RESUMO
Ischemia and hypoxia activate astrocytes into reactive types A1 and A2, which play roles in damage and protection, respectively. However, the function and mechanism of A1 and A2 astrocyte exosomes are unknown. After astrocyte exosomes were injected into the lateral ventricle, infarct volume, damage to the blood-brain barrier (BBB), apoptosis and the expression of microglia-related proteins were measured. The dual luciferase reporter assay was used to detect the target genes of miR-628, and overexpressing A2-Exos overexpressed and knocked down miR-628 were constructed. qRT-PCR, western blotting and immunofluorescence staining were subsequently performed. A2-Exos obviously reduced the infarct volume, damage to the BBB and apoptosis and promoted M2 microglial polarization. RT-PCR showed that miR-628 was highly expressed in A2-Exos. Dual luciferase reporter assays revealed that NLRP3, S1PR3 and IRF5 are target genes of miR-628. After miR-628 was overexpressed or knocked down, the protective effects of A2-Exos increased or decreased, respectively. A2-Exos reduced pyroptosis and BBB damage and promoted M2 microglial polarization through the inhibition of NLRP3, S1PR3 and IRF5 via the delivery of miR-628. This study explored the mechanism of action of A2-Exos and provided new therapeutic targets and concepts for treating cerebral ischemia.
Assuntos
Astrócitos , Barreira Hematoencefálica , Isquemia Encefálica , Exossomos , MicroRNAs , Traumatismo por Reperfusão , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Astrócitos/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/terapia , Exossomos/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Isquemia Encefálica/patologia , Barreira Hematoencefálica/metabolismo , Masculino , Apoptose/genética , Microglia/metabolismo , Microglia/patologia , CamundongosRESUMO
AIMS: Matrix metalloproteinases 9 (MMP9) plays a role in the destruction of blood-brain barrier (BBB) and cell death after cerebral ischemic/reperfusion (I/R). Esculentoside H (EH) is a saponin found in Phytolacca esculenta. It can block JNK1/2 and NF-κB signal mediated expression of MMP9. In this study, we determined whether EH can protect against cerebral I/R injury by inhibiting MMP9 and elucidated the underlying mechanism. MAIN METHODS: Male SD rats were used to construct middle cerebral artery occlusion (MCAO) models. We determined the effect of EH on MMP9 inhibition, BBB destruction, neuronal death, PANoptosis, infarct volume, and the protective factor TLE1. Adeno-associated virus (AAV) infection was used to establish TLE1 gene overexpression and knockdown rats, which were used to determine the function. LY294002 was used to determine the role of PI3K/AKT signaling in TLE1 function. KEY FINDINGS: After EH treatment, MMP9 expression, BBB destruction, neuronal death, and infarct volume decreased. We found that TLE1 expression decreased obviously after cerebral I/R. TLE1-overexpressing rats revealed distinct protective effects to cerebral I/R injury. After treatment with LY294002, the protective effect was inhibited. The curative effect of EH also decreased when TLE1 was knocked down. SIGNIFICANCE: EH alleviates PANoptosis and protects BBB after cerebral I/R via the TLE1/PI3K/AKT signaling pathway. Our findings reveal a novel strategy and new target for treating cerebral I/R injury.
Assuntos
Barreira Hematoencefálica , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Saponinas , Transdução de Sinais , Animais , Masculino , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Saponinas/farmacologia , Saponinas/uso terapêutico , Infarto da Artéria Cerebral Média , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Metaloproteinase 9 da Matriz/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologiaRESUMO
Diabetes-associated cognitive dysfunction (DACD) is a complication of diabetes mellitus that leads to an increased risk of cognitive impairment and dementia. However, the molecular mechanism underlying DACD has not been elucidated, and a promising therapy for this disease remains to be established. Hydrogen sulfide (H2S), a significant antioxidative and anti-inflammatory gasotransmitter, has emerged as a neuroprotective agent. In this study, we investigated the protective effects of H2S on DACD in a streptozotocin (STZ)-induced diabetic rat model. We applied the Morris water maze to evaluate spatial learning and memory abilities. We used Western blotting and immunohistochemical staining to investigate the expression of the Nrf-2/HO-1 axis and the NLRP3 inflammasome. After NaHS (H2S donor) administration, diabetic rats exhibited improved spatial learning and memory retrieval abilities in the Morris water maze. In STZ-induced diabetic rats, the protein expression levels of the Nrf-2/HO-1 axis, the NLRP3 inflammasome and subsequent inflammatory cytokines in the hippocampal region were elevated compared to those in control rats. Exogenous H2S triggered Nrf-2/HO-1 antioxidant activity and inhibited NLRP3 inflammasome activation and proinflammatory cytokine expression. These findings suggested that exogenous H2S has neuroprotective effects by modulating the Nrf-2/HO-1 axis and the NLRP3 inflammasome pathway, which were found to be associated with DACD. H2S treatment may be a promising therapeutic strategy for preventing the progression of tissue damage caused by DACD.
