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ObjectiveTo observe the effects of Xiangsha Liu Junzitang （XSLJZ） on gastric emptying rate and expression levels of corticotropin-releasing factor （CRF） and urocortin 2 （UCN2） in rats with functional dyspepsia （FD） due to spleen deficiency. MethodForty-eight 10-day-old male SD rats were randomly divided into the normal group （n=8） and iodoacetamide （IA） group （n=40）， and they separately received 2% sucrose solution and 0.1% sucrose solution containing IA for six successive days. Following the removal of mother rats， the three-week-old IA-treated rats were randomized into five groups， namely the model group， mosapride group， and low-， middle-， and high-dose XSLJZ groups， with eight rats in each group. At the age of six weeks， rats in all groups expert for the normal group were modeled by the modified multiple platform method for 14 d. Afterwards， the ones in normal group and model group were treated with 10 mL·kg-1 distilled water， and those in the treatment groups with 1.6×10-3 g·kg-1 mosapride and 2.8， 5.6， and 11.2 g·kg-1 XSLJZ by gavage， respectively， for 14 d. The grasping ability and gastric emptying rate were determined. The histological changes in gastric antrum were observed by hematoxylin-eosin （HE） staining. The protein and mRNA expression levels of CRF and UCN2 in gastric antrum were detected by Western blot and real-time fluorescent quantitative polymerase chain reaction （Real-time PCR）. ResultNo obvious change or organic lesion was observed in gastric antrum of rats in each group. Compared with the normal group， the model group exhibited lowered gastric emptying rate and grasping ability （P<0.01）， up-regulated CRF protein and mRNA expression in gastric antrum （P<0.01）， and down-regulated UCN2 protein and mRNA expression （P<0.05， P<0.01）. Compared with the model group， XSLJZ at the middle and high doses enhanced the grasping ability and gastric emptying rate （P<0.05， P<0.01） and down-regulated CRF mRNA expression to varying degrees （P<0.05， P<0.01）. XSLJZ at the high dose decreased CRF protein expression （P<0.05） and up-regulated UCN2 protein and mRNA expression （P<0.01）. ConclusionThe mechanism of XSLJZ in invigorating spleen and promoting gastric motility of FD rats may be related to its reduction of CRF and elevation of UCN2 in gastric antrum.
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Background: Preterm labor classically defined as delivery before completed 37 gestational weeks. Urocortin a biomarker that have raised recent research interest is a 40-amino acid neuropeptide related to the corticotrophin-releasing factor molecular family. Interestingly urocortin is produced by gestational tissue such as amnion and chorion predictability of preterm labor by biomarker assay could enhance management levels particularly in cases of preterm labor that are considered a frequent clinical scenario in obstetric practice. Aim of the study was to assess and evaluate the serum levels of urocortin predictability capacity in cases that develop preterm labor.Methods: The current research clinical trial was conducted in a prospective way there was two research groups 60 study subjects had threatened preterm labor and 60 normal research study subjects that delivered at term. Comparative analysis was performed for urocortin assay conducted in both research groups in correlation to gathered clinical data obtained from both research groups.Results: Receiver operating characteristic curve (ROC) between preterm and term delivery research groups as regards plasma urocortin level (pg/ml) as a predictor of pre term delivery showing that a cut-off point level >101.3 pg/ml in which statistical sensitivity=88.33%, statistical specificity=75%, positive predictive value=77.9, negative predictive value=86.5.Conclusions: This research finding reveal that maternal serum urocortin is an effective biomarker in predictability of preterm labor; however future research studies should be multicentric in fashion putting in consideration the racial and ethnic differences besides the impact of BMI on maternal serum urocortin indices.
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Aim To observe the effects of UCN on TGF-β1 and CTGF in rats with DCM, and to explore the protective effects of UCN on DCM and its possible signaling pathway. Methods The rats were divided into five groups: Control group, DCM group, UCN group, UCN + Astressin group, and UCN + Triciribine group. After 12 weeks of feeding and 4 weeks of treatment, blood glucose, urinary sugar, urine volume and the levels of TGF-β1, CTGF in serum were determined, and the expression of TGF-β1, CTGF, Akt, GSK-3β, p-Akt and p-GSK-βof cardiomyocytes were also determined. Results The myocardial HE staining in DM rats was consistent with DCM. The levels of TGF-β1 and CTGF in serum and myocardium of rats with DCM increased significantly, while the levels of p-Akt and p-GSK-3J3 in myocardium of rats with DCM decreased significantly ( P < 0. 05 ). The levels of TGF-β1 and CTGF in UCN group decreased, both as-tressin and triciribine could inhibit the role of UCN, and the expression of p-Akt and p-GSK-3(3 in myocardial cells in UCN group increased significantly, then astressin could inhibit the role of UCN ( P < 0. 01). Conclusions The protective effects of UCN on DCM might be related to the activation of Akt/GSK-30 signaling pathway after binding with CRH-R receptor, and then decreasing the expression of TGF-β1 and CTGF.
