Nogo-B is a major family member of the reticulon protein family 4. It is widely expressed in the central nervous system and peripheral tissues, and is mainly located in endoplasmic reticulum and cell membrane. Previous studies have revealed that Nogo-B plays a key role in vascular injury, tissue repair and inflammation process. It also may be critical for apoptosis of tumor cells and central diseases. Further investigation of the molecular characteristics and biological function of Nogo-B might be of great help to understand its role in diverse diseases.
Animals ; Apoptosis ; Cell Membrane ; physiology ; Endoplasmic Reticulum ; physiology ; Humans ; Inflammation ; Myelin Proteins ; physiology ; Nogo Proteins

Nogo-B receptor (NgBR) is a specific receptor of Nogo-B, a member of reticulon 4 protein family. It is widely expressed in many tissues and mainly located in cell membrane and endoplasmic reticulum. Previous studies have revealed that NgBR is involved in a variety of physiological and pathophysiological processes, such as dolichol synthesis, lipid metabolism, cholesterol trafficking, insulin resistance, vascular remodeling and angiogenesis, tumorigenesis and nervous system diseases. Further studies on the molecular characteristics and biological function of NgBR might be of great significance to understand its role in diverse diseases and provide possible clinical strategies for the treatment of diseases.
Carrier Proteins/metabolism* ; Endoplasmic Reticulum/metabolism* ; Lipid Metabolism ; Nogo Proteins/metabolism* ; Receptors, Cell Surface/metabolism*

OBJECTIVETo observe the effects of electroacupuncture (EA) on the expressions of Nogo-A and the ultrastructure in the cerebral cortex at different time points after the cerebral ischemia-reperfusion in rats.

METHODSOne hundred and thirty male Sprague Dawley (SD) rats were randomly divided into the EA group (n = 30), the sham-EA group (n = 30), the model group (n = 30), the sham-operation group (n = 30), and the blank group (n = 10). The modified ZeaLonga method was used to prepare the left middle cerebral artery occlusion (MCAO) model in the first three groups. After the operation Baihui (DU20) and Dazhui (DU14) were daily needled in the EA group. One inch beside Baihui (DU20) and Dazhui (DU14) were daily needled in the sham-EA group. Rats in the model group were only treated with MCAO ischemia/reperfusion. Rats in the sham-operation group only received surgical wound. No treatment was given to rats in the blank group. The ultrastructures of ischemic cells and the intervention of the Nogo-A expressions were observed using the immunohistochemical staining and the transmission electron microscope 1, 7, and 28 days after EA.

RESULTS(1) In the EA group, the damage of ultrastructures of neurons, gliocytes, and blood brain barrier in the ischemic region was alleviated when compared with that of the sham-EA group and the model group. (2) On the 1st, 7th and 28th day after the cerebral ischemia-reperfusion, the expressions of Nogo-A in the ischemic cortex in the EA group was lower when compared with those in the sham-EA group and the model group at the corresponding time points, showing significant difference (P < 0.05). But there was no statistical difference between the sham-EA group and the model group at the same time point (P > 0.05).

CONCLUSIONThe mechanism of EA for protecting cerebral ischemia/reperfusion might be closely associated with alleviating the damage on the ultrastructures of brain cells, and down-regulating the expressions of Nogo-A.

Acupuncture Points ; Animals ; Brain Ischemia ; metabolism ; pathology ; therapy ; Cerebral Cortex ; metabolism ; ultrastructure ; Electroacupuncture ; Male ; Myelin Proteins ; metabolism ; Nogo Proteins ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology ; therapy

BACKGROUNDOne of the reasons for poor neuroregeneration after central nervous system injury is the presence of inhibitory factors such as Nogo. Here, we tested the inhibition of Nogo by RNA interference both in vitro and in vivo, using recombinant adenovirus-mediated transfection of short hairpin RNAs, to explore a new method of treatment for spinal cord injury.

METHODSWe designed and cloned two Nogo-specific short hairpin RNAs and an unrelated short hairpin RNA, packaged the clones into adenovirus, and amplified the recombinant virus in 293 cells. We then tested the inhibition of Nogo expression both in vitro in adenovirus-transfected oligodendrocytes and in vivo in spinal cord tissue from adenovirus-transfected spinal cord injury model rats. We tested Nogo expression at the mRNA level by reverse-transcription PCR and at the protein level by Western blotting and immunohistochemistry.

RESULTSIn vitro, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 51% and 49%, respectively, compared with Nogo expression in cells transfected with the unrelated control small hairpin RNA (P < 0.005). Similarly, Nogo protein expression decreased by 50% and 48%, respectively (P < 0.005). In vivo, in spinal cord injury model rats, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 45% and 40%, respectively, compared with Nogo expression in spinal cord injury model rats transfected with the unrelated control short hairpin RNA (P < 0.005). The Nogo protein level was similarly decreased.

