RESUMEN
BACKGROUND: The impairment in the autophagy-lysosomal pathway (ALP) and the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome represent two molecular events leading to neurodegeneration and neuroinflammation in Alzheimer's disease (AD), a devastating neurodegenerative disorder without a cure. Previously we demonstrated the cognitive-enhancing effect of a combined electroacupuncture (EA) therapy termed TNEA in a transgenic mouse model of AD, involving activation of transcription factor EB (TFEB), a master regulator of ALP. However, whether and how TNEA inhibits NLRP3 inflammasome via TFEB-mediated ALP in AD remains to be investigated. METHODS: 5xFAD mice overexpressing amyloid-ß (Aß) were treated with TNEA or EA on its composing acupoints (GB13 and GV24). The changes in the signaling pathways regulating NLRP3 inflammasome, the association of NLRP3 inflammasome with ALP, and the roles of TFEB/TFE3 in mice brains were determined by immunoblots, immunohistochemistry and AAV-mediated knockdown assays. RESULTS: TNEA inhibits the activation of NLRP3 inflammasome and the release of active interleukin 1ß (IL1B) in the hippocampi of 5xFAD mice. Mechanistically, TNEA promoted the autophagic degradation of inflammasome components via activating both TFEB and TFE3 by modulating kinases including AMPK and AKT. The composing acupoints in TNEA showed synergistic effects on regulating these molecular events and memory improvement. CONCLUSION: Our findings suggest that TNEA attenuates AD-associated memory impairment via promoting TFEB/TFE3-mediated autophagic clearance of Aß and NLRP3 inflammasome, and partially reveal the molecular basis of combined acupoints therapy originated from ancient wisdom.
Asunto(s)
Enfermedad de Alzheimer , Inflamasomas , Ratones , Animales , Inflamasomas/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/terapia , Enfermedad de Alzheimer/metabolismo , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Autofagia , Péptidos beta-Amiloides/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genéticaRESUMEN
Alterations in formaldehyde (FA) homeostasis are associated with the pathology of Alzheimer's disease (AD). In vivo tracking of FA flux is important for understanding the underlying molecular mechanisms, but is challenging due to the lack of sensitive probes favoring a selective, rapid, and reversible response toward FA. In this study, we re-engineered the promiscuous and irreversible phenylhydrazines to make them selective and reversible toward FA by tuning their nucleophilicity. This effort resulted in PFM309, a selective (selectivity coefficient KFA,methylglyoxal = 0.06), rapid (t1/2 = 32 s at [FA] = 200 µM), and reversible fluorogenic probe (K = 6.24 mM-1) that tracks the FA flux in both live cells and live mice. In vivo tracking of the FA flux was realized by PFM309 imaging, which revealed the gradual accumulation of FA in the live mice brain during normal aging and its further increase in AD mice. We further identified the age-dependent loss of catabolism enzymes ALDH2 and ADH5 as the primary mechanism responsible for formaldehyde excess. Activating ALDH2 with the small molecular activator Alda1 significantly protected neurovascular cells from formaldehyde overload and consequently from impairment during AD progress both in vitro and in vivo. These findings revealed PFM309 as a robust tool to study AD pathology and highlight ALDH2 as a potential target for AD drug development.
Asunto(s)
Enfermedad de Alzheimer , Envejecimiento , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Animales , Formaldehído/metabolismo , RatonesRESUMEN
Grb2-associated-binding protein 1 (Gab1) is a docking/scaffolding molecule known to play an important role in cell growth and survival. Here, we report that Gab1 is decreased in cholinergic neurons in Alzheimer's disease (AD) patients and in a mouse model of AD. In mice, selective ablation of Gab1 in cholinergic neurons in the medial septum impaired learning and memory and hippocampal long-term potentiation. Gab1 ablation also inhibited SK channels, leading to an increase in firing in septal cholinergic neurons. Gab1 overexpression, on the other hand, improved cognitive function and restored hippocampal CaMKII autorphosphorylation in AD mice. These results suggest that Gab1 plays an important role in the pathophysiology of AD and may represent a novel therapeutic target for diseases involving cholinergic dysfunction.
