RESUMO
Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.
Assuntos
Bombyx/genética , Comportamento Alimentar/fisiologia , Proteínas de Insetos/genética , Receptores de Superfície Celular/genética , Percepção Gustatória/genética , Animais , Sequência de Bases , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Cromossomos de Insetos/química , Grão Comestível/parasitologia , Frutas/parasitologia , Edição de Genes/métodos , Expressão Gênica , Engenharia Genética/métodos , Loci Gênicos , Células HEK293 , Homozigoto , Humanos , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Morus/parasitologia , Folhas de Planta/parasitologia , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect Bombyx mori using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific nanos (nos) promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting transformer 2 (tra2), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for B. mori genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.
Assuntos
Bombyx/genética , Sistemas CRISPR-Cas , Cromossomos de Insetos/genética , Edição de Genes/métodos , Cromossomos Sexuais/genética , Processos de Determinação Sexual , Animais , Feminino , MasculinoRESUMO
Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes We successfully replaced the â¼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.
Assuntos
Animais Geneticamente Modificados , Bombyx , Fibroínas , Engenharia Genética , Aranhas/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bombyx/genética , Bombyx/metabolismo , Fibroínas/biossíntese , Fibroínas/genéticaRESUMO
Butylene fipronil (BFPN) is a phenylpyrazole insecticide, acting at the γ-aminobutyric acid (GABA) receptor. Here, we show that BFPN inducedcytotoxicity in PC12 murinenervous cells, which lacks GABA receptor. Treatment with BFPN for 48 hours significantly enhanced G0/G1 arrest and induced apoptosis. BFPN decreased the expression of cyclin-dependent kinase (CDK4 and CDK6) and increased P16 and cyclin D1. Simultaneously, Bcl-2 protein was declined while Bax and cytochrome c were significantly enhanced in BFPN-treated groups. The apoptotic enzymes caspase-8, -9, and -3 were also activated by BFPN. Furthermore, treatment with BFPN significantly stimulated reactive oxygen species (ROS) generation, and pretreatment with antioxidant diphenyleneiodonium, substantially reduced cell death. Overall, these results suggest that BFPN is effective to induce G0/G1-phase arrest and apoptosis in PC12 murine nervous cell. Stimulating ROS generation and activation of P16-CDK4/6-cyclin D1 and mitochondrial apoptotic pathway may participate in the cytotoxicity of BFPN.
Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pirazóis/farmacologia , Animais , Caspases/metabolismo , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Citocromos c/metabolismo , Células PC12 , RatosRESUMO
Intensive investigations have been conducted on the effect of sole drought or salinity stress on the growth of plants. However, there is relatively little knowledge on how plants, particularly woody species, respond to a combination of these two stresses although these stresses can simultaneously occur in the field. In this study, mulberry, an economically important resource for traditional medicine, and the sole food of domesticated silkworms was subjected to a combination of salt and drought stress and analyzed by physiological methods and TMT-based proteomics. Stressed mulberry exhibited significant alteration in physiological parameters, including root/shoot ratio, chlorophyll fluorescence, total carbon, and ion reallocation. A total of 577 and 270 differentially expressed proteins (DEPs) were identified from the stressed leaves and roots, respectively. Through KEGG analysis, these DEPs were assigned to multiple pathways, including carbon metabolism, photosynthesis, redox, secondary metabolism, and hormone metabolism. Among these pathways, the sucrose related metabolic pathway was distinctly enriched in both stressed leaves and roots, indicating an important contribution in mulberry under stress condition. The results provide a comprehensive understanding of the adaptive mechanism of mulberry in response to salt and drought stress, which will facilitate further studies on innovations in terms of crop performance.
Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Morus/genética , Proteínas de Plantas/genética , Proteoma/genética , Estresse Salino , Morus/metabolismo , Morus/fisiologia , Proteínas de Plantas/metabolismo , Proteoma/metabolismoRESUMO
Protocatechualdehyde (PCA) extracted from Phellinus gilvus exhibits anti-cancer activity in human colorectal carcinoma cells (HT-29). However, the underlying mechanisms remain poorly understood. We performed an in vitro study involving MTT, flow cytometry, RT-PCR, and western blot analyses to investigate the effects of PCA treatment on cell proliferation, cell cycle distribution, apoptosis, and expression of several cell cycle-related genes in HT-29 cells. The treatment enhanced S-phase cell cycle and apoptosis in HT-29 cells in a dose-dependent manner. Western blot results showed that PCA treatment decreased the expression levels of cyclin A, cyclin D1, and p27(KIP1) but increased those of cyclin-dependent kinase 2 (CDK2) in HT-29 cells. Furthermore, the expression levels of B-cell lymphoma/leukemia-2 (Bcl-2) and B-cell lymphoma/leukemia-xL (Bcl-xL) were down-regulated, whereas the levels of BH3-interacting domain death agonist (Bid), Bcl-2 homologous antagonist/killer (Bak), and cytosolic cytochrome c were significantly upregulated. Thus, the enzymes caspases-9, -3, -8, and -6 were found to be activated in HT-29 cells with PCA treatment. These results indicate that PCA-induced S-phase cell cycle arrest and apoptosis involve p27(KIP1)-mediated activation of the cyclin-A/D1-Cdk2 signaling pathway and the mitochondrial apoptotic pathway.
Assuntos
Apoptose/efeitos dos fármacos , Benzaldeídos/farmacologia , Catecóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Benzaldeídos/química , Catecóis/química , Proliferação de Células/efeitos dos fármacos , Ciclina A1/metabolismo , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células HT29 , HumanosRESUMO
AIMS: The purpose of this study was to investigate the possible mechanism(s) of saponins from Panax japonicus (SPJ) on alcohol-induced hepatic damage in mice. METHODS: SPJ were identified by high performance liquid chromatography-evaporative light scattering detection-mass spectrometry (LC-ELSD-MS). Non-toxic concentrations of SPJ were assayed on alcohol-induced hepatic injury in male ICR mice and human hepatic cells. The protective effects were evaluated by biochemical values, histopathological observations and the relative gene expression. Results. In vitro, SPJ showed significant hydroxyl radical scavenging capacity. In vivo, SPJ (50 mg/kg) could rectify the pathological changes of aspartate transaminase, alanine transaminase, malondialdehyde, reduced glutathione (GSH), glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) caused by alcohol metabolism to normal levels except for hepatic GSH and CAT. In hepatic cells, the results were in agreement with foregoing results determined in mice after pretreatment of SPJ (100 microg/ml). RT-PCR results showed that CAT, GPX and SOD mRNA decreased by alcohol metabolism were reversed, in which GPX3, SOD1 and SOD3 could return to a normal level, but CAT, GPX1 and SOD2 mRNA were still evidently lower than the control. Histopathological observations provided supportive evidence for biochemical analyses. CONCLUSIONS: SPJ plays an important role in the protection of the structure and function of hepatic mitochondria and karyon by directly scavenging reactive oxygen species/free radicals and up-regulating the expression of antioxidant enzymes (SOD, GPX and CAT), especially to GPX3, SOD1 and SOD3.
Assuntos
Glutationa Peroxidase/genética , Hepatopatias Alcoólicas/prevenção & controle , Fígado/efeitos dos fármacos , Panax/química , Saponinas/farmacologia , Superóxido Dismutase/genética , Regulação para Cima/efeitos dos fármacos , Animais , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Glutationa Peroxidase/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saponinas/química , Saponinas/isolamento & purificação , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
AIMS: The purpose of this study was to investigate the protective effects of sericin protein (SP) on alcohol-induced hepatic injury in mice and the possible mechanisms. METHODS: SP (0.375, 0.75 and 1.50 g/kg body weight) was dissolved in distilled water and given to mice by gavage 1 hour before the alcohol (56% wt/vol, 14.2 ml/kg b.w.) treatment for 30 days, then blood, urine and liver were collected, processed and used for alcohol concentration mensuration, various biochemical estimations and histopathological examination. RESULTS: The concentration of alcohol evidently decreased in serum and increased in urine in SP treated mice as compared to alcohol-administered animals. Chronic alcohol administration resulted in significantly increase in the levels of transaminase (AST and ALT) and malondialdehyde (MDA) but decrease of glutathione (GSH), glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD) in the serum and liver. Hepatic triglyceride (TG) also increased. When mice ingested high doses of SP (0.75 and 1.50 g/kg b.w.) the levels of antioxidant enzymes in the serum were restored to normal. However, hepatic CAT and GSH were still below normal, although a trend of significant increases was observed in comparison with alcohol treatment group. CONCLUSIONS: The results indicated that SP was able to hasten the alcohol elimination through urine directly and enhance the ethanol oxidation rate in liver. Simultaneously, SP may exert a protective effect against lipid peroxidation by scavenging reactive oxygen species and elevating the activity of antioxidant enzymes, in consequence prevented the peroxidative deterioration of structural lipids in membranous organelles, especially mitochondria and karyon.
Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/prevenção & controle , Sericinas/uso terapêutico , Consumo de Bebidas Alcoólicas/patologia , Sequência de Aminoácidos , Animais , Bombyx , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Sericinas/genéticaRESUMO
BACKGROUND: Sericin peptide (SP) has shown a powerful anti-oxidant property in a host of studies. The present study was designed to investigate the possible protective effects of SP against alcohol-induced gastric lesions in mice and to explore the potential mechanisms. METHODS: Animals were randomly divided into 5 groups: control, alcohol (56%, 14.2 ml/kg), SP-treated mice (0.2, 0.4, 0.8 g/kg). Mice were pretreated with SP before administering alcohol, the concentration of ethanol in serum and urine, the contents of malondialdehyde (MDA), glutathione (GSH) and the glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD) activities in the gastric mucosa were measured, subsequently, the pathological evaluation of stomach was also observed. RESULTS: Of the animals pre-treated with SP (0.4, 0.8 g/kg), the concentration of ethanol in serum was significantly decreased, while increased in urine as compared to the alcohol-administered alone animals. Alcohol administration caused severe gastric damage as indicated by markedly increased MDA levels and decreased antioxidants, such as reduced GSH, GSH-PX and SOD in the gastric tissue while the CAT activity was not altered. On SP administration there was a reversal in these values towards normal. Histopathological studies confirmed the beneficial role of SP, which was in accordance with the biochemical parameters. CONCLUSIONS: SP could protect gastric mucosa from alcohol-induced mucosal injury. These gastroprotective effects might be due to increasing 'first-pass metabolism' in the stomach and hastening ethanol elimination directly through the urine. SP might also play an important role in the protection of the structure and function of gastric mitochondria, at least partly based on their anti-oxidant effect.
Assuntos
Citoproteção , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Sericinas/farmacologia , Aminoácidos/análise , Animais , Etanol/sangue , Etanol/urina , Mucosa Gástrica/patologia , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Sericinas/análise , Superóxido Dismutase/metabolismoRESUMO
Phellinus igniarius, which is called Sanghuang in Chinese, is a fungal herb widely used in Traditional Chinese Medicine to treat stomachache, inflammation and tumors. Recent studies have demonstrated the antitumor, anti-diabetic, anti-inflammatory and immunity-modulating activities of P. igniarius. In the present study, we investigated that ameliorating effect of the aqueous extract of P. igniarius fruiting body (SH) on dextran sodium sulfate (DSS)-induced colitis in C57BL/6 mice. Treatment with SH (250 and 400 mg/kg) for 8 weeks effectively alleviated the pathological indicators of colitis such as bodyweight reduction, disease activity index score, shortening of colon length and abnormal colon histology. The plasma levels of lipopolysaccharide (LPS) and inflammatory factors such as interleukin-6 (IL-6), IL-1ß and tumor necrosis factor (TNF)-α were all significantly reduced. Supplementation of SH (10 mg/L) also inhibited LPS-elicited IL-1ß production by RAW264.7 macrophages. Real-time PCR and western blot showed that treatment with SH significantly inhibited the phosphorylation of nuclear factor kappa B inhibitor alpha (IκBα) and decreased the expression of IL-6/IL-1ß-maturation genes such as apoptosis-associated speck-like protein (ASC3) and caspase-1 in the colon of DSS-induced colitis mice. These results suggest that SH is adequate for the treatment of colitis. Inhibiting the expression and release of inflammatory factors may participate in the colitis-ameliorating effect of SH.
