RESUMEN

Structural and functional alterations in brain microvascular endothelial cells (BMECs) caused by oxygen-glucose deprivation (OGD) are involved in the pathogenesis of various brain disorders. AlkB homolog 5 (ALKBH5) is a primary m6A demethylase that regulates various cell processes, but its distinct roles in BMEC function remain to be clarified. In the present study, in mouse middle cerebral artery occlusion (MCAO) model, knockout of ALKBH5 reduced neurological deficits, infarct volumes and tissue apoptosis caused by ischemia/reperfusion injury. Evans blue leakage and decreased expression of the tight junction protein ZO-1 and Occludin were also attenuated by ALKBH5 knockout. During the exploration of the underlying mechanisms of the role of ALKBH5 in BMECs, we found that the expression of ALKBH5 was induced at both the mRNA and protein levels by hypoxia; however, its protein stability was impaired by OGD treatment. Knockdown of ALKBH5 expression increased total m6A levels and alleviated OGD-induced BMEC injury. At the same time, the selective ALKBH5 inhibitor Cpd 20m also exhibited a protective effect on cell injury. In contrast, overexpression of ALKBH5 increased the sensitivity of BMECs to OGD. Interestingly, the m6A sequencing data revealed that knockdown of ALKBH5altered the expression of many genes via m6A upregulation. The gene expression alterations were verified by real-time PCR. Taken together, our results suggest that ALKBH5, as well as its target genes, plays important roles in the regulation of brain microvascular endothelial cell function through its RNA demethylase activity.

Asunto(s)

Desmetilasa de ARN, Homólogo 5 de AlkB , Células Endoteliales , Glucosa , Ratones Noqueados , Animales , Ratones , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Células Endoteliales/metabolismo , Glucosa/deficiencia , Encéfalo/metabolismo , Encéfalo/patología , Masculino , Microvasos/patología , Microvasos/metabolismo , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Infarto de la Arteria Cerebral Media/patología , Adenosina/análogos & derivados , Adenosina/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología

RESUMEN

Background: The regulation of divalent metal transporter-1 (DMT1) by insulin has been previously described in Langerhans cells and significant neuroprotection was found by insulin and insulin-like growth factor 1 treatment during experimental cerebral ischemia in acute ischemic stroke patients and in a rat 6-OHDA model of Parkinson's disease, where DMT1 involvement is described. According to the regulation of DMT1, previously described as a target gene of NF-kB in the early phase of post-ischemic neurodegeneration, both in vitro and in vivo, and because insulin controls the NFkB signaling with protection from ischemic cell death in rat cardiomyocytes, we evaluated the role of insulin in relation to DMT1 expression and function during ischemic neurodegeneration. Methods: Insulin neuroprotection is evaluated in differentiated human neuroblastoma cells, SK-N-SH, and in primary mouse cortical neurons exposed to oxygen glucose deprivation (OGD) for 8 h or 3 h, respectively, with or without 300 nM insulin. The insulin neuroprotection during OGD was evaluated in both cellular models in terms of cell death, and in SK-N-SH for DMT1 protein expression and acute ferrous iron treatment, performed in acidic conditions, known to promote the maximum DMT1 uptake as a proton co-transporter; and the transactivation of 1B/DMT1 mouse promoter, already known to be responsive to NF-kB, was analyzed in primary mouse cortical neurons. Results: Insulin neuroprotection during OGD was concomitant to the down-regulation of both DMT1 protein expression and 1B/DMT1 mouse promoter transactivation. We also showed the insulin-dependent protection from cell death after acute ferrous iron treatment. In conclusion, although preliminary, this evaluation highlights the peculiar role of DMT1 as a possible pharmacological target, involved in neuroprotection by insulin during in vitro neuronal ischemia and acute ferrous iron uptake.