Asunto(s)
Enfermedad de Alzheimer/patología , Corteza Cerebral/patología , Neuronas Colinérgicas/fisiología , Cognición/fisiología , Regulación de la Expresión Génica/genética , Fosfoproteínas/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Proteínas Adaptadoras Transductoras de Señales , Anciano de 80 o más Años , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/diagnóstico por imagen , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/citología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Mutación/genética , Fosfoproteínas/genética , Presenilina-1/genética , Presenilina-1/metabolismoRESUMEN
BACKGROUND: The improvement of rice cultivars plays an important role in yield increase. However, little is known about the changes in starch quality and mineral elements during the improvement of rice cultivars. This study was conducted to investigate the changes in starch quality and mineral elements in japonica rice cultivars. RESULTS: Twelve typical rice cultivars, applied in the production in Jiangsu province during the last 60 years, were grown in the paddy fields. These cultivars were classified into six types according to their application times, plant types and genotypes. The nitrogen (N), phosphorus (P) and, and potassium (K) were mainly distributed in endosperm, bran and bran, respectively. Secondary and micromineral nutrients were distributed throughout grains. With the improvement of cultivars, total N contents gradually decreased, while total P, K and magnesium contents increased in grains. Total copper and zinc contents in type 80'S in grains were highest. The improvement of cultivars enhanced palatability (better gelatinisation enthalpy and amylose content), taste (better protein content) and protein quality (better protein components and essential amino acids). Correlation analysis indicated the close relationship between mineral elements and starch quality. CONCLUSION: The mineral elements and starch quality of grains during the improvement of japonica rice cultivars are improved. © 2017 Society of Chemical Industry.
Asunto(s)
Minerales/análisis , Oryza/química , Almidón/análisis , Endospermo/química , Endospermo/embriología , Endospermo/metabolismo , Magnesio/análisis , Minerales/metabolismo , Nitrógeno/análisis , Nitrógeno/metabolismo , Oryza/clasificación , Oryza/embriología , Oryza/metabolismo , Fósforo/análisis , Fósforo/metabolismo , Potasio/análisis , Potasio/metabolismo , Semillas/química , Semillas/clasificación , Semillas/embriología , Semillas/metabolismo , Almidón/metabolismoRESUMEN
A novel functional [2]rotaxane containing two alkenyl bonds was designed, synthesized and characterized by 1H, 13C NMR spectroscopy and HRESI mass spectrometry. The introduction of alkenyl bonds endowed the [2]rotaxane a fascinating ability to react with versatile functional groups such as alkenyl and thiol functional groups. The reversible shuttling movement of the macrocycle between two different recognition sites on the molecular thread can be driven by external acid and base. This kind of rotaxane bearing functional groups provides a powerful platform for preparing stimuli-responsive polymers.
RESUMEN
Accumulating evidence suggests that formation of peroxynitrite (ONOO(-)) in the cerebral vasculature contributes to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO(-) biology, efficient tools that can realize the real-time tracing of endogenous ONOO(-) fluxes are indispensable. While a few ONOO(-) fluorescent probes have been reported, direct visualization of ONOO(-) fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switch-on probe (NP3) for ONOO(-) imaging. NP3 exhibits good specificity, fast response, and high sensitivity toward ONOO(-) both in vitro and in vivo. Moreover, NP3 is two-photon excitable and readily blood-brain barrier penetrable. These desired photophysical and pharmacokinetic properties endow NP3 with the capability to monitor brain vascular ONOO(-) generation after injury with excellent temporal and spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO(-) formation in ischemia progression in live mouse brain by use of two-photon laser scanning microscopy. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.
Asunto(s)
Traumatismos Cerebrovasculares/metabolismo , Células Endoteliales/metabolismo , Colorantes Fluorescentes/química , Ácido Peroxinitroso/metabolismo , Animales , Traumatismos Cerebrovasculares/patología , Células Endoteliales/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacocinética , Ratones , Estructura Molecular , Ácido Peroxinitroso/químicaRESUMEN
The present study was designed to investigate the role of autophagy-lysosome signaling in the brain after application of nanoparticles. Here, lipid nanoparticles (LNs) induced elevations of Atg5, P62, LC3 and cathepsin B in mice brain. The transmission electron microscopy revealed a dramatic elevation of lysosome vacuoles colocalized with LNs cluster inside the neurons in mice brain. Immunoblot data revealed abnormal expression of cathepsin B in brain cortex following LNs injection, whereas its expression was further elevated in Atg5(+/-) mice. The importance of Atg5 in the LNs-induced autophagy-lysosome cascade was further supported by our finding that neurovascular response was exaggerated in Atg5(+/-) mice. In addition, the siRNA knockdown of Atg5 significantly blunted the increasing of LC3 and P62 in LNs-treated Neuro-2a cells. Taken together, we propose that LNs induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway. FROM THE CLINICAL EDITOR: These authors investigated autophagy-lysosome signaling in the mouse brain after application of lipid nanoparticles and report that these nanoparticles induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway.