CONCLUSIONSWe were successful in specifically downregulating Nogo at the mRNA and protein levels by adenovirus-mediated delivery of short hairpin RNAs, both in vitro and in vivo. This confirms the effectiveness of RNA interference for the inhibition of Nogo gene expression and the efficiency of using adenovirus for delivery. Thus gene therapy may be an effective treatment for spinal cord injury.

Adenoviridae ; genetics ; Animals ; Blotting, Western ; Humans ; Immunohistochemistry ; Myelin Proteins ; genetics ; metabolism ; Nogo Proteins ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; therapy

Biomedical Research ; trends ; Computers ; Humans ; Implants, Experimental ; Myelin Proteins ; antagonists & inhibitors ; immunology ; Nerve Regeneration ; immunology ; physiology ; Nogo Proteins ; Patient Education as Topic ; Spinal Cord Injuries ; therapy

OBJECTIVETo study the effects of Jisuikang (Chinese characters) on Nogo-NgR gene expression, and to explore the protective effects and mechanism of Jisuikang (Chinese characters) on spinal cord injury in rats.

METHODSOne hundred eighty female rats were randomly assigned to 6 groups(30 rats per group). Sham group: T10 lamina was resected only and spinal cord was untreated. Model group: spine cord injury (SCI) was created with a modified impinger of Allen's by impacting on the T10 spinal cord. Prednisolone group: Prednisolone (0.06 g/kg) was given by intragastric administration at a time interval of 24 hours after operation. The Jisuikang (Chinese characters) high, moderate and low dose groups: Jisuikang (Chinese characters) was supplied with different dose (50 g/kg, 25 g/kg, 12.5 g/kg) by intragastric administration in rats after operation,for the first time at 30 min after surgery. Animals were killed 3, 7, 14 days after surgery. The expression levels of Nogo-A and NgR were observed by Western Blot and Real-time PCR.

RESULTSThe expression of Nogo-A and NgR was at the basic level at all time points in sham group. Compared with model group, the protein expression levels of Nogo-A and NgR in sham, prednisolone, Jisuikang (Chinese characters) moderate dose groups were statistically significant at all time points (P < 0.05). No difference was found in Jisuikang (Chinese characters) high and low dose groups (P > 0.05). Three days after surgery, the mRNA levels of Nogo-A and NgR in treatment group were significantly lower than that in model group (P < 0.01); 7 days after surgery,Nogo-A and NgR mRNA expression were dramatically upregulated and peaked; 14 days after operation, the expression was decreased, but still significantly higher than that in other treatment groups (P < 0.01). Prednisolone and Jisuikang (Chinese characters) moderate dose groups showed the most significant effects among all groups,but there was no statistically significant difference between two groups (P > 0.05).

CONCLUSIONThe decoction Jisuikang (Chinese characters) can promote the nerve cell regeneration by regulating Nogo-A and NgR gene expression, activating Nogo- NgR signaling pathways after acute spinal cord injury.

Animals ; Female ; GPI-Linked Proteins ; analysis ; genetics ; physiology ; Medicine, Chinese Traditional ; Myelin Proteins ; analysis ; genetics ; physiology ; Nerve Regeneration ; drug effects ; Nogo Proteins ; Nogo Receptor 1 ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; analysis ; genetics ; physiology ; Signal Transduction ; drug effects ; Spinal Cord Injuries ; drug therapy ; metabolism

OBJECTIVEThis study examined the effect of high concentration of phenylalanine (Phe) on Nogo-66 receptor (NgR) expression in the cortical neurons of rats in vitro in order to investigate whether NgR is involved in the etiology of Phe-induced brain damage.

METHODSNeurons from the cerebral cortex of embryonic rats were cultured for 3 days and then were treated with 0.9 mM Phe. After 12, 24 and 48 hrs of Phe treatment, mRNA and protein expression of NgR was detected by real-time PCR and Western blot respectively. Growth cones and growth axons of neurons were detected by immunofluorescence and immunohistochemistry respectively after 12 and 24 hrs of Phe treatment.

RESULTSThe length of growth axons of neurons was significantly shorter after 12 and 24 hrs of Phe treatment compared with the control group without Phe treatment (P<0.05). Growth cones collapse occurred in 12.5+/-9.7% and 24.1+/-4.5% of neurons respectively after 12 and 24 hrs of Phe treatment but only in 3.5+/-1.5% in the control group (P<0.01). The protein level of NgR after 12, 24 and 48 hrs of Phe treatment was up-regulated, with 9.0, 9.4 and 12.6 times as the control. mRNA level of NgR in the Phe treatment group did not differ from control.

CONCLUSIONSHigh concentration of Phe can induce an increased NgR protein expression in cortical neurons, and the increased NgR expression may contribute to the growth cones collapse and the inhibitory activities of axon regeneration after injury.