Assuntos
Disfunção Cognitiva , Diabetes Mellitus Experimental , Sulfeto de Hidrogênio , Ratos , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/uso terapêutico , Sulfeto de Hidrogênio/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/etiologiaRESUMO
BACKGROUND: Ischemic stroke represents a major factor causing global morbidity and death. Bone marrow mesenchymal stem cell (BMSC)-derived exosomes (Exos) have important effects on treating ischemic stroke. Here, we investigated the therapeutic mechanism by which BMSC-derived exosomal miR-193b-5p affects ischemic stroke. METHODS: luciferase assay was performed to evaluate the regulatory relationship of miR-193b-5p with absent in melanoma 2 (AIM2). Additionally, an oxygen-glucose deprivation/reperfusion (OGD/R) model was constructed for the in vitro assay, while a middle cerebral artery occlusion (MCAO) model was developed for the in vivo assay. After exosome therapy, lactate dehydrogenase and MTT assays were conducted to detect cytotoxicity and cell viability, while PCR, ELISA, western blotting assay, and immunofluorescence staining were performed to detect changes in the levels of pyroptosis-related molecules. TTC staining and TUNEL assays were performed to assess cerebral ischemia/reperfusion (I/R) injury. RESULTS: In the luciferase assay, miR-193b-5p showed direct binding to the 3'-untranslated region of AIM2. In both in vivo and in vitro assays, the injected exosomes could access the sites of ischemic injury and could be internalized. In the in vitro assay, compared to normal BMSC-Exos, miR-193b-5p-overexpressing BMSC-Exos showed greater effects on increasing cell viability and attenuating cytotoxicity; AIM2, GSDMD-N, and cleaved caspase-1 levels; and IL-1ß/IL-18 generation. In the in vivo assay, compared to normal BMSC-Exos, miR-193b-5p-overexpressing BMSC-Exos showed greater effects on decreasing the levels of these pyroptosis-related molecules and infarct volume. CONCLUSION: BMSC-Exos attenuate the cerebral I/R injury in vivo and in vitro by inhibiting AIM2 pathway-mediated pyroptosis through miR-193b-5p delivery.
Assuntos
AVC Isquêmico , Melanoma , Células-Tronco Mesenquimais , MicroRNAs , Humanos , Piroptose , MicroRNAs/genética , MicroRNAs/metabolismo , AVC Isquêmico/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Background: The high levels of serum calcium and cholesterol are the important risk factors of cardiovascular disease (CVD), which frequently influence each other during the development of CVD. However, few studies have examined their temporal relationship to confirm the precursor, and it is still largely unknown whether and how their temporal relationship would influence the development of CVD. This study aimed to establish the temporal relationship between the changes in serum calcium and cholesterol using the longitudinal cohort data, and examine whether this temporal relationship influenced the arterial elasticity indicated by brachial-ankle pulse wave velocity (baPWV). Methods: This is a cohort study with a sample of 3,292 Chinese participants (aged 20-74 years) with 5.7 years follow-up. Serum calcium and cholesterol were measured at baseline and follow-up survey. The cross-lagged path analysis was used to examine their temporal relationship, and mediation analysis was performed to evaluate the potential mediating effect. Results: The cross-lagged path coefficients (ß2 values) from baseline serum calcium to follow-up cholesterol was significantly greater than the path coefficients (ß1 values) from baseline cholesterol to follow-up serum calcium (ß2 = 0.110 vs. ß1 = 0.047; P = 0.010) after adjusting for the multiple covariates. The path coefficients from baseline serum calcium to follow-up cholesterol in the participants with high baPWV was significantly greater than the participants with low baPWV (ß2 = 0.155 for high baPWV and ß2 = 0.077 for low baPWV, P = 0.028 for the difference between the ß2 values). Moreover, cholesterol partially mediated the association between the higher serum calcium and greater subsequent baPWV values, the percentage of the total effect mediated by cholesterol was estimated at 21.7%. Conclusion: Our findings indicate that increased serum calcium precedes increased in serum cholesterol, and this temporal relationship may contribute to the development of higher baPWV levels.