Asunto(s)
Encéfalo/metabolismo , Catepsina B/metabolismo , Lípidos/química , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/deficiencia , Nanopartículas/química , Animales , Proteína 5 Relacionada con la Autofagia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
Controlled-release nitrogen fertilizer (CRNF) has been expected to save labor input, reduce environmental pollution, and increase yield in crop production. However, the economic feasibility is still controversial due to its high cost. To clarify the suitable application strategy of CRNF in promoting the yield, nitrogen use efficiency and income on wheat grown in paddy soil, four equal N patterns were designed in 2017-2021 with polymer-coated urea (PCU) and common urea as material, including PCU applied once pre-sowing (M1), PCU applied 60% at pre-sowing and 40% at re-greening (M2), 30% PCU and 30% urea applied at pre-sowing, 20% PCU and 20% urea applied at re-greening (M3), and urea applied at four stage (CK, Basal:tillering:jointing:booting=50%:10%:20%:20%). In addition, M4-M6, which reduced N by 10%, 20% and 30% respectively based on M3, were designed in 2019-2021 to explore their potential for N-saving and efficiency-improving. The results showed that, compared with CK, M1 did not significantly reduce yield, but decreased the average N recovery efficiency (NRE) and benefits by 1.63% and 357.71 CNY ha-1 in the four years, respectively. M2 and M3 promoted tiller-earing, delayed the decrease of leaf area index (LAI) at milk-ripening stage, and increased dry matter accumulation post-anthesis, thereby jointly increasing spike number and grain weight of wheat, which significantly increased yield and NRE compared with CK in 2017-2021. Due to the savings in N fertilizer costs, M3 achieved the highest economic benefits. With the 20% N reduction, M5 increased NRE by 16.95% on average while decreasing yield and net benefit by only 6.39% and 7.40% respectively, compared with M3. Although NRE could continue to increase, but the yield and benefits rapidly decreased after N reduction exceeds 20%. These results demonstrate that twice-split application of PCU combined with urea is conducive to achieving a joint increase in yield, NRE, and benefits. More importantly, it can also significantly improve the NRE without losing yield and benefits while saving 20% N input.
RESUMEN
Clinical epidemiology has indicated that the endothelial injury is a potential contributor to the pathogenesis of ischemic neurovascular damage. In this report, we assessed S-nitrosylation and nitration of Keap1 to identify downstream nitric oxide redox signaling targets into endothelial cells during ischemia. Here, oxygen-glucose deprivation (OGD) exposure initiates the nuclear import of Keap1 in endothelial cells, which interacted with nuclear-localized Nrf2, as demonstrated through co-immunoprecipitation and immunocytochemical assay. Paralleling the ischemia-induced nuclear import of Keap1, increased nitrotyrosine immunoreactivity in endothelial cells was also observed. Consistently, the addition of peroxynitrite provoked nuclear import of Keap1 and a concomitant Nrf2 nuclear import in the endothelial cells. Importantly, pharmacological inhibition of nitrosative stress by melatonin partially inhibited the OGD-induced constitutive nuclear import of Keap1 and subsequently disturbance of Nrf2/Keap1 signaling. Moreover, the effect of melatonin on nitration and S-nitrosylation of keap1 was examined in endothelial cells with 6 hr OGD exposure. Here, we demonstrated that OGD induced tyrosine nitration of Keap1, which was blocked by melatonin treatment, while there were no significant changes in S-nitrosylation of Keap1. The specific amino acid residues of Keap1 involved in tyrosine nitration were identified as Y473 by mass spectrometry. Moreover, the protective role of melatonin against damage to endothelial tight junction integrity was addressed by ZO-1 expression, paralleled with the restored heme oxygenase-1 levels during OGD. Together, our results emphasize that upon nitrosative stress, the protective effect of melatonin on endothelial cells is likely mediated at least in part by inhibition of ischemia-evoked protein nitration of Keap1, hence contributing to relieve the disturbance of Nrf2/Keap1 antioxidative signaling.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isquemia/metabolismo , Melatonina/farmacología , Estrés Fisiológico/efectos de los fármacos , Análisis de Varianza , Antioxidantes/metabolismo , Antioxidantes/farmacología , Línea Celular , Células Endoteliales/metabolismo , Glucosa/metabolismo , Histocitoquímica , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Microscopía Fluorescente , Factor 2 Relacionado con NF-E2/metabolismo , Nitratos/metabolismo , Oxígeno/metabolismo , Estrés Fisiológico/fisiología , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Nitrogen (N) is an essential nutrient element required for plant growth, and the development of wheat varieties with high nitrogen use efficiency (NUE) is an urgent need for sustainable crop production. However, the molecular