Assuntos
Basidiomycota/química , Colite/induzido quimicamente , Colite/tratamento farmacológico , Sulfato de Dextrana/efeitos adversos , Animais , Caspase 1/genética , Colite/genética , Colite/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Interleucina-1beta/biossíntese , Interleucina-6/sangue , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/sangueRESUMO
OBJECTIVE: To study the protective effects of Panax japonics (PJ) on alcohol-induced gastric lesion in mice and the possible mechanisms. METHOD: Male ICR mice were randomized into six groups: normal, control, PJ (1.5, 3.0, 6.0 g x kg(-1)) and Yinduoan (1.5 g x kg(-1)). The mice were pretreated with PJ before administering ethanol to observe the effect on the concentration of ethanol in serum and urine. The contents of MDA, GSH and GSH-PX, CAT and SOD activities were measured in serum and gastric mucosa, and subsequently, the pathological evaluation of stomach was also observed. RESULT: The concentration of ethanol in serum was evidently decreased after PJ (1.5, 3.0 g x kg(-1)) was administrated because the ethanol was eliminated fleetly through urine. Synchronously the PJ reduced the content of MDA and increased the GSH increased in serum and gastric, besides, it increased the enzymatic activities of GSHPX, CAT and SOD, and the ethanol-induced gastric mitochondria structure injury were ameliorated so as to make the function to normal. CONCLUSION: Based on these observations, one could conclude that the PJ is a potent protective agent against ethanol-induced gastric damages. One mechanism may be related with inhibiting the absorbability of ethanol at gastrointestinal tract, decreasing the concentration of ethanol in serum, and accelerating the ethanol elimination through urine so as to alleviate the ethanol-induced damage to gastrointestinal mucosal, enhancing the first-pass metabolism in stomach, and particularly increasing the antioxidant levels in serum and gastric. These gastroprotective effects might be, at least partly, through ameliorating the gastric mitochondria structure.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Panax/química , Substâncias Protetoras/farmacologia , Gastropatias/prevenção & controle , Animais , Catalase/sangue , Catalase/metabolismo , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/uso terapêutico , Etanol , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Glutationa/sangue , Glutationa/metabolismo , Glutationa Peroxidase/sangue , Glutationa Peroxidase/metabolismo , Masculino , Malondialdeído/sangue , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Fitoterapia , Plantas Medicinais/química , Substâncias Protetoras/isolamento & purificação , Substâncias Protetoras/uso terapêutico , Distribuição Aleatória , Gastropatias/sangue , Gastropatias/induzido quimicamenteRESUMO
Type 2 diabetes mellitus (T2DM) is currently considered a worldwide epidemic and finding effective therapeutic strategies against this disease is highly important. A deoxynojirimycin-polysaccharide mixture (DPM) has previously been shown to exert hypoglycemic effects on alloxan- or streptozotocin (STZ)-induced diabetic mice. The purpose of the present study was to evaluate the therapeutic effects and underlying mechanism(s) of DPM on T2DM induced by high fat diet following low-dose STZ treatment in mice. After daily oral treatment of diabetic mice with DPM (150 mg/kg b.w.) for 90 d, significant decline in blood glucose, pyruvate, triglyceride (TG), aspartate transaminase (AST), alanine transaminase (ALT), creatinine (Cr), lipid peroxide (LPO) and malondialdehyde (MDA) levels as well as evident increases in high density lipoprotein (HDL-c) and hepatic glycogen concentrations were observed. In the first stage, in which DPM was administered for 60 d, blood insulin levels did not undergo significant change but a significant decrease in the HOMA-IR index was detected. By contrast, the HOMA-IR index increased significantly in T2MD controls. In the second stage, in which DPM treatment was continued for another 30 d, insulin