RESUMO
Autophagy dysregulation is implicated in cadmium (Cd)-induced nephrotoxicity. The mammalian target of rapamycin complex 1 (mTORC1) is a negative regulator of autophagy, but its role in Cd-induced autophagy inhibition and possible regulatory mechanisms remains poorly understood. In the present study, Cd exposure activated mTORC1 in primary rat proximal tubular (rPT) cells, and two mTORC1 inhibitors (rapamycin and torin 1) were separately utilized to inhibit Cd-induced mTORC1 activation. Data showed that Cd-inhibited autophagic flux was markedly restored by two mTORC1 inhibitors, respectively, as evidenced by immunoblot analysis of autophagy marker proteins and tandem red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3) fluorescence microscopy assay. Importantly, Cd exposure triggered the recruitment of mTORC1 onto lysosome membrane assessed by immunofluorescence co-localization analysis, which was obviously inhibited by rapamycin or torin 1. Moreover, Cd-induced lysosomal alkalization, suppressed vacuolar ATPases (V-ATPases) protein levels and impaired lysosomal degradation capacity were markedly reversed by rapamycin or torin 1. In summary, these findings demonstrate that Cd recruits mTORC1 to lysosome membrane to induce its activation, which results in lysosomal dysfunction and resultant autophagy inhibition in rPT cells.
Assuntos
Autofagia/efeitos dos fármacos , Cádmio/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Túbulos Renais Proximais/citologia , Lisossomos/metabolismo , RatosAssuntos
Endoscopia do Sistema Digestório/métodos , Hemorragia Gastrointestinal/etiologia , Hemangiossarcoma/patologia , Neoplasias Intestinais/patologia , Cirrose Hepática Alcoólica/diagnóstico , Angiografia/métodos , Biópsia por Agulha , Diagnóstico Diferencial , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/terapia , Hemangiossarcoma/complicações , Hemangiossarcoma/diagnóstico , Humanos , Imuno-Histoquímica , Neoplasias Intestinais/complicações , Neoplasias Intestinais/diagnóstico , Cirrose Hepática Alcoólica/complicações , Masculino , Pessoa de Meia-Idade , Doenças Raras , Medição de Risco , Tomografia Computadorizada por Raios X/métodosRESUMO
BACKGROUND: Ginsenoside Rg1 is regarded as one of main bioactive compounds responsible for pharmaceutical actions of ginseng with little toxicity and has been shown to have possibly neuroprotective effects. However, the mechanism of its neuroprotection for acute ischemic stroke is still elusive. The purpose of present study is thus to assess the neuroprotective effects of the ginsenoside Rg1 against neurological injury in a mice model of cerebral ischemia/reperfusion (I/R), and then to explore the mechanisms for these neuroprotective effects. METHODS: Mices were pretreated with ginsenoside Rg1 20,40?mg?kg?1?d?1, ig, for 7d, respectively, then subjected to cerebral ischenmia (middle cerebral artery occlusion) for 2?h and reperfusion for 22?h. The infarct volume and the neurological deficit were determined by TTC staining and Longa?s scoring, respectively. The protein expression of brain-derived neurotrophic factor (BDNF) was analyzed by Immunohistochemistry and Western blot, respectively. Interleukin-1? (IL-1?), tumor necrosis factor alpha (TNF-?) and interleukin-6 (IL-6) expression in serum was measured by ELISA kit. High-performance liquid chromatography (HPLC) was used to explore the contents of Glu and Asp. RESULTS: Compared with the ischemia/reperfusion group, ginsenoside Rg1 40?mg/kg group has significantly reduced infarct volume, neurological deficit scores (P?.05), Interleukin-1? (IL-1?), tumor necrosis factor alpha (TNF-?) and interleukin-6 (IL-6) contents in serum (p?.01, respectively), the contents of Glu and Asp (P?.01). Immunohistochemistry and Western blot indicated that ginsenoside Rg1 40?mg/kg group significantly increased brain-derived neurotrophic factor (BDNF) protein expression in the CA1 regions of the hippocampus (p?.01). CONCLUSIONS: Ginsenoside Rg1 40?mg/kg has protective effects on cerebral injury induced by ischeamia/reperfusion, which might be related to the increased in the expression of BDNF in the hippocampal CA1 region, the down-regulation of the expression of IL-1??IL-6 and TNF-? in serum, the decreases in the contents of Glu and Asp in the brain tissue.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Ginsenosídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Western Blotting , Isquemia Encefálica/patologia , Fator Neurotrófico Derivado do Encéfalo/genética , Região CA1 Hipocampal/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/complicações , Interleucina-1beta/genética , Interleucina-6/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/patologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND AND AIMS: Image quality can be guaranteed with the conventional dosage of fluorescein sodium in probe-based confocal laser endomicroscopy (pCLE). However, yellow discoloration of the skin seriously affects daily life and simultaneously increases the risk of adverse events such as allergic reactions. The aim of this study was to test whether a lower dosage of fluorescein sodium can provide satisfactory image quality and to compare the diagnostic accuracy of gastric intestinal metaplasia (GIM) through a randomized blind controlled trial. METHODS: Consecutive patients were randomly assigned to different doses of fluorescein sodium. Image quality was determined by the endoscopists' subjective assessments and signal-to-noise ratio (SNR) assessment systems. Skin discoloration was tested using a neonatal transcutaneous jaundice detector. In addition, consecutive patients with a known or suspected diagnosis of GIM were examined by pCLE with the lower dose and the traditional dose. RESULTS: Only 0.01 mL/kg dose of 10% fluorescein sodium led to a significant decrease in image quality (P < .05), and a dose of 0.02 mL/kg had the highest SNR value (P < .05). There were no significant differences in skin discoloration between the 0.01 mL/kg and 0.02 mL/kg doses (P = .148) and no statistical difference in the diagnostic accuracy of pCLE for GIM between the 0.02 mL/kg and 0.10 mL/kg doses (P > .05). The kappa values for the correlation between pCLE and histopathology were 0.867 (95% confidence interval, 0.782-0.952) and 0.891 (95% confidence interval, 0.811-0.971). CONCLUSIONS: The 0.02 mL/kg dose of 10% fluorescein sodium seems to be the best dose for pCLE in the upper GI tract, with comparable image quality with the conventional dose and insignificant skin discoloration. This dose is also very efficient for the diagnosis of GIM.