Asunto(s)

Proteínas de Transporte de Catión , Muerte Celular , Regulación hacia Abajo , Insulina , Neuronas , Animales , Insulina/metabolismo , Insulina/farmacología , Humanos , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genética , Ratones , Muerte Celular/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Línea Celular Tumoral , Fármacos Neuroprotectores/farmacología , Hierro/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Glucosa/metabolismo , Compuestos Ferrosos/farmacología

RESUMEN

Corin is a type II transmembrane serine protease mainly expressed in the heart. Recently, corin was detected in the kidney and was reported to be associated with multiple kidney diseases. To date, its effect on acute kidney injury (AKI) has not been clarified. Here, we found that corin was constitutively expressed in renal tubules, especially in proximal and distal tubular epithelial cells. The expression of corin was dramatically reduced in ischemia/reperfusion injury (IRI)-induced AKI mouse model and oxygen-glucose deprivation (OGD)-induced human proximal tubular epithelial (HK-2) cells injury model, suggesting a potential role of corin in AKI. Corin deficient mice exhibited aggravated renal injury in AKI, as indicated by higher elevation of serum creatinine (SCr) and blood urea nitrogen (BUN), more severe tubular damage, and increased cell death versus wild type mice, demonstrating a protective effect of corin on AKI. In vitro overexpression of corin didn't directly alleviate hypoxia-induced HK-2 cells death, revealing that the protective effect of corin against AKI is not due to direct protection of tubular epithelial cells but may be through indirect protection. Microarray analysis showed enhanced inflammatory chemokines signaling and leukocyte chemotaxis in corin-/- mice after AKI, identifying an important role of corin in halting leukocyte chemotaxis and inflammatory response. Consistently, corin-/- mice after AKI displayed increased tubulointerstitial neutrophils and macrophages infiltration, as well as higher inflammatory mediators in kidneys. Taken together, our study indicates that tubular corin exerts a protective effect against AKI through negative regulation of chemotaxis signaling and inflammation in the kidney.

Asunto(s)

Lesión Renal Aguda , Daño por Reperfusión , Ratones , Animales , Humanos , Lesión Renal Aguda/metabolismo , Riñón/metabolismo , Túbulos Renales/metabolismo , Daño por Reperfusión/metabolismo , Antiinflamatorios/efectos adversos , Ratones Endogámicos C57BL , Apoptosis , Serina Endopeptidasas/metabolismo

RESUMEN

Cerebral ischemia is a severe neurological disability related to neuronal apoptosis and cellular stress response. Circular RNAs (circRNAs) are emerging regulators of cerebral ischemia. Herein, this study proposed to probe the action of circ_0000115 in cerebral ischemia injury. The mouse neuroblastoma cells N2a and HT22 underwent oxygen-glucose deprivation (OGD) were used as a model of in vitro cerebral ischemia. Levels of genes and proteins were detected by qRT-PCR and western blotting. Cell proliferation and apoptosis were determined by EdU assay and flow cytometry. Western blotting was used to detect the protein level of pro-inflammatory factors. The oxidative stress injury was evaluated by detecting reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) generation. Dual-luciferase reporter and RIP assays were used to confirm the target relationship between miR-1224-5p and circ_0000115 or nitric oxide synthase 3 (NOS3). OGD exposure decreased circ_0000115 and NOS3 expression, and increased miR-1224-5p in N2a and HT22 cells in a time-dependent manner. Circ_0000115 silencing attenuated OGD-induced apoptosis, oxidative stress and inflammation in N2a and HT22 cells. Mechanistically, circ_0000115 directly sponged miR-1224-5p, which targeted NOS3. Furthermore, rescue experiments showed that miR-1224-5p overexpression abolished the neuroprotective effect of circ_0000115 in N2a and HT22 cells under OGD treatment. Besides that, silencing of miR-1224-5p protected N2a and HT22 cells against OGD-evoked injury, which was counteracted by NOS3 knockdown. Circ_0000115 protects N2a and HT22 cells against OGD-evoked neuronal apoptosis, inflammation, and oxidative stress via the miR-1224-5p/NOS3 axis, providing an exciting view of the pathogenesis of cerebral ischemia.

Asunto(s)

Apoptosis , Isquemia Encefálica , Inflamación , MicroARNs , Neuronas , Estrés Oxidativo , ARN Circular , MicroARNs/genética , MicroARNs/metabolismo , Animales , ARN Circular/genética , ARN Circular/metabolismo , Apoptosis/genética , Ratones , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Neuronas/metabolismo , Neuronas/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Línea Celular Tumoral , Glucosa/metabolismo , Glucosa/deficiencia , Especies Reactivas de Oxígeno/metabolismo , Óxido Nítrico Sintasa de Tipo III

