absent in OVX females. Nicotine withdrawal produced an increase in anxiety-like behavior and stress-associated genes in intact females that is distinct from changes produced by nicotine exposure. The latter effects were absent in OVX females, suggesting that stress produced by withdrawal is ovarian-hormone mediated. These findings have important implications towards understanding tobacco use liability among females.

Evaluation of the effect of natural peptide 'Urocortin' on corticotrophin releasing factor (CRF) receptor expression in ND7/23 cells

CRF receptors are involved in the stress management of the cells and are believed to have a cytoprotective role in the body. CRF receptors have been reported to be potential drug targets for the treatment of neurodegenerative disorders. The cell line used in the study is ND7/23 (mouse neuroblastoma and rat dorsal root ganglion neuron hybridoma). The aim of the study was to confirm the expression of CRF receptors in ND7/23 cells and to determine if urocortin (Ucn) can enhance the expression of CRF receptors. ND7/23 cells were cultured in RPMI 1640 media and cells grown after the second passage were used for the experiments. RNA was extracted from the cells and amplified by RT-PCR to confirm the presence of CRF receptors. The cells were then subjected to oxidative stress by hydrogen peroxide (0.00375%) and divided into two groups i.e. control and Ucn (10-8 μM) treated. Later RNA was extracted from both group of cells and PCR was performed. Finally, densitometry analysis was conducted on the agarose gel to determine the quantity of PCR product formed. PCR experiment confirmed the expression of both CRF-R1 and CRF-R2 in the cell line, but CRF-R1 was found to be expressed more strongly. Densitometry analysis of the PCR product and calculation of the relative expression of CRF receptors indicated a higher level of expression of CRF receptors in samples treated with Ucn as compared to those that were kept untreated. The results indicate that Ucn may be useful for the management of neuro-degenerative disorders and further studies may be carried out to establish its use as a therapeutic agent.

Quantitative comparison of mRNA level of corticotropin- releasing factor peptide family in normal kidney and corresponding clear cell renal cell carcinoma specimens.

466 Background: Urocortin (Ucn), a 40 amino acid peptide, belongs to the corticotropin releasing factor (CRF) family and exerts its actions mainly in the periphery through activation of two CRF receptors (CRFRs), CRFR1 and CRFR2 and a binding protein (CRFBP/CRHBP). Both receptors are G-protein coupled and binding of Ucn leads to activation of cAMP-dependent protein kinases via elevation of cAMP levels. Ucn and Urocortin III (UcnIII) are involved in conditions such as regulation of local inflammation, angiogenesis, and inhibition of proliferation. Suppression of neovascularization and inhibition of tumor cell cycling by Urocortins is modulated through CRFRs. Activation of CRFRs by e.g. Ucn inhibits angiogenesis via reduction of vascular endothelial growth factor (VEGF) and suppresses the cell proliferation. Here we characterized the whole CRF family on mRNA levels quantitatively comparing the normal and clear cell carcinoma of the kidney (ccRCC). Methods: In this study we measured the mRNA level of Ucn, UcnIII, CRFR1, CRFR2 and CRHBP in 78 RCC samples and paired histologically normal appearing tissues using quantitative PCR. Statistical analyses were carried out using univariate logistic regression analysis. Results: We found tumour specific down regulation of mRNA expression of UcnIII, CRFR1, CRFR2 and CRHBP in samples of RCCs with clear cell histology compared to paired normal tissues (P&lt;0.01 for all targets). Only Ucn did not show any expression difference between two groups (p=0.17). Conclusions: For the first time we showed the expression profile of the whole CRF peptide family in ccRCC compared to normal kidney on mRNA level. Our data underlines the malfunction of this actually strong protective and anticancer system in renal malignancy and shows the involvement of CRF system in carcinogenesis in kidney. More studies are necessary to find out the detailed roles of the CRF system in renal carcinoma.

