RESUMO
BACKGROUND: Oral submucous fibrosis (OSF) is a premalignant and fibrosing disease, which is closely associated with the habit of chewing areca nut. Panax notoginseng Buck F. H. Chen is an often used antifibrotic and antitumor agent. To treat areca nut-induced OSF, we have developed a chewable tablet, in which one of the major medicines is total Panax notoginseng saponins (PNS). In this study, we have investigated the antifibrotic effect and mechanism of PNS on areca nut-induced OSF in vitro. METHODS: Through human procollagen gene promoter luciferase reporter plasmid, hydroxyproline assay, gelatin zymography, qRT-PCR, ELISA, and Western blot, the influences of PNS on areca nut extract (ANE)-induced cell growth, collagen accumulation, procollagen gene transcription, MMP-2/-9 activity, MMP-1/-13 and TIMP-1/-2 expression, cytokine secretion, and the activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/Smads pathways were detected. RESULTS: Panax notoginseng saponins could inhibit the ANE-induced abnormal growth and collagen accumulation of oral mucosal fibroblasts in a concentration-dependent manner. PNS (25 µg/ml) could significantly inhibit the ANE-induced expression of Col1A1 and Col3A1, augment the ANE-induced decrease of MMP-2/-9 activity, inhibit the ANE-induced increase of TIMP-1/-2 expression, and decrease the ANE-induced transcription and release of CTGF, TGFß1, IL-6, and TNFα. PNS (25 µg/ml) also significantly inhibited the ANE-induced activation of AKT and ERK/JNK/p38 MAPK pathways in oral mucosal fibroblasts and the ANE-induced activation of TGFß/smad pathway in HaCaT cells. CONCLUSION: Panax notoginseng saponins possess excellent anti-OSF activity, and its mechanism may be related to its ability to inhibit the ANE-induced activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/smad pathways.
Assuntos
Areca/efeitos adversos , Mucosa Bucal/efeitos dos fármacos , Nozes/efeitos adversos , Fibrose Oral Submucosa/patologia , Panax notoginseng , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Colágeno Tipo I/efeitos dos fármacos , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Hidroxiprolina/análise , Interleucina-6/análise , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Bucal/citologia , Fibrose Oral Submucosa/etiologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Extratos Vegetais/efeitos adversos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Smad/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Extratos Vegetais/química , Silybum marianum/química , Silimarina/análogos & derivados , Animais , Antivirais/síntese química , Antivirais/isolamento & purificação , Autofagia/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Cães , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Células Madin Darby de Rim Canino , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Plasmídeos , Multimerização Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Silimarina/síntese química , Silimarina/isolamento & purificação , Silimarina/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Células VeroRESUMO
Autophagy is involved in many human diseases, such as cancer, cardiovascular disease and virus infection, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza A virus (IAV) and coxsackievirus B3/B4 (CVB3/B4), so a drug screening model targeting autophagy may be very useful for the therapy of these diseases. In our study, we established a drug screening model based on the inhibition of the dissociation of Beclin1-Bcl2 heterodimer, an important negative regulator of autophagy, using bimolecular fluorescence complementation (BiFC) technique for developing novel autophagy inhibitors and anti-IAV agents. From 86 examples of traditional Chinese medicines, we found Syzygium aromaticum L. had the best activity. We then determined the anti-autophagy and anti-IAV activity of eugenol, the major active compound of Syzygium aromaticum L., and explored its mechanism of action. Eugenol could inhibit autophagy and IAV replication, inhibited the activation of ERK, p38MAPK and IKK/NF-κB signal pathways and antagonized the effects of the activators of these pathways. Eugenol also ameliorated the oxidative stress and inhibited the expressions of autophagic genes. We speculated that the mechanism underlying might be that eugenol inhibited the oxidative stress and the activation of ERK1/2, p38MAPK and IKK/NF-κB pathways, subsequently inhibited the dissociation of Beclin1-Bcl2 heterodimer and autophagy, and finally impaired IAV replication. These results might conversely display the reasonableness of the design of our screening model. In conclusion, we have established a drug screening model for developing novel autophagy inhibitor, and find eugenol as a promising inhibitor for autophagy and IAV infection.