RESUMEN

Background: Hypoxic-ischemic brain damage (HIBD) is a type of brain damage that is caused by perinatal asphyxia and serious damages the central nervous system. At present, there is no effective drug for the treatment of this disease. Besides, the pathogenesis of HIBD remains elusive. While studies have shown that ferroptosis plays an important role in HIBD, its role and mechanism in HIBD are yet to be fully understood. Methods: The HIBD model of neonatal rats was established using the Rice-Vannucci method. A complete medium of PC12 cells was adjusted to a low-sugar medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 12 h. Laser Doppler blood flow imaging was used to detect the blood flow intensity after modeling. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining and transmission electron microscopy were used to observe brain injury and mitochondrial damage. Immunofluorescence was applied to monitor the expression of GFAP. Real-time quantitative polymerase chain reaction, western blot, and immunofluorescence were utilized to detect the expression of messenger RNA and protein. The level of reactive oxygen species (ROS) in cells was detected using the ROS detection kit. Results: The results showed that ferrostatin-1 (Fer-1) significantly alleviated the brain injury caused by hypoxia and ischemia. Fer-1 significantly increased the expression of SLC3A2, SLC7A11, ACSL3, GSS, and GPX4 (P<0.05) and dramatically decreased the expressions of GFAP, ACSL4, TFRC, FHC, FLC, 4-HNE, HIF-1α, and ROS (P<0.05). Conclusions: Fer-1 inhibits ferroptosis and alleviates HIBD by potentially targeting the GPX4/ACSL3/ACSL4 axis; however, its specific mechanism warrants further exploration.

RESUMEN

This study explored the protective effect of astragaloside Ⅳ(AS-Ⅳ) on oxygen-glucose deprivation(OGD)-induced autophagic injury in PC12 cells and its underlying mechanism. An OGD-induced autophagic injury model in vitro was established in PC12 cells. The cells were divided into a normal group, an OGD group, low-, medium-, and high-dose AS-Ⅳ groups, and a positive drug dexmedetomidine(DEX) group. Cell viability was measured using the MTT assay. Transmission electron microscopy was used to observe autophagosomes and autolysosomes, and the MDC staining method was used to assess the fluorescence intensity of autophagosomes. Western blot was conducted to determine the relative expression levels of functional proteins LC3-Ⅱ/LC3-Ⅰ, Beclin1, p-Akt/Akt, p-mTOR/mTOR, and HIF-1α. Compared with the normal group, the OGD group exhibited a significant decrease in cell viability(P<0.01), an increase in autophagosomes(P<0.01), enhanced fluorescence intensity of autophagosomes(P<0.01), up-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and down-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.05 or P<0.01). Compared with the OGD group, the low-and medium-dose AS-Ⅳ groups and the DEX group showed a significant increase in cell viability(P<0.01), decreased autophagosomes(P<0.01), weakened fluorescence intensity of autophagosomes(P<0.01), down-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and up-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.01). AS-Ⅳ at low and medium doses exerted a protective effect against OGD-induced autophagic injury in PC12 cells by activating the Akt/mTOR pathway, subsequently influencing HIF-1α. The high-dose AS-Ⅳ group did not show a statistically significant difference compared with the OGD group. This study provides a certain target reference for the prevention and treatment of OGD-induced cellular autophagic injury by AS-Ⅳ and accumulates laboratory data for the secondary development of Astragali Radix and AS-Ⅳ.

Asunto(s)

Proteínas Proto-Oncogénicas c-akt , Daño por Reperfusión , Ratas , Animales , Células PC12 , Proteínas Proto-Oncogénicas c-akt/genética , Glucosa/uso terapéutico , Oxígeno/metabolismo , Beclina-1/genética , Beclina-1/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Autofagia , Apoptosis , Daño por Reperfusión/tratamiento farmacológico

RESUMEN

Involving in the immune response after cerebral infarction, astrocytes could secrete large amounts of pro- and anti-inflammatory factors. The aim of this study is to investigate the effect of Wnt3a intervention on the inflammatory response of oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R) astrocyte model, and to provide a new target for immunoprotective treatment of cerebral infarction. We constructed the OGD/R rat astrocyte model, the astrocytes were treated by different concentrations of glucose (25, 50, 100 mM) intervened with/without Wnt3a (25 µg/ml). Microscope was used to observe the cell survival in rat astrocytes. The relative expression of inflammatory factors (TNF-a, IL-6, HIF-a) in rat astrocytes was detected by qRT-PCR. The expression of inflammatory factors such as TNF-a, IL-6 and HIF-a in rat astrocytes was increased after OGD/R treatment. The Wnt3a intervention promoted cell survival and decreased the expression of inflammatory factors in rat astrocytes induced by OGD/R. There is a neuroprotective effect that Wnt3a intervention could reduce inflammatory response in the OGD/R rat astrocyte model.

