RESUMO
Herbal medicines are increasingly being utilized to treat a wide variety of disease processes. Gypenosides (Gyp) are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis in human hepatoma cells. However, the molecular mechanism underlying the Gyp-induced apoptotic process is unclear. In this study, we found that Gyp induced apoptosis in human hepatoma Huh-7, Hep3B and HA22T cell lines as evidenced by morphological changes, 4',6'-diamidino-2-phenylindole staining and in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling assay. Our data demonstrated that Gyp-induced apoptotic cell death was accompanied by up-regulation of Bax, Bak and Bcl-X(L), and down-regulation of Bcl-2 and Bad, while it had no effect on the level of Bag-1 protein. Moreover, Gyp treatment caused the release of mitochondrial cytochrome c to cytosol and sequential activation of caspases, including caspase-1, -9 and -3, then leading to cleavage of poly-ADP-ribose polymerase. Furthermore, the Gyp-induced apoptosis was markedly blocked by the broad-spectrum caspase inhibitor, z-VAD-fmk. Taken together, these results suggest that treatment of human hepatoma cells with Gyp induced apoptosis through the up-regulation of Bax and Bak, and down-regulation of Bcl-2, release of mitochondrial cytochrome c and activation of caspase cascade.
Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saponinas/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Western Blotting , Proteínas de Transporte/metabolismo , Caspase 3 , Sobrevivência Celular , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Citosol/metabolismo , Regulação para Baixo , Humanos , Fatores de Tempo , Regulação para Cima , Proteína de Morte Celular Associada a bclRESUMO
BACKGROUND: Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl(4))-induced liver injury rats. METHODS: Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated. RESULTS: Oral administration of MGP significantly alleviated DMN- or CCl(4)-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression. CONCLUSIONS: The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.
Assuntos
Gleiquênias/química , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Cirrose Hepática/metabolismo , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Citocinas/biossíntese , Dimetilnitrosamina/efeitos adversos , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/tratamento farmacológico , Masculino , Tamanho do Órgão/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Ratos , Baço/efeitos dos fármacosRESUMO
We have previously reported that gypenosides induce apoptosis in human hepatocarcinoma Huh-7 cells through a mitochondria-dependent caspase-9 activation cascade. In order to further explore the critical events leading to apoptosis in gypenosides-treated cells, the following effects of gypenosides on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MPT), and the subcellular distribution of Bcl-2 and Bax. We show that gypenosides-induced apoptosis was accompanied by the generation of intracellular ROS, disruption of MPT, and inactivation of ERK, as well as an increase in mitochondrial Bax and a decrease of mitochondrial Bcl-2 levels. Ectopic expression of Bcl-2 or treatment with furosemide attenuated gypenosides-triggered apoptosis. Treatment with ATA caused a drastic prevention of apoptosis and the gypenosides-mediated mitochondrial Bcl-2 decrease and Bax increase, but failed to inhibit ROS generation and MPT dysfunction. Incubation with antioxidants significantly inhibited gypenosides-mediated ROS generation, ERK inactivation, MPT and apoptosis. Moreover, an increase of the intracellular calcium ion (Ca(2+)) concentration rapidly occurred in gypenosides-treated Huh-7 cells. Buffering of the intracellular Ca(2+) increase with a Ca(2+) chelator BAMTA/AM blocked the gypenosides-elicited ERK inactivation, ROS generation, Bcl-2/Bax redistribution, mitochondrial dysfunction, and apoptosis. Based on these results, we propose that the rise in intracellular Ca(2+) concentration plays a pivotal role in the initiation of gypenosides-triggered apoptotic death.