mechanism of NUE between diverse wheat varieties in response to N application remains unclear. To reveal the possible molecular mechanisms underlying this complex phenomenon, we investigated the transcriptional and metabolic changes of flag leaves of two wheat near-isogenic lines (NILs) differing in NUE under two N fertilizer treatments. Comparative transcriptome analysis indicated that the expression levels of the genes responsible for carbon and nitrogen metabolism were significantly higher in high-NUE wheat. The metabolome comparison revealed that the activity of the tricarboxylic acid (TCA) cycle was enhanced in high-NUE wheat, while reduced in low-NUE wheat after the N fertilizer application. Additionally, amino acid metabolism increased in both wheat NILs but more increased in high-NUE wheat. In summary, more upregulated transcripts and metabolites were identified in high-NUE wheat, and this study provides valuable new insights for improving NUE in wheat.
Asunto(s)
Transcriptoma , Triticum , Triticum/metabolismo , Nitrógeno/metabolismo , Fertilizantes , Perfilación de la Expresión GénicaRESUMEN
AIMS: Cerebrovascular impairment contributes to the pathogenesis of Alzheimer's disease (AD). However, it still lacks effective intervention in clinical practice. Here, we investigated the efficacy of electroacupuncture (EA) in cerebrovascular repair in 3xTg-AD mice and its mechanism. METHODS: 3xTg-AD mice were employed to evaluate the protective effect of EA at ST36 acupoint (EAST36). Behavioral tests were performed to assess neurological disorders. Laser speckle contrast imaging, immunostaining, and Western blot were applied to determine EAST36-boosted cerebrovascular repair. The mechanism was explored in 3xTg mice and endothelial cell cultures by melatonin signaling modulation. RESULTS: EAST36 at 20/100 Hz effectively alleviated the olfactory impairment and anxiety behavior and boosted cerebrovascular repair in AD mice. EAST36 attenuated cerebral microvascular degeneration in AD mice by modulating endothelial cell viability and injury. Consequently, the Aß deposits and neural damage in AD mice were reversed after EAST36. Mechanistically, we revealed that EAST36 restored melatonin levels in AD mice. Melatonin supplement mimicked the EAST36 effect on cerebrovascular protection in AD mice and endothelial cell cultures. Importantly, blockage of melatonin signaling by antagonist blunted EAST36-induced cerebrovascular recovery and subsequent neurological improvement. CONCLUSIONS: These findings provided strong evidence to support EAST36 as a potential nonpharmacological therapy against cerebrovascular impairment in AD. Further study is necessary to better understand how EAST36 treatment drives melatonin signaling.
Asunto(s)
Enfermedad de Alzheimer , Electroacupuntura , Melatonina , Ratones , Animales , Enfermedad de Alzheimer/terapia , Enfermedad de Alzheimer/tratamiento farmacológico , Melatonina/uso terapéutico , Electroacupuntura/métodos , Modelos Animales de Enfermedad , Ratones TransgénicosRESUMEN
BACKGROUND: The cerebral microvascular occlusion elicits microvascular injury which mimics the different degrees of stroke severity observed in patients, but the mechanisms underlying these embolic injuries are far from understood. The Fas ligand (FasL)-Fas system has been implicated in a number of pathogenic states. Here, we examined the contribution of microglia-derived FasL to brain inflammatory injury, with a focus on the potential to suppress the FasL increase by inhibition of the P2X(7)-FasL signaling with pharmacological or genetic approaches during ischemia. METHODS: The cerebral microvascular occlusion was induced by microsphere injection in experimental animals. Morphological changes in microglial cells were studied immunohistochemically. The biochemical analyses were used to examine the intracellular changes of P2X(7)/FasL signaling. The BV-2 cells and primary microglia from mice genetically deficient in P2X(7) were used to further establish a linkage between microglia activation and FasL overproduction. RESULTS: The FasL expression was continuously elevated and was spatiotemporally related to microglia activation following microsphere embolism. Notably, P2X(7) expression concomitantly increased in microglia and presented a distribution pattern that was similar to that of FasL in ED1-positive cells at pathological process of microsphere embolism. Interestingly, FasL generation in cultured microglia cells subjected to oxygen-glucose deprivation-treated neuron-conditioned medium was prevented by the silencing of P2X(7). Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility. Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7). Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult. CONCLUSION: FasL functions as a key component of an immunoreactive response loop by recruiting microglia to the lesion sites through a P2X(7)-dependent mechanism. The specific modulation of P2X(7)/FasL signaling and aberrant microglial activation could provide therapeutic benefits in acute and subacute phase of cerebral microembolic injury.
