Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the "bubble" structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to pyruvate kinase M2 (PKM2)-dependent conventional caspase-3/gasdermin E (GSDME) cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knockdown significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-programmed death-1 (anti-PD-1). In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.
Pyroptosis/immunology* ; Humans ; Endoplasmic Reticulum/immunology* ; Animals ; Nogo Proteins/antagonists & inhibitors* ; Mice ; Cell Line, Tumor ; Xanthones/pharmacology* ; Neoplasms/pathology* ; Mice, Nude

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*

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 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

OBJECTIVETo observe the effect of electric acupuncture (EA) on the Nogo receptors (NgR) protein expression in the cerebral cortex, the medulla oblongata, and the spinal cord of cerebral ischemia-reperfusion (I/R) stroke-prone renovascular hypertensive rats (RHRSP) with middle cerebral artery occlusion (MCAO) at different time points, and to investigate its possible mechanisms for remote-organ injury of acute cerebral infarction (ACI).

METHODSThe RHRSP model was duplicated in male SPF grade SD rats. Then the MCAO model was prepared by a thread stringing method. Rats were divided into the hypertension group,the sham-operation group, the MCAO group, the EA group, and the sham-acupoint group by random number table method, 60 in each group. Rats in the MCAO group only received MCAO reperfusion treatment. Those in the sham-operation group only received surgical trauma. Baihui (DU20) and Dazhui (DU14) were needled in the EA group, once daily for a total of 28 days.The needles were acupunctured at the skin one cun distant from Baihui (DU20) and Dazhui (DU14) and then the same EA treatment was performed in the sham-acupoint group. At day 1, 7, 14, 28 after treatment, six rats were executed from each group, and their right cortex and medulla oblongata, and the left spinal cord were isolated. The infarct volume was detected by Nissl's staining method. The NgR expression was detect by Western blot.

RESULTS(1) In the cortex area: compared with the hypertension group,the NgR expression increased in the MCAO group at day 1,7,14,and 28 after MCAO (P < 0.05). Compared with the MCAO group, the NgR expression of the EA group and the sham-acupoint group were equivalent at 1 day af ter MCAO (P > 0.05). At day 7, 14,and 28 after MCAO, the NgR expression decreased in the EA group (P < 0.05), it was quite similar to that in the sham-acupoint group (P > 0.05). (2) In the medulla oblongata area: compared with the hypertension group, the NgR expression was equivalent in the sham-operation group. the MCAO group,the EA group, and the sham-acupoint group at 1 day after MCAO (P > 0.05). At day 7.14, and 28 after MCAO, the NgR expression increased in the MCAO group (P < 0.05). Compared with the MCAO group,the NgR expression decreased in the EA group at day 7, 14, and 28 after MCAO (P < 0.05), whereas it was similar in the sham-acupoint group (P > 0.05). (3) In the spinal cord area: compared with the hypertension group, the NgR expression was equivalent in the sham-operation group, the MCAO group,the EA group, and the sham-acupoint group at day 1 and 7 after MCAO (P > 0.05). At day 14 and 28 after MCAO, the NgR expression increased in the MCAO group (P < 0.05). Compared with the MCAO group, the NgR expression decreased in the EA group at day 14 and 28 after MCAO (P < 0.05), whereas it was equivalent in the sham-acupoint group (P > 0.05).

CONCLUSIONSIncreased NgR expression in the cerebral cortex, the medulla oblongata, and the spinal cord of cerebral infarct rats was an important reason for involving remote-organ injury of ACI. The protective effect of EA on hypertensive I/R cerebral injury rats might be closely related to down-regulating central nervous system myelin growth inhibition mediated factors Nogo-A receptor NgR protein expression.

Animals ; Cerebral Infarction ; metabolism ; therapy ; Disease Models, Animal ; Electroacupuncture ; GPI-Linked Proteins ; metabolism ; Hypertension, Renal ; metabolism ; therapy ; Male ; Medulla Oblongata ; metabolism ; Myelin Proteins ; metabolism ; Nogo Receptor 1 ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; metabolism ; Spinal Cord ; 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

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

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

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