RESUMO
Leucine-rich α2-glycoprotein1 (LRG1), a pleiotropic protein, plays a pathogenic role in multiple human diseases. However, its pathophysiological function in ischemia/reperfusion injury remains unclear. In this study, we discussed the function and mechanism of LRG1 in acute ischemic stroke from both basic and clinical research points of view. Mice underwent transient middle cerebral artery occlusion (tMCAO) surgery 2 weeks after LRG1 was overexpressed by the delivery of adeno-associated virus (AAV). For wild-type mice, both the protein and the transcript of LRG1 in the brain tissue were elevated after tMCAO. Meanwhile, the serum levels of LRG1 were decreased after tMCAO. The neuronal injury was shown aggravated in the AAV-LRG1 group (AAV-LRG1 mice with tMCAO) through infarction volume, neurological score, HE, and Nissl staining. Meanwhile, LRG1 significantly enhanced apoptosis and autophagy during tMCAO, as detected by caspase3, Bax, Bcl-2, LC3II/LC3I, Beclin1, p62, and a TUNEL assay. Furthermore, by overexpression of LRG1, the protein of ALK1 was upregulated and the TGFß-smad1/5 signaling pathway was activated upon tMCAO. We also showed that patients with acute cerebral infarction had lower serum levels of LRG1 compared to healthy controls. In addition, LRG1 levels were associated with infarction volume, stroke severity, and prognosis in patients with supratentorial infarction. Taken together, the data from this study revealed that LRG1 promoted apoptosis and autophagy through the TGFß-smad1/5 signaling pathway by up-regulating ALK1, which exacerbates ischemia/reperfusion injury.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Isquemia Encefálica/metabolismo , Glicoproteínas/sangue , Glicoproteínas/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND/AIMS: This study investigated the role of the microRNA miR-298 and its target Act1 in ischemic stroke. METHODS: Cell viability was assessed with the 3-(4,5-dimethythiazol-2- yl)-2,5-diphenyl tetrazolium bromide assay. Apoptotic cells were detected by flow cytometry, and mRNA and protein expression were assessed by quantitative real-time PCR and western blotting, respectively. The regulatory relationship between miR-298 and Act1 was evaluated with the luciferase assay. To clarify the role of Act1 following ischemic stroke, the transcript was knocked down by short interfering RNA. The in vitro findings were validated in a mouse model of middle cerebral artery occlusion by administration of miR-298 mimic. RESULTS: Act1 was upregulated whereas miR-298 was downregulated in ischemic stroke. miR-298 overexpression by transfection of a mimic suppressed Act1 protein levels in vitro and in vivo, and the luciferase assay showed that miR-298 directly binds to the 3' untranslated region of the Act1 transcript. miR-298 overexpression enhanced cell apoptosis and autophagy and exacerbated ischemic infarction and neurological deficits, effects that were exerted via negative regulation of Act1/c-Jun N-terminal kinase (JNK)/nuclear factor (NF)-κB signaling and downstream autophagy pathways. CONCLUSIONS: Upregulation of miR-298 following ischemic stroke promotes brain injury in vitro and vivo by inhibiting the Act1/JNK/NF-κB signaling cascade and the downstream autophagy pathway. Therapeutic strategies that target miR-298 could be beneficial for the treatment of ischemic stroke.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , MicroRNAs/metabolismo , Traumatismo por Reperfusão/patologia , Acidente Vascular Cerebral/patologia , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antagomirs/metabolismo , Apoptose , Linhagem Celular , Modelos Animais de Doenças , Células HEK293 , Humanos , Infarto da Artéria Cerebral Média/complicações , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , NF-kappa B/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/etiologia , Transdução de Sinais , Acidente Vascular Cerebral/complicaçõesRESUMO
AIMS: The aim of the study is to evaluate the neuroprotective effects of olfactory ensheathing cells (OECs) with the overexpression of nuclear receptor-related factor 1 (Nurr1) and neurogenin 2 (Ngn2) in experimental models of Parkinson's disease (PD) and to elucidate the potential mechanism underlying the neuroprotective effects of OECs-Nurr1-Ngn2. MATERIALS AND METHODS: In vitro study, OECs-Nurr1-Ngn2 conditioned medium (CM) was added to MPP+-treated PC12 cells for 24h, and then the viability of PC12 cells, oxidative stress and apoptosis were detected. In vivo study, 48 male Sprague-Dawley (SD) rats were randomly divided into four groups. OECs/VMCs and OECs-Nurr1-Ngn2/VMCs groups were transplanted with 2×105 cells each of OECs or OECs-Nurr1-Ngn2 and VMCs into the right striatum one week after a unilateral 6-OHDA lesion. Control and PD groups were injected with 0.9% NaCl and 0.2% ascorbic acid into the same region. Rotational behavior was determined at 2, 4, 6 and 8weeks after injection or implantation in all groups. Neuronal differentiation markers, oxidative stress- and apoptosis-related indicators were detected at 8weeks post-grafting. KEY FINDINGS: OECs-Nurr1-Ngn2 increased the viability of PC12 cells, inhibited oxidative stress and apoptosis, and these effects could be reversed by pre-treatment of k252a, a TrkB receptor inhibitor. The behavioral deficits of PD rat were ameliorated by the transplantation of OECs-Nurr1-Ngn2/VMCs. SIGNIFICANCE: These results suggest that OECs-Nurr1-Ngn2 exhibits substantial neuroprotective, anti-oxidant, and anti-apoptotic effects against PD via the up-regulation of the neurotrophic factor-TrkB pathway.