Asunto(s)
Proteína Ligando Fas/biosíntesis , Embolia Intracraneal/metabolismo , Microglía/metabolismo , Microesferas , Receptores Purinérgicos P2X7/fisiología , Animales , Muerte Celular , Células Cultivadas , Proteína Ligando Fas/metabolismo , Embolia Intracraneal/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus. METHODS: SD rats were randomly divided into three groups: control group was given distilled water, lead-exposed groups were given 0.5 g/L (low-dose) or 2.0 g/L(high-dose) lead acetate solution in drinking water. The rat pups started to drink the lead content water until 60 d maturity. The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry. The expressions of Beclin 1, LC3, LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry. RESULTS: Compared with control group, the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats (P<0.01). Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group (P<0.05 or P<0.001). The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals. CONCLUSION: The disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Hipocampo/metabolismo , Intoxicación por Plomo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Autofagia/efectos de los fármacos , Beclina-1 , Catepsina B/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/patología , Intoxicación por Plomo/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Background: Imaging amyloid-beta (Aß) deposits with high fidelity in naturally aging brains is crucial for the early diagnosis of Alzheimer's disease (AD). However, this is impeded by the lack of highly sensitive probes. Methods: By conducting computational modelling to quantitatively fine-tune the twisted intramolecular charge transfer (TICT) tendency of Thioflavin T (ThT) analogues, we developed an ultrasensitive probe AH-2. AH-2 retained the binding affinity and binding mode of ThT towards Aß deposits, and exhibited ca 10-fold less background fluorescence and 5-10 folds of improved signal-to-background contrast upon binding Aß deposits. These desirable features endowed AH-2 the sensitivity to detect Aß deposition in naturally aging wild-type mice. Results: AH-2 imaging revealed that Aß puncta signals appeared near the nuclei in young mice and spread through the intracellular and extracellular compartments in older mice. Moreover, Aß deposits were observed to emerge earlier in mice cerebral cortex than in the hippocampus region. Given this desirable sensitivity and good spatiotemporal resolution, AH-2 was successfully applied in the preclinical evaluation of Aß-targeted treatment by melatonin. Conclusions: We expect that AH-2 is promising for early diagnosis of AD and will serve as a sensitive tool for studying Aß-related AD pathology.
Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Animales , Diagnóstico Precoz , Colorantes Fluorescentes/química , Ratones , Ratones Transgénicos , Imagen Óptica/métodosRESUMEN
OBJECTIVE: To investigate the protective effect of electroacupuncture (EA) at "Zusanli"(ST36)and "Weiwanxiashu"(EX-B3) on capillary function around the renal tubule and renal tubule structure in diabetic mice based on two-photon microscopy (TPM) imaging, so as to providing visualizable evidence for the regulatory effect of EA on diabetic renal vascular microcirculation. METHODS: Spontaneous type â ¡ diabetes mellitus mice (db/db) were employed for this study. Twenty db/db mice were randomly divided into model group (n=10) and EA group (n=10), and 10 db/m mice used as the control group. EA was applied to bilateral ST36 and EX-B3 for 20 min/time, 6 times a week for 6 weeks. The body weight was recorded and the fasting blood glucose measured before and after the intervention. The urine production and water consumption of mice in each cage were recorded after EA. The renal in vivo imaging method based on TPM was established to display the morphological structure of renal tubules, and the mouse renal blood flow velocity was detected by injecting 500 kDa dextran-fluorescein into femoral vein after the intervention. RESULTS: Compared with the control group, the proportion of mice with decreased body mass in the model group was increased, accounting for 40%, while that in the control group was 0%; and fasting blood glucose, urine production and water consumption were significantly increased in the model group (P<0.001, P<0.000 1). A renal in vivo imaging method based on TPM was successfully established, which can be applied to quantitatively analyze the renal blood flow and renal tubular diameter of mice. Based on this method, the results showed that compared with the control group, the blood flow velocity of peritubular capillary in the model group was significantly decreased (P<0.000 1, P<0.001), renal tubular cells were slightly exfoliated and the diameter of renal tubular was significantly increased (P<0.000 1). Compared with the model group, EA reduced the body weight loss ratio from 40% to 0%, and significantly decreased the fasting blood glucose, urine production and water consumption (P<0.01, P<0.000 1, P<0.001), and the blood flow velocity of peritubular capillary in the EA group was significantly increased (P<0.001, P<0.05) and tubule dilatation significantly alleviated (P<0.0 1). CONCLUSION: EA at ST36 and EX-B3 can ameliorate renal vascular microcirculation disorder to relieve the renal structure damage and improve renal function in diabetes mice.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Electroacupuntura , Animales , Glucemia , Diabetes Mellitus Experimental/diagnóstico por imagen , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 2/diagnóstico por imagen , Diabetes Mellitus Tipo 2/terapia , Ratones , Microcirculación , MicroscopíaRESUMEN
