RESUMO
By means of a computational method based on Density Functional Theory (DFT), using commercially available software, a novel method for simulating equilibrium geometry harmonic vibrational frequencies is proposed. Finasteride, Lamivudine, and Repaglinide were selected as model molecules to study the adaptability of the new method. Three molecular models, namely the single-molecular, central-molecular, and multi-molecular fragment models, were constructed and calculated by Generalized Gradient Approximations (GGAs) with the PBE functional via the Material Studio 8.0 program. Theoretical vibrational frequencies were assigned and compared to the corresponding experimental data. The results indicated that the traditional single-molecular calculation and scaled spectra with scale factor exhibited the worst similarity for all three pharmaceutical molecules among the three models. Furthermore, the central-molecular model with a configuration closer to the empirical structure resulted in a reduction of mean absolute error (MAE) and root mean squared error (RMSE) in all three pharmaceutics, including the hydrogen-bonded functional groups. However, the improvement in computational accuracy for different drug molecules using the central-molecular model for vibrational frequency calculation was unstable. Whereas, the new multi-molecular fragment interception method showed the best agreement with experimental results, exhibiting MAE and RMSE values of 8.21 cm-1 and 18.35 cm-1 for Finasteride, 15.95 cm-1 and 26.46 cm-1 for Lamivudine, and 12.10 cm-1 and 25.82 cm-1 for Repaglinide. Additionally, this work provides comprehensive vibrational frequency calculations and assignments for Finasteride, Lamivudine, and Repaglinide, which have never been thoroughly investigated in previous research.
Assuntos
Finasterida , Lamivudina , Conformação Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Preparações Farmacêuticas , Teoria Quântica , VibraçãoRESUMO
Chronic stress has a profound effect on health in both animals and humans. Dexamethasone (Dex), a synthetic glucocorticoid, is used to induce chronic stress in many studies. The impact of chronic stress on epithelial cells of hindgut of ruminants is still unknown. In this study, we investigated the effect of chronic stress induced by long term injection of low dosage of Dex on the colonic epithelium of goats. The results showed that Dex exposure increased the number of TUNEL-positive cells, upregulated caspase-3 and caspase-8 enzyme activity, but decreased protein expression of cell proliferation markers proliferating cell nuclear antigen (PCNA) and Cyclin D2(CCND2). It also activated TLR-4 and NF-κB pathway and increased the transcription levels of vital inflammatory cytokines such as interleukin-10 (IL-10), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase 2 (iNOS2). Chronic stress down-regulated the methylation level of total DNA, suggesting a mechanism for the transcriptional activation of genes, such as claudin-1, claudin-4, ZO-1, and cell cycle-related genes. Taken together, long-term injection of a low dosage of Dex caused damage to the colon epithelium accompanied with the inhibition of cell proliferation and the activation of cell apoptosis and inflammation. However, a general up-regulation of genes expression induced by Dex is due to a lower level of genomic DNA methylation.
Assuntos
Apoptose/efeitos dos fármacos , Colo/patologia , Dexametasona/efeitos adversos , Células Epiteliais/metabolismo , Cabras/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Receptor 4 Toll-Like/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Citocinas/genética , Citocinas/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Mediadores da Inflamação/metabolismo , Masculino , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.