Asunto(s)

Glucosa , Oxígeno , Ratas , Animales , Glucosa/metabolismo , Oxígeno/farmacología , Oxígeno/metabolismo , Astrocitos/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacología , Infarto Cerebral/metabolismo

RESUMEN

Hypoxic ischemic encephalopathy (HIE) is among the leading causes of neonatal mortality, and currently there is no effective treatment. Ginsenoside Rb1 (GsRb1) is one of the principal active components of ginseng, and has protective benefits against oxidative stress, inflammation, hypoxic injury, and so on. However, the role and underlying mechanism of GsRb1 on HIE are unclear. Here, we established the neonatal rat hypoxic-ischemic brain damage (HIBD) model in vivo and the PC12 cell oxygen-glucose deprivation (OGD) model in vitro to investigate the neuroprotective effects of GsRb1 on HIE, and illuminate the potential mechanism. Our results showed that GsRb1 and the ferroptosis inhibitor liproxstatin-1 (Lip-1) could significantly restore System Xc activity and antioxidant levels as well as inhibit lipid oxidation levels and inflammatory index levels of HIBD and OGD models. Taken together, GsRb1 might inhibit ferroptosis to exert neuroprotective effects on HIE through alleviating oxidative stress and inflammation, which will set the foundation for future research on ferroptosis by reducing hypoxic-ischemic brain injury and suggest that GsRb1 might be a promising therapeutic agent for HIE.

Asunto(s)

Ferroptosis , Hipoxia-Isquemia Encefálica , Fármacos Neuroprotectores , Animales , Ratas , Animales Recién Nacidos , Ratas Sprague-Dawley , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Inflamación/tratamiento farmacológico , Oxígeno/uso terapéutico , Encéfalo

RESUMEN

AIMS: A network pharmacological analysis combined with experimental validation was used to investigate the neuroprotective mechanism of the natural product Tetramethylpyrazine(TMP). BACKGROUND: Protecting neurons is critical for acute ischemic stroke treatment. Tetramethylpyrazine is a bioactive component extracted from Chuanxiong. The neuroprotective potential of TMP has been reported, but a systematic analysis of its mechanism has not been performed. OBJECTIVE: Based on the hints of network pharmacology and bioinformatics analysis, the mechanism by which TMP alleviates oxygen-glucose deprivation-induced neuronal damage through inhibition of the HIF-1α/BNIP3 pathway was verified. METHOD: In this study, we initially used network pharmacology and bioinformatics analyses to elucidate the mechanisms involved in TMP's predictive targets on a system level. The HIF-1α/BNIP3 pathway mediating the cellular response to hypoxia and apoptosis was considered worthy of focus in the bioinformatic analysis. An oxygen-glucose deprivation (OGD)-induced PC12 cell injury model was established for functional and mechanical validation. Cell viability, lactate dehydrogenase leakage, intracellular reactive oxygen species, percentage of apoptotic cells, and Caspase-3 activity were determined to assess the TMP's protective effects. Transfection with siRNA/HIF-1α or pcDNA/HIF-1α plasmids to silence or overexpress hypoxia-inducible factor 1α(HIF-1α). The role of HIF-1α in OGD-injured cells was observed first. After that, TMP's regulation of the HIF-1α/BNIP3 pathway was investigated. The pcDNA3.1/HIF-1α-positive plasmids were applied in rescue experiments. RESULT: The results showed that TMP dose-dependently attenuated OGD-induced cell injury. The expression levels of HIF-1α, BNIP3, and the Bax/Bcl-2 increased significantly with increasing OGD duration. Overexpression of HIF-1α decreased cell viability, increased BNIP3 expression, and Bax/Bcl-2 ratio; siRNA-HIF-1α showed the opposite effect. TMP treatment suppressed HIF-1α, BNIP3 expression, and the Bax/Bcl-2 ratio and was reversed by HIF-1α overexpression. CONCLUSION: Our study shows that TMP protects OGD-damaged PC12 cells by inhibiting the HIF-1α/BNIP3 pathway, which provides new insights into the mechanism of TMP and its neuroprotective potential.