Peroxynitrite contributes to diverse cellular stresses in the pathogenesis of ischemic complications. Here, we investigate the downstream effector signaling elements of nitrosative stress which regulate ischemia-like cell death in endothelial cells and protective effect of melatonin. When the mitochondrial membrane potential (ΔΨm) of oxygen-glucose deprivation (OGD)-treated cells was assessed using the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol -carbocyanine iodide, we observed spontaneous changes in peroxynitrite formation. Concomitantly, western blot and confocal microscopy analyses indicated that prolonged OGD exposure initiates the release of mitochondrial HtrA2 and dramatically decreases phosphoprotein enriched in astrocytes (PED or PEA-15) protein levels. Consistently, cultured endothelial cells treated with peroxynitrite (1-50 µm) exhibited a concentration-dependent release of mitochondrial HtrA2 and concomitant PED degradation in vitro. Notably, HtrA2 activation coincided with increased nitrotyrosine immunoreactivity in microvessels of rats following microsphere embolism. Additionally, the protective effect of PED overexpression in OGD-induced apoptosis was abolished by transfection with the PED(S104A/S116A) mutant. Furthermore, the effect of melatonin, an potential antioxidant, on endothelial apoptotic cascade was examined in OGD-evoked nitrosative stress. Our data showed that the application of melatonin provided significant protection against OGD-induced peroxynitrite formation and mitochondrial HtrA2 release, accompanied with a decrease in degradation PED and x-linked inhibitor of apoptosis protein, which is associated with activation of the caspase cascade. Taken together, the protective effect of melatonin is likely mediated, in part, by inhibition of peroxynitrate-mediated nitrosative stress, which in turn relieves imbalance of mitochondrial HtrA2-PED signaling and endothelial cell death.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Células Endoteliales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Isquemia Encefálica/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Citometría de Flujo , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Melatonina , Potencial de la Membrana Mitocondrial , Microscopía Confocal , Proteínas Mitocondriales/genética , Proteínas del Tejido Nervioso/genética , Ácido Peroxinitroso/farmacología , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Ratas , Ratas Wistar , Serina Endopeptidasas/genética , Factores de Empalme Serina-Arginina , Transducción de Señal/efectos de los fármacosRESUMEN
The cellular mechanisms that underlie the diverse nitrosative stress-mediated cellular events associated with ischemic complications in endothelial cells are not yet clear. To characterize whether autophagic elements are associated with the nitrosative stress that causes endothelial damage after ischemia injury, an in vitro sustained oxygen-glucose deprivation (OGD) and an in vivo microsphere embolism model were used in the present study. Consistent with OGD-induced peroxynitrite formation, a rapid induction of microtubule-associated protein 1 light chain 3 (LC3)-I/II conversion and green fluorescent protein-LC3 puncta accumulation were observed in endothelial cells. The Western blot analyses indicated that OGD induced elevations in lysosome-associated membrane protein 2 and cathepsin B protein levels. Similar results were observed in the microvessel insult model, following occlusion of the microvessels using microsphere injections in rats. Furthermore, cultured endothelial cells treated with peroxynitrite (1-50 µm) exhibited a concentration-dependent change in the pattern of autophagy-lysosome signaling. Intriguingly, OGD-induced autophagy-lysosome processes were attenuated by PEP-19 overexpression and by a small-interfering RNA (siRNA)-mediated knockdown of eNOS. The importance of nitrosative stress in ischemia-induced autophagy-lysosome cascades is further supported by our finding that pharmacological inhibition of nitrosative stress by melatonin partially inhibits the ischemia-induced autophagy-lysosome cascade and the degradation of the tight junction proteins. Taken together, the present results demonstrate that peroxynitrite-mediated nitrosative stress at least partially potentiates autophagy-lysosome signaling during sustained ischemic insult-induced endothelial cell damage.