Influence of Jianpi Huashi granule on CRF and CRFR1 expression in the brain-gut axis of rats with diarrhea-predominant irritable bowel syndrome

目的：从结肠、海马及下丘脑中促肾上腺皮质激素释放因子（corticotropin releasing factor,CRF）和CRF受体1（CRF receptor 1,CRFR1）角度探讨健脾化湿颗粒改善腹泻型肠易激综合征（diarrhea-predominant irritable bowel syndrome,D-IBS）模型大鼠结肠运动和内脏敏感性的作用机制.方法：采用番泻叶灌胃结合束缚应激法建立D-I B S大鼠模型,应用健脾化湿颗粒进行干预,采用酶联免疫法（ELISA）检测大鼠结肠中CRF含量,采用免疫组织化学法检测结肠中CRFR1及海马、下丘脑中CRF,CRFR1阳性表达,采用RT-PCR法检测结肠、海马中CRF m RNA和CRFR1 m RNA的表达水平.结果：与正常组相比,模型组结肠中CRF含量（67.1±3.8 vs 36.0±3.0）,海马、下丘脑中CRF阳性表达（0.23±0.02 vs 0.09±0.01,0.17±0.02 v s 0.09±0.01）明显升高（P〈0.01）;结肠、海马、下丘脑中C R F R1阳性表达（0.17±0.01 vs 0.03±0.01,0.20±0.02 vs 0.09±0.01,0.19±0.02 vs 0.07±0.01）明显升高（P〈0.01）;结肠、海马中C R F m RNA和CRFR1 m RNA的表达（结肠：0.89±0.04 vs 0.09±0.01,1.09±0.09 vs 0.21±0.04;海马：0.56±0.01 vs 0.15±0.05,1.26±0.14 vs 0.23±0.06）显著升高（P〈0.01）.与模型组相比,各治疗组结肠、海马中C R F（51.0±3.4,54.6±4.1,45.1±4.7,43.3±3.9 vs 67.1±3.8;0.18±0.02,0.19±0.02,0.15±0.02,0.11±0.01 vs 0.23±0.02）显著下降（P〈0.01）,阳性对照组、中、高剂量组下丘脑中CRF（0.15±0.02,0.13±0.01,0.12±0.01 vs 0.17±0.02）下降显著（P〈0.05,P〈0.0 1）;阳性对照组、中、高剂量组结肠、海马、下丘脑中C R F R1表达（结肠：0.10±0.01,0.08±0.01,0.05±0.01 vs 0.17±0.01;海马：0.16±0.01,0.14±0.02,0.13±0.01 vs 0.20±0.02;下丘脑：0.15±0.02,0.13±0.01,0.11±0.01 vs 0.19±0.02）下降显著（P〈0.05,P〈0.01）;结肠中CRF m RNA表达（0.63±0.04,0.76±0.06,0.32±0.06,0.13±0.03 v s 0.89±0.04）及中、高剂量组海马中CRF m RNA表达（0.76±0.11,0.67±0.10 v s 1.09±0.09�

Behavior and brain gene expression changes in mice exposed to preimplantation and prenatal stress.

Preimplantation culture of mouse embryos has been suggested to result in reduced anxiety-like behavior in adulthood. Here, we investigated the effects of in vitro fertilization (IVF), embryo culture, and different diets on anxiety-like behavior using the elevated plus maze (EPM). We hypothesized that exposure to suboptimal conditions during the preimplantation stage would interact with the suboptimal diet to alter behavior. The expression of genes related to anxiety was then assessed by quantitative real-time polymerase chain reaction in various brain regions. When fed a normal diet during gestation and a moderately high-fat Western diet (WD) postnatally, naturally conceived (NC) and IVF mice showed similar anxiety-like behavior on the EPM. However, when fed a low-protein diet prenatally and a high-fat diet postnatally (LP/HF), NC mice showed a modest increase in anxiety-like behavior, whereas IVF mice showed the opposite: a strongly reduced anxiety-like behavior on the EPM. The robust reduction in anxiety-like behavior in IVF males fed the LP/HF diets was, intriguingly, associated with reduced expression of MAO-A, CRFR2, and GABA markers in the hypothalamus and cortex. These findings are discussed in relation to the developmental origin of health and disease hypothesis and the 2-hit model, which suggests that 2 events, occurring at different times in development, can act synergistically with long-term consequences observed during adulthood.

Sex dependent reduction by prenatal stress of the expression of 5HT1A receptors in the prefrontal cortex and CRF type 2 receptors in the raphe nucleus in rats: reversal by citalopram

