Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

Effect of neuregulin on expressions of apoptosis and nuclear factor kappa B in hippocampus of Alzheimer's disease model rats / 中华行为医学与脑科学杂志

ObjectiveTo investigate the effect of neuregulin1β (NRG1β) on the learning memory abilities and the neuronal apoptosis and the expressions of nuclear factor kappa B (NFκB) in experimental Alzheimer's disease model in rats induced with beta-amyloid protein1-40 (Aβ1-40) injection.To explore the mechanisms of NRG in improving the capabilities of learning and memory.MethodsThirty adult healthy male wistar rats were randomly divided into control group (n =10),model group (n =10) and treated group (n =10).Alzheimer's disease models were established by stereotactically injecting Aβ1-40 into the left lateral ventricle,and treated by injecting NRG1β(0.3 μg · kg-1 ) into the right lateral ventricle.The learning and memory abilities of rats were evaluated with Y-electric maze before the experiment and 7 days after making Alzheimer's disease models and 14 days after treatment.HE staining was used to observe the structure of hippocampal neurons.The neuronal apoptosis of hippocampus was investigated by TUNEL assay.The expressions of NFκB in hippocampal neurons were determined with immunohistochemistry technique.ResultsCompared with control group (57.50 ± 1.58,7.20 ±1.03 ),the model group rats ( 59.50 ± 2.79,7.50 ± 1.08 ) showed low cognitive ability ( t =20.36,5.28,P ＜0.05 ),the hippocampal pyramidal cells of rats in the model group were sparse and disturbed pyramidal cells,noticeable neuron loss.The number of neuronal apoptosis and the expressions of NFκB increased significantly than those in control group (P＜0.05).Compared with model group (79.10 ±4.12,4.40 ±0.69),NRG1β strikingly improved cognitive ability ( 67.70 ± 4.90,5.80 ± 0.63 ) and normal cell structure ( t =5.63,4.69,P ＜ 0.05 ).The expressions of NFκB (25.90 ± 6.67 ) reduced while the number of neuronal apoptosis ( 23.50 ± 3.89 ) decreased markablely than those ( 41.10 ±7.95,29.30 ± 7.24) in model group(t =4.63,2.23,P ＜ 0.05).ConclusionNRG1β might decrease the neuronal apoptosis by inhibiting NFκB expressions,so that to improve the learning and memory abilities of experimental dementia rats.

Neuregulinas 1-alfa e 1-beta na regeneração de nervos periféricos / Neuregulins 1-alpha and 1-beta on the regeneration the peripheral nerves

OBJETIVO: Avaliar o efeito das neuregulinas 1-alfa e 1-beta na regeneração de nervos ciáticos de camundongos C57BL/6J, adultos, machos, através da técnica de tubulização. MÉTODOS: Utilizaram-se 18 animais, divididos em 3 grupos, implantando-se prótese de polietileno em falhas de 4,0 mm no nervo ciático esquerdo: grupo 1 contendo apenas colágeno purificado (Vitrogen®); grupo 2, colágeno associado a neuregulina 1-alfa; grupo 3 com colágeno e neuregulina 1-beta. O grupo controle foi formado por 6 segmentos de nervos ciáticos direitos. Após 4 semanas, os animais foram sacrificados; extraiu-se segmento do ponto médio do nervo regenerado no interior das próteses, padronizaram-se cortes histológicos e confecção das lâminas para análise histomorfométrica. Confrontaram-se os resultados estatisticamente. RESULTADOS: Os animais tratados com neuregulinas tiveram maior número de axônios mielinizados, com diferença estatisticamente significante quando comparados ao grupo colágeno. Não houve diferença estatística entre os grupos de neuregulinas 1-alfa e 1-beta. CONCLUSÃO: a adição de neuregulinas proporcionou aumento significativo do número de fibras mielinizadas.

OBJECTIVE: to evaluate the effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. METHODS: eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen®); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group consisted of six segments of right sciatic nerves. After four weeks, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted; histological sections were standardized, and slides were made up for histomorphometric analysis. RESULTS: the results were statistically compared using the Tukey multiple comparisons test and the Student's t test. The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1-beta groups. CONCLUSION: the addition of neuregulins provided a significant increase in the number of myelinized fibers.