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Objective To investigate the effect of Urocortin I pretreatment on heart function and ATP in rat heart during myocardial ischemia-reperfusion injury. Methods Part I. 48 SD rats were randomly divided into four groups(n=12):normal group(group N1),ischemia-reperfusion group(group IR),urocortin I pretreatment group (group U1),and 5-HD+urocortin I group (group HU1). Group N1 received continuous perfusion for 155 min ,group IR experienced 40 min ischemia and 60 min reperfusion ,group U1 received urocortin I for 30 min before ischemia,and group HU1 received 5-HD for 5 min before urocortin I pretreatment. Changes of heart rate (HR),left ventricular end-diastolic pressure(LVEDP),left ventricular developed pressure(LVDP),maximum dp/dt(dp/dtmax)and microstructure of myocardium were evaluated. Part Ⅱ. The isolated and cultivated myocytes were randomly divided into four groups:group N2,HR,U2 and HU2. Group N2 was cultured continuously for 155 min,while other groups experienced 40 min-anoxia and 60 min reoxygenation. Urocortin I was added to group U2 for 30 min before anoxia. 5-HD was added to group HU2 before adding urocortin I. The content of ATP at the end of reoxygenation was detected. Results Urocortin I pretreatment restrained the decline in HR,LVDP,and dp/dt and the rise in LVEDP during ischemia-reperfusion injury. Furthermore ,the content of ATP was preserved and the microstructure of myocardium was sustained during anoxia/reperfusion. Conclusions Urocortin I pretreatment can not only enhance cardiac contractility of the isolated heart ,but also preserve the content of ATP through the opening of mitoKATP channel,thus maintaining the microstructures of myocytes and improving heart function after ischemia-reperfusion injury.
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Objective To investigate the effect of Urocortin I pretreatment on heart function and ATP in rat heart during myocardial ischemia-reperfusion injury. Methods Part I. 48 SD rats were randomly divided into four groups(n=12):normal group(group N1),ischemia-reperfusion group(group IR),urocortin I pretreatment group (group U1),and 5-HD+urocortin I group (group HU1). Group N1 received continuous perfusion for 155 min ,group IR experienced 40 min ischemia and 60 min reperfusion ,group U1 received urocortin I for 30 min before ischemia,and group HU1 received 5-HD for 5 min before urocortin I pretreatment. Changes of heart rate (HR),left ventricular end-diastolic pressure(LVEDP),left ventricular developed pressure(LVDP),maximum dp/dt(dp/dtmax)and microstructure of myocardium were evaluated. Part Ⅱ. The isolated and cultivated myocytes were randomly divided into four groups:group N2,HR,U2 and HU2. Group N2 was cultured continuously for 155 min,while other groups experienced 40 min-anoxia and 60 min reoxygenation. Urocortin I was added to group U2 for 30 min before anoxia. 5-HD was added to group HU2 before adding urocortin I. The content of ATP at the end of reoxygenation was detected. Results Urocortin I pretreatment restrained the decline in HR,LVDP,and dp/dt and the rise in LVEDP during ischemia-reperfusion injury. Furthermore ,the content of ATP was preserved and the microstructure of myocardium was sustained during anoxia/reperfusion. Conclusions Urocortin I pretreatment can not only enhance cardiac contractility of the isolated heart ,but also preserve the content of ATP through the opening of mitoKATP channel,thus maintaining the microstructures of myocytes and improving heart function after ischemia-reperfusion injury.