Alterations in the serotonergic transmission and activity of corticotropin-releasing factor (CRF) family may underlie anxiety and depressive disorders. These could be corrected by treatment with SSRIs. The objective of the current study is to determine whether the increased anxiety of prenatally stressed (PS) rats of both sexes is associated with changes in 5HT1A and CRF type 2 receptors (5HT1AR and CRFR2) in the prefrontal cortex (PFC)-dorsal raphe nuclei (DRN) axis, and how these are affected by chronic treatment with citalopram (10 mg/kg/day). We focussed on GABAergic cells that co-express parvalbumin and/or neuropeptide Y, and 5HT1AR in the medial prefrontal cortex (mPFC) and on cells that express 5HT, parvalbumin, 5HT1AR or CRFR2 in the DRN. Immunohistochemistry with fluorescent antibodies demonstrated sex differences in the expression of 5HT1AR and CRFR2 protein. Prenatal stress selectively reduced the expression of 5HT1AR on GABAergic cells in the mPFC in males and that of CRFR2 in the DRN of females. Citalopram treatment for 5 weeks abolished the increase in anxiety in both sexes, restored the intensity of expression of 5HT1AR in the mPFC in males and increased their expression in the mPFC and DRN in females. Citalopram reduced CRFR2 expression in control and PS males but increased it in PS females. Male and female rats show differences in the expression of 5HT1AR and CRFR2 protein that are selectively reduced by prenatal stress. Reversal by citalopram of the changes in the expression of these receptors induced by prenatal stress support their role in the aetiology of anxiety.

Effect of restraint stress on intestinal mucosal barrier function in middle cerebral artery occulusion rats

Objective To investigate the effect of restrain stress on intestinal mucosal barrier function in middle cerebral artery occulusion (MCAO) rats and its mechanism.Methods Male SD rats were randomly divided into sham group (J),sham-operated plus restraint group (JS),MCAO group (M),and MACO plus restraint group (MS) (n =12 each).Improved Longa suture method was used to establish MCAO model in rats,and rat fixator to creat restraint stress model.Serum diamineoxidase (DAO),D-lactic acid (D-LA),endotoxin (LPS),and corticotropin releasing factor (CRF) levels,tight junction proteins in ileum tissue,CRF,CRF-r1 and CRF-r2 expression levels in ileum tissue,and CRF,tumor necrosis factor (TNF)-α and CRP expression levels in the brain tissues were measured.Results The serum DAO,LPS and CRF levels in MS group were (693.96 ± 55.38) U/L,(147.04 ± 2.93) μg/L,and (194.03 ± 19.80) ng/L respectively,significantly higher than in M group (all P ＜ 0.05).The levels of tight junction proteins ZO-1,occluding,claudin-1 and claudin-2 levels in the ileum tissue of MS group were 0.52 ± 0.05,0.58 ± 0.03,0.57 ± 0.05 and 0.35 ± 0.07 respectively,significantly higher than in M group (all P ＜ 0.05).The expression levels of claudin-3 and claudin-4 in MS group were 0.59 ± 0.40 and 0.58 ± 0.03 respectively,significantly higher than M group (all P ＜ 0.05).The CRF and CRF-r1 expression levels in ileum tissue,and CRF and CRP expression levels in brain tissues of MS group were 173.80 ± 34.34,169.56 ± 26.99,115.27 ± 17.79 and 135.27 ± 40.67 respectively,significantly higher than M group (all P ＜ 0.05).Conclusion Restrain stress aggravated intestinal mucosa barrier damage in MCAO rats. Key words: Restraint stress; Intestinal mucosal barrier; Rat

Effects of Bifidobacterium infantis on intestinal corticotropin releasing factor receptors and mast cells activation in a murine model of irritable bowel syndrome

Objective To investigate the effects of Bifidobacterium infatn ison the expression of in -testinal corticotropin releasing factor （ CRF） receptors and how the peripheral CRF receptors activate mast cells in a murine model of irritable bowel syndrome （IBS）.Methods Thirty BALB/c male mice were ran-domly divided into three groups including control group , model group and Bifidobacterium infantis group. The mouse model of IBS was established by using chronic restraint stress .Mice in Bifidobacterium infantis group received daily intragastrical administration of Bifidobacterium infantis for 14 days.Mice in control and model groups were treated with equal volume of saline .Then all mice were killed after the assessment of weight and abdominal withdrawal reflex （ AWR） .The levels of histamine , tryptase and tumor necrosis fac-tor-α（ TNF-α） in serum samples were detected by ELISA .The expression of CRF in colonic mucosa was analyzed by immunohistochemistry .The expression of CRF-R1 and CRF-R2 in mast cells and the number of mast cells in colonic mucosa were detected by double immunofluorescence staining assay .The expression of CRF-R1 and CRF-R2 at mRNA level in colon were detected by reverse transcription polymerase chain reac-tion （ RT-PCR） .Results Compared with control group , the levels of histamine , tryptase and TNF-αin pe-ripheral blood samples , the expression of CRF-R1 and CRF-R2 at mRNA level , and the number of mast cells, CRF-R1＋mast cells and CRF-R2＋mast cells in colonic mucosa were increased significantly in model group （P〈0.05）, but were remarkably down-regulated with the treatment ofB ifidobacterium infantis （P〈0.05）.Conclusion Bifidobacterium infantis could reduce the activation of mast cells in a murine model of IBS by inhibiting the expression of CRF-R1 and CRF-R2 in intestinal mast cells . Key words: Irritable bowel syndrome; Probiotics; Corticotropin releasing factor; Mast cell