RESUMEN

Controlling axonal mitochondria is important for maintaining normal function of the neural network. Oxygen-glucose deprivation (OGD), a model used for mimicking ischemia, eventually induces neuronal cell death similar to axonal degeneration. Axonal mitochondria are disrupted during OGD-induced neural degeneration; however, the mechanism underlying mitochondrial dysfunction has not been completely understood. We focused on the dynamics of mitochondria in axons exposed to OGD; we observed that the number of motile mitochondria significantly reduced in 1 h following OGD exposure. In our observation, the decreased length of stationary mitochondria was affected by the following factors: first, the halt of motile mitochondria; second, the fission of longer stationary mitochondria; and third, a transformation from tubular to spherical shape in OGD-exposed axons. Motile mitochondria reduction preceded stationary mitochondria fragmentation in OGD exposure; these conditions induced the decrease of stationary mitochondria in three different ways. Our results suggest that mitochondrial morphological changes precede the axonal degeneration while ischemia-induced neurodegeneration.

Asunto(s)

Glucosa , Oxígeno , Ratas , Animales , Oxígeno/metabolismo , Glucosa/metabolismo , Ganglios Espinales/metabolismo , Ratas Sprague-Dawley , Axones/metabolismo , Células Cultivadas , Mitocondrias/metabolismo

RESUMEN

The beneficial effects of photobiomodulation (PBM) on function recovery after stroke have been well-established, while its molecular and cellular mechanisms remain to be elucidated. The current study was designed to investigate the effect of PBM on synaptic proteins and astrocyte polarization of photothrombotic (PT)-stroke induced rats in vivo, and explore the possible effect of PBM treatment on oxygen-glucose deprivation (OGD)-induced neurotoxic astrocytic polarization in vitro. We reported that 2-min PBM treatment (808 nm) for 7 days significantly increased synaptic proteins and neuroprotective astrocytic marker S100 Calcium Binding Protein A10 (S100A10) and inhibited neurotoxic astrocytic marker C3d in the peri-infarct region after ischemic stroke. Cell culture studies of primary cortical neurons and N2a cells showed that single-dose PBM treatment could increase cellular viability, regulate the apoptotic proteins (Caspase 9, Bcl-xL and BAX) and preserve synaptic proteins following OGD exposure. Additionly, PBM decreased the levels of C3d, inducible nitric oxide synthase (iNOS) and interleukin 1ß (IL-1ß) on astrocytes exposed to OGD. In summary, we demonstrated that PBM could inhibit neurotoxic astrocytic polarization, preserve synaptic integrity and protect neurons against stroke injury both in vitro and in vivo.

Asunto(s)

Astrocitos , Accidente Cerebrovascular , Ratas , Animales , Astrocitos/metabolismo , Accidente Cerebrovascular/radioterapia , Accidente Cerebrovascular/metabolismo , Neuronas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Glucosa/metabolismo , Oxígeno/metabolismo

RESUMEN

This study explored the protective effect of astragaloside Ⅳ(AS-Ⅳ) on oxygen-glucose deprivation(OGD)-induced autophagic injury in PC12 cells and its underlying mechanism. An OGD-induced autophagic injury model in vitro was established in PC12 cells. The cells were divided into a normal group, an OGD group, low-, medium-, and high-dose AS-Ⅳ groups, and a positive drug dexmedetomidine(DEX) group. Cell viability was measured using the MTT assay. Transmission electron microscopy was used to observe autophagosomes and autolysosomes, and the MDC staining method was used to assess the fluorescence intensity of autophagosomes. Western blot was conducted to determine the relative expression levels of functional proteins LC3-Ⅱ/LC3-Ⅰ, Beclin1, p-Akt/Akt, p-mTOR/mTOR, and HIF-1α. Compared with the normal group, the OGD group exhibited a significant decrease in cell viability(P<0.01), an increase in autophagosomes(P<0.01), enhanced fluorescence intensity of autophagosomes(P<0.01), up-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and down-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.05 or P<0.01). Compared with the OGD group, the low-and medium-dose AS-Ⅳ groups and the DEX group showed a significant increase in cell viability(P<0.01), decreased autophagosomes(P<0.01), weakened fluorescence intensity of autophagosomes(P<0.01), down-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and up-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.01). AS-Ⅳ at low and medium doses exerted a protective effect against OGD-induced autophagic injury in PC12 cells by activating the Akt/mTOR pathway, subsequently influencing HIF-1α. The high-dose AS-Ⅳ group did not show a statistically significant difference compared with the OGD group. This study provides a certain target reference for the prevention and treatment of OGD-induced cellular autophagic injury by AS-Ⅳ and accumulates laboratory data for the secondary development of Astragali Radix and AS-Ⅳ.