levels significantly increased in DPM-treated mice in comparison with T2DM controls. These results indicate that insulin resistance in the pre-diabetic period and the dysfunction of pancreatic ß-cells are ameliorated by DPM treatment. DPM also down-regulated protein levels of insulin receptor (IR) and gluconeogenic enzymes (pyruvate carboxylase, fructose-1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in peripheral tissues (liver and/or muscle), but enhanced the expressions of insulin in pancreas, lipoprotein lipase (LPL) and glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylase E1) in the liver. Furthermore, deoxynojirimycin (DNJ) and polysaccharide (P) were found to increase proliferation of hepatic LO-2 cells and scavenging of radicals in vitro. These results support the results of our biochemical analyses and underscore possible mechanisms underlying the protective effects of DPM on STZ-induced damage to the pancreas and the liver. Taken together, our findings suggest that DPM may be developed as an antihyperglycemic agent for the treatment of diabetes mellitus.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucosamina/análogos & derivados , Hipoglicemiantes/farmacologia , Fígado/metabolismo , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/uso terapêutico , Animais , Glicemia/análise , Western Blotting , Frutose-Bifosfatase/análise , Frutose-Bifosfatase/metabolismo , Glucoquinase/análise , Glucoquinase/metabolismo , Glucosamina/farmacologia , Glucosamina/uso terapêutico , Glucose-6-Fosfatase/análise , Glucose-6-Fosfatase/metabolismo , Hipoglicemiantes/uso terapêutico , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosfoenolpiruvato Carboxiquinase (ATP)/análise , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfofrutoquinase-1 Hepática/análise , Fosfofrutoquinase-1 Hepática/metabolismo , Piruvato Carboxilase/análise , Piruvato Carboxilase/metabolismo , Piruvato Descarboxilase/análise , Piruvato Descarboxilase/metabolismo , Distribuição AleatóriaRESUMO
We had previously shown that deoxynojirimycin-polysaccharide mixture (DPM) not only decreased blood glucose but also reversed the damage to pancreatic ß-cells in diabetic mice, and that the anti-hyperglycemic efficacy of this combination was better than that of 1-deoxynojirimycin (DNJ) or polysachharide alone. However, the mechanisms behind these effects were not fully understood. The present study aimed to evaluate the therapeutic effects of DPM on streptozotocin (STZ)-induced diabetic symptoms and their potential mechanisms. Diabetic mice were treated with DPM (150 mg/kg body weight) for 90 days and continued to be fed without DPM for an additional 30 days. Strikingly, decrease of blood glucose levels was observed in all DPM treated diabetic mice, which persisted 30 days after cessation of DPM administration. Significant decrease of glycosylated hemoglobin and hepatic pyruvate concentrations, along with marked increase of serum insulin and hepatic glycogen levels were detected in DPM treated diabetic mice. Results of a labeled (13)C6-glucose uptake assay indicated that DPM can restrain glucose absorption. Additionally, DPM down-regulated the mRNA and protein expression of jejunal Na(+)/glucose cotransporter, Na(+)/K(+)-ATPase and glucose transporter 2, and enhanced the activities as well as mRNA and protein levels of hepatic glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylas E1). Activity and expression of hepatic gluconeogenesis enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) were also found to be attenuated in diabetic mice treated with DPM. Purified enzyme activity assays verified that the increased activities of glucose glycolysis enzymes resulted not from their direct activation, but from the relative increase in protein expression. Importantly, our histopathological observations support the results of our biochemical analyses and validate the protective effects of DPM on STZ-induced damage to the pancreas. Thus, DPM has significant potential as a therapeutic agent against diabetes.
