RESUMO
Hepatitis C virus (HCV) infection is one of the major factors to trigger a sustained hepatic inflammatory response and hence hepatocellular carcinoma (HCC), but direct-acting-antiviral (DAAs) was not efficient to suppress HCC development. Heat shock protein 90 kDa (HSP90) is highly abundant in different types of cancers, and especially controls protein translation, endoplasmic reticulum stress, and viral replication. In this study we investigated the correlation between the expression levels of HSP90 isoforms and inflammatory response marker NLRP3 in different types of HCC patients as well as the effect of a natural product celastrol in suppression of HCV translation and associated inflammatory response in vivo. We identified that the expression level of HSP90ß isoform was correlated with that of NLRP3 in the liver tissues of HCV positive HCC patients (R2 = 0.3867, P < 0.0101), but not in hepatitis B virus-associated HCC or cirrhosis patients. We demonstrated that celastrol (3, 10, 30 µM) dose-dependently suppressed the ATPase activity of both HSP90α and HSP90ß, while its anti-HCV activity was dependent on the Ala47 residue in the ATPase pocket of HSP90ß. Celastrol (200 nM) halted HCV internal ribosomal entry site (IRES)-mediated translation at the initial step by disrupting the association between HSP90ß and 4EBP1. The inhibitory activity of celastrol on HCV RNA-dependent RNA polymerase (RdRp)-triggered inflammatory response also depended on the Ala47 residue of HSP90ß. Intravenous injection of adenovirus expressing HCV NS5B (pAde-NS5B) in mice induced severe hepatic inflammatory response characterized by significantly increased infiltration of immune cells and hepatic expression level of Nlrp3, which was dose-dependently ameliorated by pretreatment with celastrol (0.2, 0.5 mg/kg, i.p.). This study reveals a fundamental role of HSP90ß in governing HCV IRES-mediated translation as well as hepatic inflammation, and celastrol as a novel inhibitor of HCV translation and associated inflammation by specifically targeting HSP90ß, which could be developed as a lead for the treatment of HSP90ß positive HCV-associated HCC.
Assuntos
Carcinoma Hepatocelular , Hepatite C , Neoplasias Hepáticas , Camundongos , Animais , Hepacivirus , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Proteínas de Choque Térmico , Proteína 3 que Contém Domínio de Pirina da Família NLR , Hepatite C/complicações , Hepatite C/tratamento farmacológico , Proteínas de Choque Térmico HSP90/metabolismo , Inflamação/tratamento farmacológicoRESUMO
OBJECTIVE: To investigate the inhibitory effect of TBN on rabbit platelet aggregation in vitro and in vivo, and compare with tetramethylpyrazine (TMP). METHODS: To seek out the maximum of platelet aggregation (expressed in percentage) within 5 minutes which was induced by ADP, PAF and AA according to the Born turbidimetric method with a Platelet-Aggregometer. RESULTS: TBN significantly inhibited platelet aggregation induced by ADP, PAF and AA both in vitro and in vivo. TBN was more active than TMP. CONCLUSION: TBN has significant activity inhibiting platelet aggregation induced by ADP, PAF and AA in vitro and in vivo.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Pirazinas/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Ácidos Araquidônicos/farmacologia , Medicamentos de Ervas Chinesas/química , Masculino , Nefelometria e Turbidimetria , Fator de Ativação de Plaquetas/farmacologia , Inibidores da Agregação Plaquetária/química , Pirazinas/química , CoelhosRESUMO
Neurodegenerative disorders are one of the leading causes of death among the elderly. Therapeutic approaches with a single target have proven unsuccessful in treating these diseases. Structural combination of multi-functional compounds may lead to a molecule with multiple properties. In this study, we designed and synthesized T-006, a novel analog derived from two multi-functional neuroprotective chemicals, tetramethylpyrazine and J147. The methoxyphenyl group of J147 was replaced by tetramethylpyrazine. Bioactivity evaluation showed that T-006 at very low concentrations had multi-functional neuroprotective effects including rescuing iodoacetic acid-induced neuronal loss, preventing oxidative stress-induced neurotoxicity and reducing glutamate-induced excitotoxicity in vitro. Most importantly, T-006 significantly ameliorated memory impairments in APP/PS1 transgenic mice. These multiple functions of a single molecule suggest that T-006 is a promising novel neuroprotective agent for treating various neurodegenerative disorders, including and in particular Alzheimer's disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antioxidantes/farmacologia , Hidrazonas/farmacologia , Fármacos Neuroprotetores/farmacologia , Pirazinas/farmacologia , Animais , Antioxidantes/síntese química , Antioxidantes/uso terapêutico , Células Cultivadas , Hidrazonas/síntese química , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/uso terapêutico , Células PC12 , Pirazinas/síntese química , Pirazinas/química , Ratos , Ratos Sprague-DawleyRESUMO
INTRODUCTION: The natural product tetramethylpyrazine (TMP) has a variety of biologic activities, including neuroprotection. Nitrones are powerful free radical scavengers. We have designed and synthesized a TMP derivative, TN-2, which is armed with two nitrone moieties. AIMS: In this study, we investigated the neuroprotective effect of TN-2 against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro and in zebrafish. METHODS: PC12 cells, zebrafish and rats were exposed to 6-OHDA challenge. MTT assay, LDH release, Hoechst staining, DAF-FM staining, luciferase reporter construct transfection, and western blotting were applied to detect cell viability, apoptosis, intracellular nitric oxide (NO), NF-κB transcriptional activity and proteins expression. In zebrafish, whole-mount staining and real-time PCR were performed to quantify dopaminergic neurons and mRNA expression. Hematoxylin and eosin staining and immunohistochemistry for glial fibrillary acidic protein were used to detect the astrocyte activation in the unilateral 6-OHDA rat model. RESULTS: TN-2 but not TMP exhibited potent neuroprotective effect against 6-OHDA-induced apoptosis in PC12 cells. Moreover, TN-2 prevented dopaminergic neuron loss and suppressed mRNA expression of pro-inflammatory genes, including IL-1ß, TNF-α and COX-2, in 6-OHDA-treated zebrafish. TN-2 remarkably attenuated microglial/astrocyte activation in the unilateral 6-OHDA rat model. The mechanistic study demonstrated that TN-2 inhibited over-production of intracellular NO and protein expression of inducible nitric oxide synthase through down-regulating NF-κB activity. Additionally, the PKCα/PI3-K/Akt pathway was also involved in the neuroprotection of TN-2. CONCLUSION: These results suggest that TN-2 protected against 6-OHDA-induced neurotoxicity via modulating the NF-κB-medicated neuroinflammation and PKCα/PI3-K/Akt pathways.