Hypoactivation of CRF receptors, predominantly type 2, in the medial-posterior BNST is vital for adequate maternal behavior in lactating rats.

Maternal behavior ensures the proper development of the offspring. In lactating mammals, maternal behavior is impaired by stress, the physiological consequence of central corticotropin-releasing factor receptor (CRF-R) activation. However, which CRF-R subtype in which specific brain area(s) mediates this effect is unknown. Here we confirmed that an intracerebroventricularly injected nonselective CRF-R antagonist enhances, whereas an agonist impairs, maternal care. The agonist also prolonged the stress-induced decrease in nursing, reduced maternal aggression and increased anxiety-related behavior. Focusing on the bed nucleus of the stria terminalis (BNST), CRF-R1 and CRF-R2 mRNA expression did not differ in virgin versus lactating rats. However, CRF-R2 mRNA was more abundant in the posterior than in the medial BNST. Pharmacological manipulations within the medial-posterior BNST showed that both CRF-R1 and CRF-R2 agonists reduced arched back nursing (ABN) rapidly and after a delay, respectively. After stress, both antagonists prevented the stress-induced decrease in nursing, with the CRF-R2 antagonist actually increasing ABN. During the maternal defense test, maternal aggression was abolished by the CRF-R2, but not the CRF-R1, agonist. Anxiety-related behavior was increased by the CRF-R1 agonist and reduced by both antagonists. Both antagonists were also effective in virgin females but not in males, revealing a sexual dimorphism in the regulation of anxiety within the medial-posterior BNST. In conclusion, the detrimental effects of increased CRF-R activation on maternal behavior are mediated via CRF-R2 and, to a lesser extent, via CRF-R1 in the medial-posterior BNST in lactating rats. Moreover, both CRF-R1 and CRF-R2 regulate anxiety in females independently of their reproductive status.

우울증 생쥐 모델에서 반하후박탕가미(半夏厚朴湯加味)의 항우울 효과

Objectives: The purpose of this study was to examine the anti-depressive effects of BanHaHuBakTang-kami (BHHBT) on an animal model of depression induced by chronic immobility stress. Methods: Mice were treated daily with immobilization stress for 2 hours over a period of 21 days. To examine the effect of BHHBT, we performed behavioral, biochemical and histological analysis to measure immobility time (FST), brain neurotransmitter concentration (HPLC, ELISA), hippocampal damage (H&E staining) and CRF-R1 expression (immunohistochemistry). Results: BHHBT has reduced the immobility time of immobilization stress exposed mice in the forced swimming test. BHHBT has increased the amount of serotonin in the brain. BHHBT has increased the expression level of serotonin in the brain. BHHBT 540 mg/kg were sufficient to prevent tissue damage in the hippocampus region. BHHBT has reduced the expression level of CRF receptors in the hippocampus region. Conclusions: These results suggest that BHHBT may have anti-depressive effects on mice treated with immobilization stress by reducing immobility, increasing brain serotonin concentration and reducing CRF-R1 expression in the hippocampus region.

Strain differences in stress-induced changes in central CRF1 receptor expression

The Wistar Kyoto (WKY) rat is genetically predisposed to increased sensitivity to psychological and physical stressors. Evidence points towards the importance of corticotropin-releasing factor (CRF), a peptide secreted by the paraventricular nucleus of the hypothalamus, in this strain's aberrant response to stress. CRF binds to CRF1 and 2 receptors (CRFR1 and CRFR2) which are expressed in both hypothalamic and extra-hypothalamic brain regions. Phosphorylation of the signal transduction molecule, extracellular signal regulated kinase (ERK)1/2 has been linked with stress and the actions of CRF. Western blotting techniques were employed to examine changes in protein expression of CRFR1 and phosphorylated ERK1/2 in hypothalamic and extra-hypothalamic brain regions of open field-stressed Sprague Dawley (SD) and WKY rats. Stress exposure resulted in increased hypothalamic ERK1/2 phosphorylation and subsequent increases in CRFR1 expression in SD but not WKY rats. In extra-hypothalamic brain regions, the stressor caused decreased or unchanged ERK 1/2 phosphorylation in both strains. A potentiated increase in CRFR1 expression was noted in the frontal cortex of WKY rats following the stressor and expression of CRFR1 was reduced in the hippocampus of WKY rats. These data demonstrate region-specific differences in stress-induced changes in expression of CRF receptors and intracellular signaling molecules in stress-sensitive WKY rats and stress-resilient SD rats.