Assuntos
1-Desoxinojirimicina/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Polissacarídeos/uso terapêutico , Estreptozocina/toxicidade , Animais , Glicemia/efeitos dos fármacos , Western Blotting , Glucose/metabolismo , Insulina/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
ETHNOPHARMACOLOGY RELEVANCE: Our previous study showed that the proteoglycan P1 from Phellinus linteus (Mesima) exhibits significant anti-tumor activity against human hepatocellular carcinoma cells (HepG2); however, its molecular mechanism remains unknown. This study aims to provide insights into the mechanism of the anti-tumor activity of P1 against HepG2 cells. METHODS: We examined the effects of P1 on HepG2 cell proliferation in vitro and in vivo. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. Proteomic analysis, real-time (RT)-PCR, and Western blot were carried out to observe the expression of several cell cycle control proteins in HepG2 cells. RESULTS: Both the volume and the weight of solid tumors were significantly decreased in P1-treated mice (200mg/kg) compared with the control. The HepG2 cells in the P1-treated tumors were significantly decreased, irregularly shaped, and smaller. P1 slightly increased the body weight of the tumor-bearing mice, which indicates that P1 is nontoxic to mammals at 200mg/kg. P1 also caused a significant dose-dependent increase in S phase arrest, but no apoptosis was observed in HepG2 cells. The results of the proteomic analysis, RT-PCR, and Western blot analysis showed that significantly downregulated expression of calreticulin, cyclin D1, cyclin E, and CDK2 and upregulated expression of P27 kip1 and cyclin A in the P1-treated HepG2 cells (200 µg/ml). CONCLUSION: These results suggest that calreticulin expression and the P27 kip1-cyclin A/D1/E-CDK2 pathway were involved in P1-induced S-phase cell cycle arrest in HepG2 cells.
Assuntos
Antineoplásicos/farmacologia , Basidiomycota , Polissacarídeos Fúngicos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Calreticulina/metabolismo , Proliferação de Células/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclinas/metabolismo , Feminino , Polissacarídeos Fúngicos/uso terapêutico , Células Hep G2 , Humanos , Camundongos , Camundongos Nus , Proteômica , Fase S/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We investigated the role of 1-deoxynojirimycin (DNJ) on glucose absorption and metabolism in normal and diabetic mice. Oral and intravenous glucose tolerance tests and labeled (13)C6-glucose uptake assays suggested that DNJ inhibited intestinal glucose absorption in intestine. We also showed that DNJ down-regulated intestinal SGLT1, Na(+)/K(+)-ATP and GLUT2 mRNA and protein expression. Pretreatment with DNJ (50â mg/kg) increased the activity, mRNA and protein levels of hepatic glycolysis enzymes (GK, PFK, PK, PDE1) and decreased the expression of gluconeogenesis enzymes (PEPCK, G-6-Pase). Assays of protein expression in hepatic cells and in vitro tests with purified enzymes indicated that the increased activity of glucose glycolysis enzymes was resulted from the relative increase in protein expression, rather than from direct enzyme activation. These results suggest that DNJ inhibits intestinal glucose absorption and accelerates hepatic glucose metabolism by directly regulating the expression of proteins involved in glucose transport systems, glycolysis and gluconeogenesis enzymes.
Assuntos
1-Desoxinojirimicina/farmacologia , Diabetes Mellitus Experimental/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/biossíntese , Transportador de Glucose Tipo 2/biossíntese , Transportador de Glucose Tipo 2/genética , Glicólise/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/biossíntese , Transportador 1 de Glucose-Sódio/biossíntese , Transportador 1 de Glucose-Sódio/genética , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/genética , EstreptozocinaRESUMO
Our previous study showed that polysaccharide (P1) from Phellinus linteus exhibits a significant inhibitive activity on human colorectal carcinoma cells (HT-29). However its novel molecular mechanism remains unknown. To obtain insights into P1's mechanism of action, we examined its effects on cell proliferation in vitro and in vivo, cell cycle distribution, apoptosis, autophagy, and expression of several cell cycle interrelated proteins in HT-29 cells. Interestingly, we found that volume and weight of the solid tumor significantly decreased in P1 (200mg/kg)-treated mice compared with the control. However, slightly increased the body weight of the P1 treated tumor-bearing mice, with no significant increased ALT, AST levels in serum and LPO concentration in liver and kidney indicated that P1 has no toxicity to mammals at a dose of 200mg/kg. Furthermore, P1 caused a significantly dose-dependent increase in the S-phase cell cycle, but no apoptosis and autophagy in HT-29 cells. RT-PCR and Western blot results showed significantly down-regulated expressions of cyclin D1, cyclin E, and CDK2, as well as increased expressions of P27kip1 in P1 (100 µg/mL)-treated HT-29 cells. These results suggested that the activation of P27kip1-cyclin D1/E-CDK2 pathway is involved in P1-induced S-phase cell cycle arrest in HT-29 cells.