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas do Tecido Nervoso/genética , Neuroglia/transplante , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Bulbo Olfatório/citologia , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Transfecção , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/uso terapêutico , Células Cultivadas , Modelos Animais de Doenças , Terapia Genética/métodos , Masculino , Proteínas do Tecido Nervoso/uso terapêutico , Neuroglia/metabolismo , Neuroproteção , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/uso terapêutico , Células PC12 , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ratos , Ratos Sprague-Dawley , Transfecção/métodos , Regulação para CimaRESUMO
OBJECTIVE: This study investigated the role of miR-215 and nuclear factor-κB activator (Act)1 and their mechanisms of action in ischemic stroke. METHODS: Cell viability was examined with the 3-(4,5-dimethythiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide assay; cell apoptosis was detected by flow cytometry; and mRNA and protein expression was assessed by quantitative real-time PCR and western blotting, respectively. A mouse model of middle cerebral artery occlusion (MCAO) was treated with or without miR-215 mimic to verify the in vitro results. The relationship between miR-215 and interleukin (IL)-17 was evaluated in human peripheral blood from 29 patients. RESULTS: Act1 was upregulated whereas miR-215 was downregulated in ischemic stroke. Overexpression of miR-215 by transfection of a mimic repressed Act1 protein levels in vitro and in vivo, although the luciferase assay showed that miR-215 did not directly bind to the 3' untranslated region of Act1. MiR-215 overexpression inhibited cell apoptosis and autophagy. Increasing miR-215 levels reduced ischemic infarction and improved neurological deficit, while loss of miR-215 phenocopied the effects of IL-17. CONCLUSION: Upregulation of miR-215 exerts neuroprotection against ischemic injury by negatively regulating Act1/IL-17 receptor A signaling. These findings provide potential therapeutic targets for the treatment of ischemic stroke.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Isquemia Encefálica/metabolismo , MicroRNAs/metabolismo , Receptores de Interleucina-17/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Apoptose , Isquemia Encefálica/complicações , Linhagem Celular Tumoral , Células HEK293 , Humanos , Infarto da Artéria Cerebral Média/metabolismo , Camundongos , MicroRNAs/sangue , Transdução de Sinais , Acidente Vascular Cerebral/complicações , Regulação para CimaRESUMO
BACKGROUND: Stroke still has a high incidence with a tremendous public health burden and it is a leading cause of mortality and disability. However, biomarkers for early diagnosis are absent and the metabolic alterations associated with ischemic stroke are not clearly understood. The objectives of this case-control study are to identify serum biomarkers and explore the metabolic alterations of ischemic stroke. METHODS: Metabonomic analysis was performed using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and multivariate statistical analysis was employed to study 60 patients with or without ischemic stroke (30 cases and 30 controls). RESULTS: Serum metabolic profiling identified a series of 12 metabolites with significant alterations, and the related metabolic pathways involved glycerophospholipid, sphingolipid, phospholipid, fat acid, acylcarnitine, heme, and purine metabolism. Subsequently, multiple logistic regression analyses of these metabolites showed uric acid, sphinganine and adrenoyl ethanolamide were potential biomarkers of ischemic stroke with an area under the receiver operating characteristic curve of 0.941. CONCLUSIONS: These findings provide insights into the early diagnosis and potential pathophysiology of ischemic stroke.
Assuntos
Biomarcadores/sangue , Isquemia Encefálica/sangue , Cromatografia Líquida/métodos , Metabolômica/métodos , Acidente Vascular Cerebral/sangue , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We developed a highly efficient optical thermometer based on intensity ratio of upconversion green fluorescence of Er3+/Yb3+-codoped NaYF4 microcrystals. The sensor consists simply of a 980nm laser diode, one narrow-band interference filter, two lenses, one Si-photocell and one multimeter, while being without use of spectrometer and additional electronics. The device not only has a simple, compact structure (hence a low cost), but also displays highly efficient sensing performance, characterized by large signal-to-noise ratio due to strong fluorescence intensity, high thermal resolution and sensitivity, which have the values 1.3K and 1.24×10-2K-1, respectively, at the physiological temperature 310K. The excellent sensing performance of the device was further confirmed by the results of the measurements repeated using a spectrometer. The thermometer is highly generalized that can be applied to other luminescent materials, and shows great potential for the physiological temperature sensing in biological tissues and cells.