Assuntos
4-Butirolactona/análogos & derivados , Caspase 1/metabolismo , Células Caliciformes/efeitos dos fármacos , Homosserina/análogos & derivados , Piroptose/efeitos dos fármacos , Percepção de Quorum , 4-Butirolactona/toxicidade , Arildialquilfosfatase/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Ativação Enzimática , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Homosserina/toxicidade , Humanos , Mediadores da Inflamação/metabolismo , Mucinas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Dexamethasone (Dex), an artificially synthetic cortisol substitute, is commonly used as an anti-inflammatory drug, and is also employed to mimic the stress state experimentally. It is well known that chronic stress disturbs the gut microbiota community and digestive functions. However, no relevant studies have been conducted in ruminants. RESULTS: In this study, a low dosage of Dex (0.2 mg/kg body weight, Dex group, n = 5) was consecutively injected intramuscularly for 21 days to simulate chronic stress in growing goats. Goats were injected with saline (0.2 mg/kg body weight) as the control group (Con, n = 5). Dex-treated goats showed a higher number of white blood cells and blood glucose levels (p < 0.01), but lower dry matter intake (DMI) and body weight (p < 0.01) than those of saline-injected goats. Plasma cortisol concentration decreased significantly in response to the Dex injection compared to the control (p < 0.05). The Dex treatment did not change most ruminal volatile fatty acid (VFAs) concentrations before the morning feeding after 1-21 days of treatment (p > 0.05); however, ruminal VFA concentrations decreased dramatically 2, 4, 6, and 8 h after the morning feeding on day 21 of the Dex injections. In this study, chronic Dex exposure did not alter the community structure of microbes or methanogenes in the rumen, caecum, or colonic digesta. Only Prevotella increased on days 7 and 14 of Dex treatment, but decreased on day 21, and Methanosphaera was the only genus of methanogene that decreased. CONCLUSIONS: Our results suggest that chronic Dex exposure retards growth by decreasing DMI, which may be mediated by higher levels of blood glucose and lower ruminal VFA production. Microbiota in the digestive tract was highly resistant to chronic Dex exposure.
Assuntos
Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Dexametasona/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Cabras/microbiologia , Animais , Bactérias/genética , Bactérias/metabolismo , Glicemia/metabolismo , Ácidos Graxos Voláteis/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Cabras/sangue , Hidrocortisona/sangue , MasculinoRESUMO
The aim of this study was to investigate the effects of dexamethasone (DEX) on zinc metabolism in goats. In this study, 10 goats were randomly divided into two groups. One group was injected with dexamethasone (Dex group) and the other group was injected with saline (Con group). Dex treatment significantly decreased hepatic zinc levels (p < .01) and increased Zn transporters 1 (ZNT-1) expression (p < .05). The concentration of zinc in the cecal and colonic contents was significantly increased (p < .05). However, zinc levels were increased only in the colon tissues (p < .05) but not in the cecal tissues (p > .05). A dramatic increase in Zrt-, Irt-related proteins 14 (ZIP-14) expression (p < .05) following Dex treatment was also observed and likely induced the elevated zinc levels in the colon, and a significant reduction in Zip-14 methylation (p < .05) may be responsible for the observed increase in Zip-14 expression. Together, these results indicate that Dex influences zinc homeostasis by increasing hepatic ZNT-1 and colonic ZIP-14 expression. Additionally, these results provide valuable information for the clinical application of Dex.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colo/metabolismo , Dexametasona/efeitos adversos , Cabras/metabolismo , Fígado/metabolismo , Zinco/metabolismo , Animais , Expressão Gênica/efeitos dos fármacos , MasculinoRESUMO
Chronic stress severely threatens the welfare and health of animals and humans. In order to study the effects of chronic stress on metabolism, de novo transcriptome sequencing was used to generate the expressed sequence tag dataset for the goat, using nextgeneration sequencing technology. For this study, consecutive dexamethasone (Dex) injection was used in 10 healthy male goats (body weight 25⯱â¯1.0â¯kg) to mimic chronic stress. Ten male goats were randomly assigned into two groups, one group was injected intramuscularly with the same volume of saline as control (Con) group, and another (Dex) group was injected intramuscularly with 0.2â¯mg/kg Dex for 21â¯days. To elucidate the resulting changes in genes, transcriptome profiling of liver was conducted by analysing samples from three goats of each group using RNA-Seq. A total of 137 differentially expressed genes (DEGs) were identified between Con group and Dex group. GO classification showed rhythmic process and hormone secretion in term cellular, and chemoattractant activity in term molecular function had noticeable differences in the proportion between DEGs and all genes. By mapping the DEGs to the COG database, we found that general function prediction only, energy production and conversion, and amino acid transport and metabolism were the most frequently represented functional clusters. We mapped the unigenes to the KEGG pathway database and found most annotated genes were involved in the AMPK signalling pathway as well as pathways in cancer and insulin signalling pathway. Via KEGG enrichment analysis, we found the DEGs were significantly enriched in insulin signalling pathway, AMPK signalling pathway and adipocytokine signalling pathway. In addition, these pathways have close relationship with metabolism, which resulted in metabolic changes in which the identified DEGs may play important roles. These results provide valuable information for further research on the complex molecular mechanisms of dexamethasone in goats and will provide a foundation for future studies.