Assuntos
Fungos/química , Polissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células CACO-2 , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células HT29 , Humanos , Camundongos , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: 1-Deoxynojirimycin (DNJ) discovered from mulberry trees has been reported to be a potent inhibitor of intestinal α-glycosidases (sucrase, maltase, glucoamylase), and many polysaccharides were useful in protecting against alloxan-induced pancreatic islets damage through their scavenging ability. This study was aimed to evaluate the therapeutic effect and potential mechanism(s) of the hybrid of DNJ and polysaccharide (HDP) from mulberry leaves on alloxan-induced diabetic mice. MATERIALS AND METHODS: Daily oral treatment with HDP (150 mg/kg body weight) to diabetic mice for 12 weeks, body weight and blood glucose were determined every week, oral glucose tolerance test was performed after 4 and 8 weeks, biochemical values were measured using assay kits and gene expressions were investigated by RT-PCR. RESULTS: A significant decline in blood glucose, glycosylated hemoglobin, triglyceride, aspartate transaminase and alanine transaminase levels and an evident increase in body weight, plasma insulin level and high density lipoprotein were observed in HDP treated diabetic mice. The polysaccharide (P1) showed a significant scavenging hydroxyl radicals and superoxide anion radical effects in vitro, which indicated that P1 could protect alloxan-induced pancreatic islets from damage by scavenging the free radicals and repaired the destroyed pancreatic ß-cells. Pharmacokinetics assay showed that DNJ could be absorbed from the gastrointestinal mucosa and diffused rapidly into the liver, resulted in postprandial blood glucose decrease and alleviated the toxicity caused by sustained supra-physiological glucose to pancreatic ß-cells. RT-PCR results indicated that HDP could modulate the hepatic glucose metabolism and gluconeogenesis by up/down-regulating the expression of rate-limiting enzymes (glucokinase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in liver and up-regulating the pancreatic and duodenal homeobox factor-1 (PDX-1), insulin-1 and insulin-2 expressions in pancreas. CONCLUSION: These findings suggested that HDP has complimentary potency to develop an antihyperglycemic agent for treatment of diabetes mellitus.
Assuntos
1-Desoxinojirimicina/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Morus/química , Folhas de Planta/química , Polissacarídeos/uso terapêutico , 1-Desoxinojirimicina/farmacologia , Aloxano , Animais , Sequência de Bases , Primers do DNA , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/metabolismo , Glucoquinase/genética , Glucoquinase/metabolismo , Teste de Tolerância a Glucose , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Polissacarídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismoRESUMO
Proteoglycan (P1) purified from Phellinus linteus has been reported to have anti-disease activities. The objectives of our research were to determine the anti-tumor effect and possible mechanisms of P1 on human cancer cells. Cell inhibition assay showed that P1 has an antiproliferative effect on HepG2, HT-29, NCI-H 460 and MCF-7 human colon cancer cells, especially it was very effective in inhibiting HT-29 cells. When HT-29-bearing mice were treated with P1(100mg/kg), there was relative increase in spleen and thymus weights, the plasmatic pIgR and IgA levels were significantly increased, also there was a notable decrease in plasmatic PGE2, Reg IV, EGFR and Akt concentrations measured by ELISA. RT-PCR analysis suggested that P1-induced HT-29 apoptosis appeared to be associated with a decrease in the levels of expression of Reg IV and EGFR. These results suggest that P1 might have two potential roles in treating cancer; it acts as an immunopotentiator partly through protecting T cells from escaping PGE2 attack and enhancing the mucosal IgA response, and as a direct inhibitor by disrupting the Reg IV/EGFR/Akt signaling pathway.