Asunto(s)
Autofagia/fisiología , Isquemia Encefálica/patología , Lisosomas/metabolismo , Ácido Peroxinitroso/farmacología , Animales , Autofagia/efectos de los fármacos , Encéfalo/irrigación sanguínea , Isquemia Encefálica/metabolismo , Línea Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Glucosa/metabolismo , Humanos , Inmunohistoquímica , Embolia Intracraneal , Masculino , Melatonina/farmacología , Microscopía Fluorescente , Microesferas , Microvasos , Proteínas del Tejido Nervioso/metabolismo , Nitrosación , Oxígeno/metabolismo , Ratas , Ratas Wistar , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/fisiologíaRESUMEN
Soil salinity is a worldwide issue that affects wheat production. A comprehensive understanding of salt-tolerance mechanisms and the selection of reliable screening indices are crucial for breeding salt-tolerant wheat cultivars. In this study, 30 wheat genotypes (obtained from a rapid selection of 96 original varieties) were chosen to investigate the existing screening methods and clarify the salinity tolerance mechanisms in wheat. Ten-day-old seedlings were treated with 150 mM NaCl. Eighteen agronomic and physiological parameters were measured. The results indicated that the effects of salinity on the agronomic and physiological traits were significant. Salinity stress significantly decreased K+ content and K+/Na+ ratio in the whole plant, while the leaf K+/Na+ ratio was the strongest determinant of salinity tolerance and had a significantly positive correlation with salt tolerance. In contrast, salinity stress significantly increased Na+ concentration and relative gene expression (TaHKT1;5, TaSOS1, and TaAKT1-like). The Na+ transporter gene (TaHKT1;5) showed a significantly greater increase in expression than the K+ transporter gene (TaAKT1-like). We concluded that Na+ exclusion rather than K+ retention contributed to an optimal leaf K+/Na+ ratio. Furthermore, the present exploration revealed that, under salt stress, tolerant accessions had higher shoot water content, shoot dry weight and lower stomatal density, leaf sap osmolality, and a significantly negative correlation was observed between salt tolerance and stomatal density. This indicated that changes in stomata density may represent a fundamental mechanism by which a plant may optimize water productivity and maintain growth under saline conditions. Taken together, the leaf K+/Na+ ratio and stomatal density can be used as reliable screening indices for salt tolerance in wheat at the seedling stage.
RESUMEN
The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.
Asunto(s)
Apoptosis/efectos de los fármacos , Citotoxinas/toxicidad , Células Endoteliales/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , S-Nitrosoglutatión/toxicidad , Serina Endopeptidasas/metabolismo , Acetofenonas/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , NADPH Oxidasas/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso/metabolismo , Pirimidinonas/farmacología , Serina Endopeptidasas/genética , Transducción de Señal/efectos de los fármacos , Marcadores de Spin , Tionas/farmacologíaRESUMEN
Vascular stent interventional therapy is the main method for clinical treatment of coronary artery diseases. However, due to the insufficient biocompatibility of cardiovascular materials, the implantation of stents often leads to serious adverse cardiac events. Surface biofunctional modification to improve the biocompatibility of vascular stents has been the focus of current research. In this study, based on the structure and function of extracellular matrix on vascular injury healing, a novel fibronectin-loaded poly-l-lysine/heparin nanoparticles was constructed for stent surface modification. In vitro blood compatibility evaluation results showed that the nanoparticles-modified surface could effectively reduce platelet adhesion and activation. In vitro cellular compatibility evaluation results indicated that the nanocoating may provide adequate efficacy in promoting the adhesion and proliferation of endothelial cells and thereby accelerate endothelialization. This study provides a new approach for the surface biological function modification of vascular stents.