Asunto(s)

Ratas , Animales , Células PC12 , Proteínas Proto-Oncogénicas c-akt/genética , Glucosa/uso terapéutico , Oxígeno/metabolismo , Beclina-1/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Autofagia , Apoptosis , Daño por Reperfusión/tratamiento farmacológico

RESUMEN

While oxygen-glucose deprivation (OGD) has been widely utilized in many cell lines to mimic certain biological changes, it has yet to be validated in mesenchymal stem cells. We performed RNA sequencing on adipose-derived stem cells (ADSCs) under hypoxic and glucose-free conditions after 4 h and 8 h. A total of 335 common differentially expressed genes (DEGs) were identified in the two OGD groups compared with the normal control group, consisting of 292 upregulated and 43 downregulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that DEGs are mainly involved in metabolic processes, programmed cell death, and DNA-binding transcription activator activity. Protein‒protein interaction and hub gene analysis revealed various potential hub genes, in which response to oxygen levels, the IL-17-related biological function and the hypoxia-inducible factor 1 signaling pathway have been of vital importance. In summary, changes in transcription factor activity may play pivotal roles in oxygen-glucose deprivation. Through RNA sequencing, we have a deeper understanding of the changes in ADSCs after OGD treatment, providing more precise insight into predicting and regulating the stemness of ADSCs.

RESUMEN

Neonatal hypoxic-ischemic brain damage (HIBD) is one of the main causes of neonatal acute death and chronic nervous system impairment, but still lacks effective treatments. ZNF580/ZFP580, reported in our previous studies, may be a newly identified member of the Krüppel-like factor (KLF) family, and has anti-apoptotic effects during ischemic myocardial injury. In the present study, we showed that the expression levels of both ZFP580/ZNF580 mRNA and protein increased significantly in neonatal HIBD rats and oxygen-glucose deprivation (OGD) SH-SY5Y cell models. ZNF580 overexpression promoted neuron survival and suppressed neuron apoptosis after OGD in neuron-like SH-SY5Y cells, while interference with ZNF580 resulted in the opposite results. RNA-seq analysis identified 248 differentially-expressed genes (DEGs) between ZNF580 overexpression SH-SY5Y cells and interference-expressed SH-SY5Y cells. Gene Ontology functional enrichment analysis showed that these DEGs played significant roles in the growth, development, and regeneration of axons, DNA biosynthetic processes, DNA replication, and apoptosis. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that these DEGs were found in some pathways, including ferroptosis, glutamatergic synapses, protein processing in the endoplasmic reticulum, estrogen signaling pathways, the TGF-beta signaling pathway, and the longevity regulating pathway. The qRT-PCR validation results were consistent with RNA-seq results, which showed that HSPA5, IGFBP3, NTN4, and KLF9 increased in ZNF580-overexpressed SH-SY5Y cells and decreased in interference-expressed SH-SY5Y cells, when compared with normal cells. Together, the results suggested that ZNF580 targeted these genes to inhibit neuronal apoptosis.

Asunto(s)

Apoptosis , Hipoxia-Isquemia Encefálica , Animales , Encéfalo/metabolismo , Supervivencia Celular/fisiología , Glucosa/farmacología , Hipoxia-Isquemia Encefálica/genética , Hipoxia-Isquemia Encefálica/metabolismo , RNA-Seq , Ratas , Factores de Transcripción/metabolismo

RESUMEN

Cerebral ischemia and its sequelae, which include memory impairment, constitute a leading cause of disability worldwide. Micro-RNAs (miRNA) are evolutionarily conserved short-length/noncoding RNA molecules recently implicated in adaptive/maladaptive neuronal responses to ischemia. Previous research independently implicated the miRNA-132/212 cluster in cholinergic signaling and synaptic transmission, and in adaptive/protective mechanisms of neuronal responses to hypoxia. However, the putative role of miRNA-132/212 in the response of synaptic transmission to ischemia remained unexplored. Using hippocampal slices from female miRNA-132/212 double-knockout mice in an established electrophysiological model of ischemia, we here describe that miRNA-132/212 gene-deletion aggravated the deleterious effect of repeated oxygen-glucose deprivation insults on synaptic transmission in the dentate gyrus, a brain region crucial for learning and memory functions. We also examined the effect of miRNA-132/212 gene-deletion on the expression of key mediators in cholinergic signaling that are implicated in both adaptive responses to ischemia and hippocampal neural signaling. miRNA-132/212 gene-deletion significantly altered hippocampal AChE and mAChR-M1, but not α7-nAChR or MeCP2 expression. The effects of miRNA-132/212 gene-deletion on hippocampal synaptic transmission and levels of cholinergic-signaling elements suggest the existence of a miRNA-132/212-dependent adaptive mechanism safeguarding the functional integrity of synaptic functions in the acute phase of cerebral ischemia.