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Aim To investigate the effects of endocrinal petptide urocortin on subthalamic nucleus (STN) neuron''s discharge, also observe the convergence effect of UCN with dopamine (DA) and glutamate (GLU), so as to understand the regulation effects of UCN and its mechanism in Parkinson''s disease (PD).Methods Forty Sprague-Dawley rats were used in this experiment.Nerve electrophysiology method-microiontophoresis was used to observe the effects of UCN on STN neuron firing rates and firing wave.Astressin (AST, the blocker of CRF receptor 2), protein kinase A (PKA) were used to observe the effects of UCN whether via CRF-2R and PKA signal pathway.Moreover, given UCN during the period of DA and GLU, the effects of UCN on DA and GLU in STN neurons were determined.Results During the period of using the UCN, UCN could inhibit the firing rate of 82％ (27/33) STN neuron (P<0.01), and the firing discharge rates were reduced from(3.65±0.27)Hz to (2.05±0.33) Hz (P<0.01).However, the inhibitory effects of UCN in STN could be antagonized by AST.Given UCN during the period of microiontophoresis of inhibitory neurotransmitter (DA) and excited neurotransmitter (GLU), UCN could enhance the effects of DA and attenuate the excitatory effects of GLU (P<0.01).Conclusion UCN and GLU/DA in STN, UCN play inhibitory and regulated effects on STN neurotransmitters(DA and GLU)via CRF-2 receptor and PKA signal pathway.
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Aim To investigate the inhibitory effects of urocortin2 (UCN2) on ventral tegmental area (VTA) nervous activity of morphine addiction rats and the mechanism. Methods Morphine addiction rats and the microiontophoresis method were used to observe the effects of UCN2 on VTA neuron′s spontaneous dis-charge changing rule, as well as the inhibitory effects of UCN2 on DA neuron′s cluster spontaneous dis-charge, to identify UCN2 and DA on the same VTA neuron. Morever, the inhibitor of corticotropin-regula-ting factor′s receptor ( CRF-2 R ) and the blocker of protein kinase A ( PKA ) , AST-2 B and H89 , were used to investigate the effects of UCN2 on VTA neuron′s of morphine addiction rats. Results UCN2 could inhibit the firing rate 82% (31/38) of the tested VTA neuron ( P<0. 01 ) , the discharge frequency changed from (20. 89 ± 2. 86) Hz to (13. 66 ± 3. 93) Hz (P<0. 01). Further, the inhibitor of PKA, AST-2B and H89 could ablolish the inhibitory effects of UCN2 . Morever, the excitatory firing of VTA neurons was at-tenuated by addition of UCN2 , while AST application could inhibit the UCN2′s inhibitory effects. Conclu-sion UCN2 could regulate the effects of VTA via PKA pathway and may thereby contribute to the improvement of drug addiction.
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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.
Receptores de CRF estão envolvidos na gestão do estresse das células e são acreditados para ter um papel de cito-proteção no organismo. Os receptores do CRF têm sido relatados como alvos potenciais de fármacos para o tratamento de doenças neurodegenerativas. A linhagem celular utilizada no estudo é ND7/23 (neuroblastoma de camundongo e hibridoma de raíz dorsal do neurônio ganglionar de rato). O objetivo do estudo foi confirmar o que a expressão de receptores de CRF em células ND7/23 determinar se urocortina (Ucn) pode aumentar a expressão de receptores de CRF. Cultivaram-se células ND7/23 em meio RPMI 1640 e as células que cresceram após a segunda passagem foram usadas para os experimentos. O RNA foi extraído células e amplificado por RT-PCR para confirmar a presença de receptores de CRF. As células foram, então, submetidas a estresse oxidativo por peróxido de hidrogênio (0.00375 %) e divididas em dois grupos, ou seja, controle e tratadas com UCN (10-8 µM). Em seguida, o RNA foi extraído de ambos os grupo de células e realizou-se o PCR. Finalmente, realizou-se análise densitométrica em gel de agarose para determinar a quantidade de produto formado por PCR. O PCR confirmou a expressão de CRF-R1 e CRF-R2 na linhagem celular, mas o CRF-R1 expresso mais fortemente. A análise densitométrica do produto de PCR e o cálculo da expressão relativa de receptores de CRF indicaram um nível mais elevado de expressão de receptores de CRF em amostras tratadas com Ucn, em comparação com aqueles sem tratamento. Os resultados indicam que a Ucn pode ser útil no tratamento de doenças neurodegenerativas e mais estudos podem ser realizados para estabelecer seu uso como agente terapêutico.