Therapeutic effect and mechanism of neuregulin on cerebral ischemia reperfusion injury in mice / 中国病理生理杂志

AIM: To study the effects of neuregulin-1β (NRG-1β) on the nervous behavioral function, cerebral infarction volume, brain water content (BWC), neuroal apoptosis and aquaporin-4 (AQP-4) expression in astrocytes after cerebral ischemic reperfusion in mice. METHODS: Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion (MCAO/R) model in mice. Neuregulin-1β (2 μg/kg) was injected into the internal carotid artery for treatment. The nervous behavioral function was evaluated by Bedersons test. The cerebral infarction volume was observed with tetrazolium chloride (TTC) staining. The BWC was measured by calculating the dry-wet weight ratio. The apoptotic positive cells were counted by immunofluorescence assay. The expression of AQP-4 was determined by immunohistochemical method. RESULTS: Nervous behavioral malfunction appeared in all the mice with left middle cerebral artery occlusion (MCAO) and/or reperfusion. The infarction focus showed in the ischemic hemisphere following the injury. The BWC, the numbers of neuroal apoptotic cells and AQP-4 expression in astrocytes were higher than those in sham control group. In MCAO/R+NRG-1β treatment group, the nervous behavioral function at ischemia 24 h significantly improved, the numbers of apoptotic positive cells reduced and the infarction volume decreased significantly than those in MCAO/R group (P<0.05). The BWC and AQP-4 expression in astrocytes showed no significant difference compared with MCAO/R group (P>0.05). In the reperfusion 22 h, 46 h and 70 h groups, the five indexes mentioned above were significantly different from those in the corresponding MCAO/R groups (P<0.05). CONCLUSION: NRG-1β might reduce cerebral edema and infarction volume, and improve the nervous behavioral function via down-regulating the expression of AQP-4 in astrocytes and inhibiting the neuronal apoptosis induced by ischemia reperfusion injury.

Extracellular Signal-regulated Kinase Activation Is Required for Serine 727 Phosphorylation of STAT3 in Schwann Cells in vitro and in vivo

In the peripheral nerves, injury-induced cytokines and growth factors perform critical functions in the activation of both the MEK/ERK and JAK/STAT3 pathways. In this study, we determined that nerve injury-induced ERK activation was temporally correlated with STAT3 phosphorylation at the serine 727 residue. In cultured Schwann cells, we noted that ERK activation is required for the serine phosphorylation of STAT3 by neuropoietic cytokine interleukin-6 (IL-6). Serine phosphorylated STAT3 by IL-6 was transported into Schwann cell nuclei, thereby indicating that ERK may regulate the transcriptional activity of STAT3 via the induction of serine phosphorylation of STAT3. Neuregulin-1 (NRG) also induced the serine phosphorylation of STAT3 in an ERK-dependent fashion. In contrast with the IL-6 response, serine phosphorylated STAT3 induced by NRG was not detected in the nucleus, thus indicating the non-nuclear function of serine phosphorylated STAT3 in response to NRG. Finally, we determined that the inhibition of ERK prevented injury-induced serine phosphorylation of STAT3 in an ex-vivo explants culture of the sciatic nerves. Collectively, the results of this study show that ERK may be an upstream kinase for the serine phosphorylation of STAT3 induced by multiple stimuli in Schwann cells after peripheral nerve injury.

Effect of neuregulin on inflammatory reaction following cerebral ischemia-reperfusion injury in rats / 中华神经科杂志