Asunto(s)

Secuencia de Bases , Isquemia Encefálica/genética , Giro Dentado/metabolismo , MicroARNs/genética , Eliminación de Secuencia , Acetilcolina/metabolismo , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/metabolismo , Neuronas Colinérgicas/patología , Giro Dentado/patología , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica , Glucosa/deficiencia , Glucosa/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/metabolismo , Microtomía , Oxígeno/farmacología , Técnicas de Placa-Clamp , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Transmisión Sináptica , Técnicas de Cultivo de Tejidos

RESUMEN

Herkinorin is a novel opioid receptor agonist. Activation of opioid receptors, a member of G protein coupled receptors (GPCRs), may play an important role in Herkinorin neuroprotection. GPCRs may modulate NOD-like receptor protein 3 (NLRP3)-mediated inflammatory responses in the mechanisms of inflammation-associated disease and pathological processes. In this study, we investigated the effects of Herkinorin on NLRP3 and the underlying receptor and molecular mechanisms in oxygen-glucose deprivation/reperfusion (OGD/R)-treated rat cortex neurons. First, Western blot analysis showed that Herkinorin can inhibit the activation of NLRP3 and Caspase-1, decrease the expression of interleukin (IL)-1ß, and decrease the secretion of IL-6 and tumour necrosis factor α detected by enzyme-linked immunosorbent assay in OGD/R-treated neurons. Then we found that Herkinorin downregulated NLRP3 levels by inhibiting the activation of nuclear factor kappa B (NF-κB) pathway, reducing the phosphorylation level of p65 and IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Instead, both the mu opioid receptor (MOR) inhibitor, ß-funaltrexamine, and MOR knockdown reversed the effects of Herkinorin on NLRP3 (p < .05 or .01, n = 3 per group). Further, we found that the level of ß-arrestin2 decreased in the cell membrane and increased in the cytoplasm after Herkinorin pretreatment in OGD/R-treated neurons. In co-immunoprecipitation experiments, Herkinorin increased the binding of IκBα with ß-arrestin2, decreased the ubiquitination level of IκBα, and ß-arrestin2 knockdown reversed the effects of Herkinorin on IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Our data demonstrated that Herkinorin negatively regulated NLRP3 inflammasome to alleviate neuronal ischemic injury through inhibiting NF-κB pathway mediated primarily by MOR activation. Inhibition of the NF-κB pathway by Herkinorin may be achieved by decreasing the ubiquitination level of IκBα, in which ß-arrestin2 may play an important role.

RESUMEN

Protein O-GlcNAcylation is a dynamic post-translational protein modification that regulates fundamental cellular functions in both normal physiology and diseases. The levels of protein O-GlcNAcylation are determined by flux of the hexosamine biosynthetic pathway (HBP), which is a branch of glycolysis, and are directly controlled by a pair of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). An increase in protein O-GlcNAcylation has been shown to have protective effects on ischemia-related insults in the heart and brain. To determine whether O-GlcNAcylation plays a beneficial role in ischemia-reperfusion (IR)-induced intestinal injury, we used pharmacological manipulation of O-GlcNAc to induce loss- and gain-of-function conditions and evaluated the viability and apoptosis of intestinal epithelioid cells in an in vitro oxygen-glucose deprivation (OGD) model and tissue injury grade in a small intestinal ischemia-reperfusion (SIIR) mouse model. We found that 1) Upregulation of O-GlcNAcylation induced by glucosamine (GlcN, increase in HBP flux) or thiamet G (an OGA inhibitor) enhanced intestinal cell survival in the OGD model. In contrast, downregulation of O-GlcNAcylation induced by DON (due to a reduction in HBP flux) or OMSI-1 (an OGT inhibitor) made the cells more susceptible to hypoxia injury. 2) Reducing the increase in O-GlcNAcylation levels with a combination of either GlcN with DON or thiamet G with OMSI-1 partly canceled its protective effect on OGD-induced cell injury. 3) In the in vivo SIIR mouse model, GlcN augmented intestinal protein O-GlcNAcylation and significantly alleviated intestinal injury by inhibiting cell apoptosis. These results indicate that acute increases in protein O-GlcNAcylation confer protection against intestinal ischemia insults, suggesting that O-GlcNAcylation, as an endogenous stress sensor, could be a universal protective mechanism and could be a potential therapeutic target for intestinal ischemic disease.