Animals ; Blotting, Western ; Cerebral Cortex ; chemistry ; drug effects ; GPI-Linked Proteins ; Immunohistochemistry ; Myelin Proteins ; analysis ; genetics ; Nogo Receptor 1 ; Phenylalanine ; pharmacology ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; analysis ; genetics

OBJECTIVEThe hypoxic-ischemic encephalopathy caused by asphyxia in peripartum is a serious disease in newborn infants, with a high disability and mortality rate. Lack of regenerative ability in central nervous system after injury is considered as the fundamental cause. However, in recent years many studies have revealed that there are myelin-associated neurite growth inhibitory factors that exert inhibiting effect through the Nogo receptor (NgR). This study aimed to investigate the expression level of NgR and the possible neuroprotective effect of NEP1-40 in newborn rats with hypoxic ischemic brain damage (HIBD).

METHODEighty healthy Wistar rats aged 7 days were randomly divided into 4 groups; 8 in control group, 24 in HIBD model group, 24 in GM-1 group and 24 in NEP1-40 group. The rats of the control group and HIBD group were injected with normal saline (0.25 ml/kg) intraperitoneally, while those in NEP1-40 group and GM-1 group with NEP1-40 12.5 microg/d, GM-1 10 mg/(kg.d) for continuous 3 days of 72-hour group or 7 days of 168-hour group, respectively. In situ hybridization was adopted for detecting the expression of NgR in the brain of the rats at the time point of 24 hours, 72 hours and 7 days. Meanwhile histopathological changes of neurons and axon were detected by transmission electron microscopy (TEM). The SPSS statistical software package for Windows, version 10.0, was used to run Chi-square tests and least significance difference (LSD-t) on the data presented, and P value of less than 0.05 was regarded as statistically significant.

RESULTThe expression level of Nogo-A receptor in the control group was higher than that of the other groups at different time point (t value was 5.48, 6.11, 6.96, 8.24, 5.99 and 5.34, respectively, and all P values were less than 0.05). There were no significant differences in Nogo-A receptor level among the HIBD group, the GM-1 group and the NEP1-40 at 24 hours (t was 1.48, 2.76 and 1.29, respectively, and all P > 0.05), while the expression of Nogo-A receptor of NEP1-40 at 72 hours and 7 days was lower than that of the HIBD group and the GM-1 group at the same time point, respectively (all P < 0.05). Repair of neurons in damaged brain to some extent was found after GM-1 treatment and satisfactory repair of neurons and axon regeneration was obtained with NEP1-40 administration as shown by TEM.

CONCLUSIONHypoxic ischemic brain damage can down-regulate the expression of Nogo-A receptor in the central nervous system. NEP1-40 contributes to the regeneration of axon and repair of brain damage, thus exerts neuroprotective effect.

Animals ; Animals, Newborn ; Brain ; drug effects ; metabolism ; pathology ; GPI-Linked Proteins ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; Myelin Proteins ; pharmacology ; Nogo Receptor 1 ; Peptide Fragments ; pharmacology ; Rats ; Rats, Wistar ; Receptors, Cell Surface ; Receptors, Peptide ; metabolism

Animals ; Animals, Newborn ; Blotting, Western ; Brain ; metabolism ; Disease Models, Animal ; Hypoxia, Brain ; metabolism ; Immunohistochemistry ; Myelin Proteins ; genetics ; immunology ; metabolism ; Nogo Proteins ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVETo screen and identify the proteins that interact with connexin 26 (CX26) and to analyze the expressions of these proteins in cochlea so as to explore the proteins that relate to the trafficking, assembly, localizing and gap junction functions of CX26.

METHODSThe whole coding region of GJB2 (CX26) gene was amplified from normal human genomic DNA by polymerase chain reaction (PCR) and then directionally subcloned into the vector pGBKT7 plasmid of the Match Maker Ga14 Two-Hybrid System 3 as a target to screen the interactive proteins of CX26 from the human fetal brain cDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by repeated yeast two hybrid method between CX26 and everyone of the preys respectively. The DNAs of the insert of the identified positive clone were sequenced and BLAST analyzed against the GenBank. Lastly, the mRNA of the gene encoding the identified protein was analyzed in the murine inner ear by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe insert of one positive clone contained 867 bp with the former 525 bp being coding region. The DNA sequence and the open reading frame of the insert were identical to the 525 bp before the stop codes (including the stop codes) and the 238 bp after the stop codes of RTN4 gene which encoded Nogo protein. And the 174 amino acid residues encoded by the insert were those of the C-terminal of Nogo protein: Nogo-A, Nogo-B and Nogo-C. RTN4 mRNA expressed in the murine inner ear was confirmed by RT-PCR method.

CONCLUSIONThe C-terminal of Nogo protein interacts with CX26. Nogo protein expresses in the inner ear and may take part in the trafficking of CX26 or CX26 gap junction function.

Animals ; Base Sequence ; Connexin 26 ; Connexins ; genetics ; metabolism ; Ear, Inner ; metabolism ; Gene Expression ; Humans ; Mice ; Molecular Sequence Data ; Myelin Proteins ; genetics ; metabolism ; Nogo Proteins ; Protein Binding ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Two-Hybrid System Techniques