Subject(s)
Adrenocorticotropic Hormone/pharmacokinetics , Urocortins/analysis , Neurodegenerative Diseases/classification , NeuroblastomaABSTRACT
Objective To study the regulatory effect of urocortin (UCN) on ischemia/reperfusion (I/R)-induced myocardial autophagy, so as to explore the myocardial protection mechanism of UCN. Methods Cardiac I/R model was established with rats and hypoxia/reoxygenation(H/R) model was also established with neonatal rat cardiomyocytes. The injury was created by ischemic/hypoxia for 1 h plus reperfusion/reoxygenation for 2 h, and UCN pretreatment was given 1 h before ischemia/hypoxia. The I/R or H/R-induced myocardial injury, myocardial autophagy and autophagy-related gene expression were observed 2 h after reperfusion/reoxygenation. Results UCN pretreatment greatly reduced I/R-induced myocardial damage by decreasing the infarct size, serum creatine kinase (CK) and lactate dehydrogenase (LDH) concentration, increasing the vitality of H/R cardiomyocytes in vitro, and reducing LDH level in the culture supernatant. Moreover, UCN pretreatment also inhibited H/R-induced myocardial autophagy by reducing the ratio of LC3BII/LC3B I and inhibiting expression of autophagy-related genes (Beclin1 and Bnip3). Conclusion UCN can inhibit I/R-induced myocardial autophagy, which may play an important role in the protection against I/R injury.
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AIM To investigate the effects and mechanism of cardiomyocyte hypertrophy induced by urocortin. METHODS The cardiomyocytes were divided into 8 groups: normal control, urocortin, staurosporine(Sta), verapamil(Ver), H89, urocortin+Sta, urocortin+Ver, and urocortin+H89 groups. The cardiomyocytes diameter was measured by computer photograph analysis system. The protein synthetic rate was obtained through measuring the incorporation of [~3H]-leucine into myocyte protein by liquid scintillation method. The total protein content was assayed by Lowry method. The expression of atrial natriuretic peptide (ANP) was determined by Western blot. [Ca~(2+)]_i transient was measured by Till image system by cell loading Fura-2/AM. RESULTSUrocortin group enhanced cardiomyocyte volume, protein synthesis, total protein content and expression of ANP by 30.9%, 36.3%, 35.5% and 34.7%;urocortin+Sta group decreased cardiomyocyte diameter, protein synthesis, total protein content and expression of ANP by 16.5%, 22.1%, 18.1% and 21.3%;urocortin+H89 group decreased the cardiomyocyte diameter, the protein synthesis,total protein content and expression of ANP by 16.6%, 21.5%, 19.5% and 20.6%;urocortin+Ver decreased the cardiomyocyte diameter, the protein synthesis, total protein content and the expression of ANP by 17.1%, 20.9%, 17.9% and 19.9%;Sta, H89 and Ver could decrease the [Ca~(2+)]_i transient induced by urocortin. CONCLUSION The hypertrophic effect of urocortin in rat neonatal cardiomyocytes is mediated via activation of protein kinase C and protein kinase A pathway and L-type calcium channels.
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Objective:To investigate the effects of Urocortin-postconditioning on mitochondria-dependentapoptotic pathway.Methods: 24 Wistar rats were randomly divided into 3 groups(n=8):Control group(CON),I/R group(I/R),Urocortin group(UCN).The left ventricular samples in the Control group were collected as pre-ischemia controls through thoracotomy.After isolated heart Langendoff and Neely models were established,the rat hearts in the I/R group and the UCN group were continuously perfused with Krebs-Henseleit (K-H)buffersolution for 30 min,then the rat hearts were arrested by cardioplegia,St.Thomas-2 solution(STH-2)for 30min.After that,the rat hearts in the I/R group were reperfused with K-H buffer solution for 90 min.The rat hearts in the UCN group were reperfused with K-H buffer solution containing 10-8 mol/L Urocortin for 90 min.The releasing of Cyto C protein into cytosol was detected by Western blot.The expression of Caspase-3 protein in cardiomyocyte was detected by immunohistochemical assay.The apoptosis index (AI)of cardiomyocyte was detected by TUNEL.Results:Compared with Control group,the expression ofCaspase-3 and Cyto Cprotein in the I/R group and the UCN group were significantly increased(P
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Corticotropin releasing factor (CRF) is known to be involved in the stress response and in some degenerative brain disorders. In addition, CRF has a role as a neuromodulator in adult cerebellar circuits. Data from developmental studies suggest a putative role for CRF as a trophic factor during cerebellar development. In this study, we investigated the trophic role for CRF family of peptides by culturing cerebellar neurons in the presence of CRF, urocortin or urocortin II. Primary cell cultures of cerebella from embryonic day 18 mice were established, and cells were treated for either 1, 5 or 9 days with Basal Medium Eagles complete medium alone or complete medium with 1 micrometer CRF, urocortin, or urocortin II. The number of GABA-positive neurons in each treatment condition was counted at each culture age for monitoring the changes in neuronal survival. Treatment with 1 micrometer CRF or 1 micrometer urocortin increased the survival of GABAergic neurons at 6 days in vitro and 10 days in vitro, and this survival promoting effect was abolished by treatment with astressin in the presence of those peptides. Based on these data, we suggest that CRF or urocortin has a trophic role promoting the survival of cerebellar GABAergic neurons in cultures.