Asunto(s)

Glucosa/deficiencia , Hipoxia/metabolismo , Intestino Delgado/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Oxígeno/metabolismo , Daño por Reperfusión/metabolismo , Animales , Línea Celular , Hipoxia/patología , Hipoxia/prevención & control , Intestino Delgado/patología , Masculino , Ratones , Ratones Endogámicos ICR , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control

RESUMEN

BACKGROUND: Traumatic Spinal Cord Injury (SCI) is a severe condition usually accompanied by an inflammatory process that gives rise to uncontrolled local apoptosis and a subsequent unfavorable prognosis. One reason for this unfavorable outcome could be the activation of the NLRP3 inflammasome. OBJECTIVE: MCC950 is a specific inhibitor of NLRP3 that further inhibits the formation of the NLRP3 inflammasome. The purpose of this study was to determine whether the NLRP3 inflammasome was associated with the severity of local apoptosis and whether MCC950 could prevent neuronal apoptosis following SCI. METHODS: In this study, primary cortical neurons were cultured in vitro. With or without pretreatment/ posttreatment with MCC950, neurons were subjected to Oxygen-Glucose Deprivation (OGD) for 2 h and then reperfusion for 20 h. Immunofluorescence was used to determine the expression of NLRP3, ASC, and cleaved caspase-1 in neurons. In vivo, SCI model mice were established with a 5 g weight-drop method. MCC950 was intraperitoneally injected at 0, 2, 4, 6, 8, 10, and 12 days after SCI. Basso Mouse Scale (BMS) scores and footprint assays were used to assess motor function. Paw withdrawal threshold and tail-flick latency were used to assess somatosensory function. H&E, Nissl, and TUNEL staining were used to measure histological changes and apoptosis at 3 days after SCI, and scar formation was observed by Masson staining and GFAP immunohistochemical analysis at 28 days after SCI. RESULTS: Immunofluorescence analysis confirmed that MCC950 inhibited OGD-induced activation of the NLRP3 inflammasome in neurons. Behavioral tests, Masson staining, and GFAP immunohistochemical analysis showed that MCC950-treated mice had improved neuronal functional recovery and reduced scar formation at 28 days after SCI. H&E, Nissl, and TUNEL staining confirmed that there were more living neurons and fewer apoptotic neurons in MCC950-treated mice than control mice at 3 days after SCI. CONCLUSION: These results reveal that MCC950 exerts neuroprotective effects by reducing neuronal apoptosis, preserving the survival of the remaining neurons, attenuating the severity of the damage, and promoting the recovery of motor function after SCI.

Asunto(s)

Apoptosis/efectos de los fármacos , Furanos/farmacología , Indenos/farmacología , Traumatismos de la Médula Espinal/metabolismo , Sulfonamidas/farmacología , Animales , Etiquetado Corte-Fin in Situ , Inflamasomas/metabolismo , Inflamación/metabolismo , Masculino , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Recuperación de la Función

RESUMEN

Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALR: autophagic lysosome reformation; ATG7: autophagy related 7; CTSB: cathepsin B; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HPCA: hippocalcin; i.c.v: intracerebroventricular; KEGG: kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; LSDs: lysosomal storage disorders; MAP2: microtubule-associated protein 2; MCAO: middle cerebral artery occlusion; mCTSB: mature CTSB; mCTSD: mature CTSD; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PBS: phosphate-buffered saline; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; proCTSD: pro-cathepsin D; RAPA: rapamycin; RNA-seq: RNA sequencing; RPS6KB/p70S6K: ribosomal protein S6 kinase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM: Structured Illumination Microscopy; SNAP25: synaptosomal-associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYT1: synaptotagmin I; TBST: tris-buffered saline Tween-20; TEM: transmission electron microscopy; TFEB: transcription factor EB; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; TUBB3: tubulin, beta 3 class III.

Asunto(s)

Autofagia/fisiología , Isquemia/metabolismo , Lisosomas/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Regulación hacia Arriba/fisiología , Animales , Autofagosomas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratas Wistar