OBJECTIVE: High-altitude hypoxia exposure often damages hippocampus-dependent learning and memory. Nogo-A is an important axonal growth inhibitory factor. However, its function in high-altitude hypoxia and its mechanism of action remain unclear. METHODS: In an in vivo study, a low-pressure oxygen chamber was used to simulate high-altitude hypoxia, and genetic or pharmacological intervention was used to block the Nogo-A/NgR1 signaling pathway. Contextual fear conditioning and Morris water maze behavioral tests were used to assess learning and memory in rats, and synaptic damage in the hippocampus and changes in oxidative stress levels were observed. In vitro, SH-SY5Y cells were used to assess oxidative stress and mitochondrial function with or without Nogo-A knockdown in Oxygen Glucose-Deprivation/Reperfusion (OGD/R) models. RESULTS: Exposure to acute high-altitude hypoxia for 3 or 7 days impaired learning and memory in rats, triggered oxidative stress in the hippocampal tissue, and reduced the dendritic spine density of hippocampal neurons. Blocking the Nogo-A/NgR1 pathway ameliorated oxidative stress, synaptic damage, and the learning and memory impairment induced by high-altitude exposure. CONCLUSION: Our results demonstrate the detrimental role of Nogo-A protein in mediating learning and memory impairment under high-altitude hypoxia and suggest the potential of the Nogo-A/NgR1 signaling pathway as a crucial therapeutic target for alleviating learning and memory dysfunction induced by high-altitude exposure. GRAPHICAL ABSTRACT available in www.besjournal.com.
Animals ; Oxidative Stress ; Hippocampus/metabolism* ; Rats ; Nogo Proteins/genetics* ; Male ; Rats, Sprague-Dawley ; Hypoxia/metabolism* ; Altitude ; Synapses ; Humans ; Altitude Sickness/metabolism*

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

OBJECTIVE: To suppress NgR gene expression in neural stem cells and observe differentiation of neural stem cells in vitro after interfered which provide nutritional support for the facial nerve repair in vivo. METHOD: PCR amplification, restriction endonuclease digestion, T4DNA ligase connections were used to connected NgR with rector pGCsi, and constructed recombinant vector (NgR shRNA). Lipofectamine 2000 were used to transfect the NSC. The expression of NgR was examined by Western Blot. The proportion of neural stem cells transformed into neurons after transfection was tested by Immunocytochemistry. Neural stem cells were planted in PLGA tubes after transfected, and were scanned by electron microscopy. RESULT: NgR shRNA plasmid was constructed and infected neural stem cells successfully. Western Blot showed that the expression of NgR decreased in neural stem cells after interference. Immunocytochemistry showed that the rate of the neural stem cells transformed into neurons after interfered was significantly higher (P < 0.01). CONCLUSION Neural stem cells were transformed into neurons after NgR shRNA plasmid infected neural stem cells, which promoted axonal regeneration more effectively and provided a efficient and stable gene platform for facial nerve repair.
Animals ; Cell Differentiation ; Cells, Cultured ; Facial Nerve ; surgery ; GPI-Linked Proteins ; genetics ; metabolism ; Myelin Proteins ; genetics ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Nogo Receptor 1 ; RNA Interference ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; genetics ; metabolism

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

OBJECTIVETo evaluate the effects of lentiviral vector-mediated RNA interfere gene Nogo receptor (NgR) of rat cortical neurons in repairing spinal cord injury.

METHODSThe recombinant-lentiviral vector with small inferring RNA siNgR199 which had been constructed was transfected into rat cortical neuron cells in vitro in 3 multiplicity of infection (MOI). The infection rate was determined with fluorescent microscope, and the target gene was detected by PCR analysis. Then, the recombinant was injected into the cortical motor area of the rats with severe spinal cord injury, and the saline was also injected into other rats with severe spinal cord injury as a match control. The functional recovery of the rats' hindlimb was assessed using BBB score and the nerve fiber of the injured region was observed by nerve tracing.

RESULTSThe rate of recombinant infecting rat cortical neuron in vitro exceeded 99%. PCR analysis confirmed that the effect of lentiviral vector-mediated RNA interfering gene NgR of rat cortical neurons in vitro was 61%. Although all rats with spinal cord injury were observed to have the hindlimb functional recovery, these rats injected with recombinant had better hindlimb functional recovery than others showing by more BBB score (P < 0.01). Moreover, it was found that some nerve fiber passed the injured spinal cord region of the rats which were injected with recombinant.

CONCLUSIONThe recombinant lentiviral vector with siNgR199 which had been constructed is able to promote the growth of nerve fiber and the functional recovery of the rats' hindlimb.

Animals ; Cells, Cultured ; Disease Models, Animal ; GPI-Linked Proteins ; Genetic Vectors ; Hindlimb ; physiopathology ; Lentivirus ; genetics ; Myelin Proteins ; Nerve Regeneration ; Neurons ; Nogo Receptor 1 ; RNA Interference ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; Receptors, Peptide ; genetics ; Spinal Cord Injuries ; genetics ; physiopathology ; therapy ; Spinal Cord Regeneration ; Transfection

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

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

BACKGROUNDWe investigated the expression and role of TN4 in the oncogenesis of human hepatocellular carcinoma (HCC) from Qidong which is a HCC risk area.

METHODSThe expression of TN4 in HCC was observed using immunohistochemical staining (IHC). TN4 levels were manipulated in human liver cancer cell SMMC7721, using pcDNA3.1 eukaryotic expression constructs designed to express the complete TN4 cDNA. The biological changes of the cells were observed before and after transfection of TN4 and the change of gene expression was analysed by atlas cDNA expression array.

RESULTSAmong 100 pairs of samples of HCC, TN4 down-regulation expression and up-regulation expression positive rate were 81% (81/100), 19% (19/100), respectively (P < 0.01). TN4 protein was mainly localized in cytoplasm and membrane. The positive rate of TN4 were 10% (3/30), 100% (70/70) in lymph node metastasis and no lymph node metastasis, respectively (P < 0.01). The growth rates of the derivative SMMC7721-TN4 cell lines were decreased in comparison with that of normal SMMC7721 cells and pcDNA-SMMC7721. Some gene expression was changed before and after transfection of TN4. At 30 days of post-implantation of SMMC7721-TN4, SMMC7721-pcDNA3, SMMC7721 group produced tumors of (301.9 +/- 143.4) mm(3), (2418.7 +/- 362.8) mm(3), (2317.4 +/- 587.8) mm(3), respectively, (P < 0.01). Tumor inhibiting rate was 82.4% in TN4 transfection group. Sections of tumors were observed for their degree of tissue necrosis and there was higher degree of necrosis in tumors of the TN4-SMMC7721 cell group than those of the SMMC7721, SMMC7721-pcDNA groups.

CONCLUSIONSTN4 may play an important role in the oncogenesis of human HCC, especially in Qidong, the HCC risk area and TN4 could be a candidate tumor suppressor gene for HCC.

Adult ; Aged ; Carcinoma, Hepatocellular ; epidemiology ; genetics ; Carrier Proteins ; genetics ; China ; epidemiology ; DNA, Complementary ; analysis ; Gene Expression ; Genes, Suppressor ; Humans ; Immunohistochemistry ; Intracellular Signaling Peptides and Proteins ; Liver Neoplasms ; epidemiology ; genetics ; Lymphatic Metastasis ; genetics ; Male ; Membrane Proteins ; genetics ; Middle Aged ; Myelin Proteins ; Nogo Proteins ; Risk Factors ; Transfection