Urocortin 2 is associated with abdominal aortic aneurysm and mediates anti-proliferative effects on vascular smooth muscle cells via corticotrophin releasing factor receptor 2

AAA (abdominal aortic aneurysm) is an important cause of sudden death in older adults, but there is no current effective drug therapy for this disease. The UCNs (urocortins1-3) and their receptors: CRFR (corticotrophin-releasing factor receptor)-1 and -2 have been implicated in various CVDs (cardiovascular diseases). We assessed the relative expression of UCN1-3in AAA by qRT-PCR (quantitative reverse transcription-PCR) and ELISA, and examined invitro how UCN2 affects human aortic VSMC (vascular smooth muscle cell) Akt phosphorylation, pro-inflammatory cytokine IL (interleukin)-6 secretion, proliferation, cell cycle and apoptosis. UCN2 and CRFR2 expression were significantly up-regulated in biopsies from the AAA body. AAA body biopsies released high amounts of UCN2 invitro. Median plasma UCN2 concentrations were 2.20 ng/ml (interquartile range 1.14-4.55 ng/ml, n=67) in AAA patients and 1.11 ng/ml (interquartile range 0.76-2.55 ng/ml, n=67) in patients with non-aneurysmal PAD (peripheral artery disease) (P=0.001). Patients with UCN2in the highest quartile had a 4.12-fold (95% confidence interval, 1.37-12.40) greater prevalence of AAA independent of other risk factors, P=0.012. Invitro, UCN2 significantly inhibited VSMC Akt phosphorylation and proliferation in a dose-dependent manner. UCN2 induced VSMC G1 cell-cycle arrest and increased IL-6 secretion over 24h. The CRFR2 antagonist astressin-2B significantly abrogated the effects of UCN2 on VSMCs. In conclusion, UCN2 is significantly associated with AAA and inhibits VSMC proliferation by inducing a G1 cell cycle arrest suggesting a plausible regulatory role in AAA pathogenesis.

MiR-449a Contributes to Glucocorticoid-Induced CRF-R1 Downregulation in the Pituitary During Stress

The hypothalamic-pituitary-adrenal axis is controlled by the feedback of glucocorticoids on the hypothalamus and pituitary. Stress increases CRF, ACTH, and glucocorticoid secretion. The expression of not only CRF mRNA in the hypothalamus and proopiomelanocortin mRNA in corticotrophs, but also CRF type 1 receptor (CRF-R1) mRNA and protein on corticotrophs are downregulated through glucocorticoids. However, the mechanisms underlying the glucocorticoid-induced CRF-R1 downregulation are not fully understood. Short RNA molecules, called microRNAs (miRNAs), are posttranscriptional regulators that usually induce translational repression or gene silencing via binding to complementary sequences within target mRNAs. We hypothesized that glucocorticoids may induce the expression of miRNAs in the pituitary, which are involved in glucocorticoid-induced downregulation of CRF-R1. We found 3 miRNAs with sequences predicted to bind to the CRF-R1 3' untranslated region (3'-UTR) by database search. Expression of 1 of these miRNAs (miR-449a) was significantly higher in the anterior pituitary of restrained rats than in that of unrestrained control rats. Expression of miR-449a was evident in many anterior pituitary cells, including corticotrophs. Although overexpression of miR-449a decreased CRF-R1 mRNA and CRF-R1 protein expression, knockdown of miR-449a attenuated dexamethasone-induced suppression of CRF-R1 mRNA and CRF-R1 protein expression in the monolayer-cultured pituitary cells. Notably, luciferase activity was significantly lower in cells cotransfected with a luciferase vector containing the CRF-R1 3'-UTR and a miR-449a vector. miR-449a expression was significantly increased by dexamethasone. Adrenalectomy attenuated restraint-induced increase in miR-449a expression in the pituitary. These results indicated that miR-449a plays an important role in stress-induced, glucocorticoid-mediated downregulation of CRF-R1 expression.