Objective To observe the expression of matrix metalloproteinase-9 (MMP-9) and the regulation of neuregulin-1β (NRG-1β) in brain tissue in rats following cerebral ischemia/reperfusion injury. Methods The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by a monofilament method from left external-internal carotid artery in 200 adult healthy male Wistar rats. The rat models in the treatment group (75 rats) and in control group (75 rats) were injected with 1.5%NRG-1β 5 μl and 0.1 mol/L PBS 5 μl, respectively, from internal carotid artery (ICA). The cerebral infarct volume was measured by TFC stain, the apoptosis was identified with in situ TUNEL method, and the expression of MMP-9 was detected by immunohistochemical and immunofluorescent double staining and Western blotting analysis. Results Cerebral ischemia-reperfusion can induce apoptosis and expression of MMP-9 in cerebral cortex and striatum. With the ischemic time prolonging, the number of apototic cells in cortex from ischemic 0, 0. 5, 1.0, 1.5 and 2. 0 h increased from 1.78 ± 0. 15,5. 78 ± 0. 51,10. 35 ± 0. 77, 21.50 ± 1.19 to 32. 00 ± 1.78, while the number of apoptotic cells in stratum from ischemic also increased significantly from 1.46±0.21, 4. 12±0.54, 7.33±0.71, 16.54 ± 1.63 to 19.03± 1.44 (t =9.31- 37.78, P < 0. 01) and the expression of MMP-9 increased significantly (t = 7.73-27.75, P < 0. 01) in the control group. With NRG-1β treatment, the number of apoptotie cells in cortex from ischemic 0, 0. 5,1.0, 1.5 and 2. 0 h reduced from 1.66±0. 11,4. 80±0. 61,5.63±0. 56, 9.75±1.22 to 13.54 ±1.26; while the number of apoptotic cells in striatum from ischemic also decreased significantly from 1.34 ± 0. 14, 3.35 ± 0. 32, 4. 55± 0. 50, 7. 63 ±1.41 to 10. 46 ± 0. 98 (t = 2. 74-18. 93, P < 0. 05), the expression of MMP-9 decreased (t = 3.85-12. 09, P < 0. 01), and the infarct volume decreased significantly (t = 4. 645-13. 043,P < 0. 01) compared with those in the control group at the same timepoint and the corresponding region. Conclusions The expression of MMP-9 is increased after cerebral ischemia/ reperfusion, and it may contribute to the inflammatory reaction. NRG-1β might down-regulate the expression of MMP-9 to inhibit apoptosis inducing by inflammatory reaction in cerebral ischemic reperfusion.

INFLUENCE OF NEUREGULIN ON NEURONAL APOPTOSIS AND EXPRESSIONS OF STAT3 AND GFAP FOLLOWING CEREBRAL ISCHEMIC REPERFUSION INJURY IN RATS / 解剖学报

Objective To observe the influence of neuregulin-1?(NRG-1?) on neuronal apoptosis and the expressions of signal transducer and activator of transcription(STAT3) and glial fiberillary acidic protein(GFAP) following cerebral ischemia/reperfusion in rats.Methods The animal models of middle cerebral artery occlusion/reperfusion(MCAO/R) was established by the intralumianl filament method from left external-internal carotid artery in adult healthy male Wistar rats.The rat models were treated by injecting 1.5% NRG-1? 5?l from internal carotid artery.The neuronal apoptosis was detected by DendEnd fluorometric TUNEL assay,and the expressions of STAT3 and GFAP were determined by immunohistochemical and immumofluorescent assays.Results The cerebral ischemia/reperfusion injury could induce neuronal apoptosis and the expressions of STAT3 and GFAP in brain tissue.In control group,the number of neuronal apoptosis increased gradually and the STAT3 and GFAP were expressed highly along ischemia times in the cortex,striatum and hippocampus areas.After treatment with NRG-1?,the number of neuronal apoptosis reduced and the expression level of STAT3 and GFAP increased when compared to those in the control group at different ischemia times and corresponding areas(P

Effect of neuregulins on mtp53 and HIF-1? in MDA-MB-231 cells / 中国病理生理杂志

AIM:To explore the effect and significance of neuregulins /ErbB2 receptor signal transduction pathway on mtp53 and hypoxia-iducible factor-1?(HIF-1?) in none-overexpression ErbB2 breast cancer cell MDA-MB-231.METHODS:The expression of neuregulin was detected by immunocytochemistry and Western blotting.MDA-MB-231 cells were treated with ErbB2 kinase inhibitor AG825.Proliferation was measured by MTT assay.The cell cycle and apoptosis were determined by flow cytometry.The expressions of mtp53 and HIF-1? were detected by Western blotting.The mRNA expression of HIF-1? was detected by RT-PCR.RESULTS:MDA-MB-231 cells expressed a relative higher level of neuregulin.In the results of Western blotting,the positive reaction band was found in 44 kD which coincides with the molecular weight of neuregulin.When MDA-MB-231 cells were treated with AG825,the proliferation was inhibited in time and dose dependent manners(P