Subject(s)
Mice , Animals , gamma-Aminobutyric Acid/metabolism , Time Factors , Receptors, Corticotropin-Releasing Hormone/metabolism , Peptides/chemistry , Neurons/metabolism , Mice, Inbred C57BL , Immunohistochemistry , Image Processing, Computer-Assisted , Corticotropin-Releasing Hormone/biosynthesis , Cerebellum/embryology , Cells, Cultured , Cell SurvivalABSTRACT
Aim To investigate the vasodilatory effect and the mechanisms of urocortin(Ucn) on the thoracic aorta of spontaneously hypertensive rats(SHR).Methods Rings cut from SHR thoracic aorta were used in vitro.The endothelium dependent and independent vasorelaxing effects of Ucn were measured.Furthermore,it was also explored whether the relaxing effects of Ucn were affected by N~((?)) nitro-L-arginine methyl ester(L-NAME), methylene blue(MB) and glybenclamide.Results Ucn(1 nmol?L~(-1)～1 ?mol?L~(-1)) caused concentration dependent relaxation in SHR thoracic aorta with endothelium and without endothelium(P
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AIM: To obverse the expression and localization of urocortin on ultrathin cryosections of syncytiotrophoblast of human term placenta with immunocytochemistry technique under transmission electron microscope. METHODS: The human term placenta tissue from Cesarean delivery and normal labor were fixed in 4% paraformaldehyde, and then divided into two parts. One part was for regular immunocytochemistry under microscope, and the other part was used to prepare ultrathin cryosections for immunocytochemistry under transmission electron microscope. RESULTS: 1.Uroncortin mainly distributed in cytoplasm of syncytiotrophoblast of human term placenta under microscope. Urocortin also appeared in cytoplasm in some stromal cells. 2. Under transmission electron microscope, the anti-urocortin gold particles were observed in cytoplasm of syncytioptrophoblast ultrathin cryosections and sited on rough-surfaced endoplasmic reticulum. The anti-urocortin gold particles also appeared on nucleus and nuclear membrane of syncytiotrophoblast. CONCLUSION: Syncytiotrophoblast of human term placenta synthesized and secreted urocortin. The internalization of urocortin within syncytiotrophoblast nuclear indicates that urocortin may act as intracrine.
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Urocortin,a novel peptide of the cortiotropin releasiong hormone(CRH)family,was discovered in 1995 in the rat brain and subsequently cloned and localized to human chromosome 2.It can bind to both CRH type 1 receptor and CRH type 2 receptor,as well as CRH binding protein.Especially to CRH type 2 receptor,urocortin binds to it with 40 times more potency than CRH does.Therefore,urocortin is thought to be an endogenous ligand for CRH type 2 receptor.Immunoreactive urocortin and urocortin mRNA were localized in many areas such as mammalian brain,cardiovascular system,placenta.It is believed that urocortinmay play important physiological roles as a neuropeptide not only in the central nervous system but also in peripheral tissues.
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AIM: To study the effects of urocortin(Ucn) on the isolated heart tissues of spontaneously hypertensive rat(SHR).METHODS: Effects of Ucn on contractile force and heart rate were observed in the SHR and Wistar rat right atrium,left atrium and right ventricle strip.RESULTS: Ucn(1-10 nmol/L) concentration-dependently increased the contractile force in the SHR and Wistar rat isolated right atrium.Ucn increased the contractile force in the SHR by(31.1?14.9)% at 3 nmol/L and by(65.7?22.4)% at 10 nmol/L,and its inotropic effect was significantly greater than that in Wistar rat(P