Assuntos
Dexametasona/efeitos adversos , Perfilação da Expressão Gênica/métodos , Cabras/genética , Fígado/química , Redes e Vias Metabólicas/efeitos dos fármacos , Adenilato Quinase/genética , Animais , Dexametasona/administração & dosagem , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Injeções Intramusculares , Fígado/efeitos dos fármacos , Masculino , Distribuição Aleatória , Análise de Sequência de RNA/métodos , Análise de Sequência de RNA/veterináriaRESUMO
AIMS: The quorum-sensing molecule N(3oxododecanoyl)lhomoserine lactone (C12-HSL), produced by the Gram negative human pathogenic bacterium Pseudomonas aeruginosa, modulates mammalian cell behavior. Our previous findings suggested that C12-HSL rapidly decreases viability and induces apoptosis in LS174T goblet cells. MAIN METHODS: In this study, the effects of 100⯵M C12-HSL on mitochondrial function and cell proliferation in LS174T cells treated for 4â¯h were evaluated by real-time PCR, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. KEY FINDINGS: The results showed that the activities of mitochondrial respiratory chain complexes IV and V were significantly increased (Pâ¯<â¯0.05) in LS174T cells after C12-HSL treatment, with elevated intracellular ATP generation (Pâ¯<â¯0.05). Flow cytometry analysis revealed significantly increased intracellular Ca2+ levels (Pâ¯<â¯0.05), as well as disrupted mitochondrial activity and cell cycle arrest upon C12-HSL treatment. Apoptosis and cell proliferation related genes showed markedly altered expression levels (Pâ¯<â¯0.05) in LS174T cells after C12-HSL treatment. Moreover, the paraoxonase 2 (PON2) inhibitor TQ416 (1⯵M) remarkably reversed the above C12-HSL associated effects in LS174T cells. SIGNIFICANCE: These findings indicated that C12-HSL alters mitochondrial energy production and function, and inhibits cell proliferation in LS174T cells, with PON2 involvement.
Assuntos
4-Butirolactona/análogos & derivados , Células Caliciformes/efeitos dos fármacos , Homosserina/análogos & derivados , Intestinos/citologia , Intestinos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , 4-Butirolactona/farmacologia , Trifosfato de Adenosina/biossíntese , Apoptose/efeitos dos fármacos , Arildialquilfosfatase/antagonistas & inibidores , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Homosserina/farmacologia , HumanosRESUMO
BACKGROUND: Dexamethasone (Dex), a synthetic glucocorticoid, is among the most commonly used drugs worldwide in animals and humans as an anti-inflammatory and immunosuppressive agent. GC has profound effects on plasma glucose level and other metabolic conditions. However, the effect of prolonged use of Dex on glucose metabolism in ruminants is still unclear. RESULTS: Ten goats were randomly assigned to two groups: the control goats were injected with saline, and the Dex-treated goats were intramuscularly injected daily for 21 d with 0.2 mg/kg Dex. The results showed that plasma glucose and insulin concentrations were significantly increased after Dex administration (P < 0.05). Additionally, the content of hepatic glycogen was also markedly increased in Dex-treated goats (P < 0.01), while the content of glycogen in dorsal longissimus was unchanged by Dex (P > 0.05). The expression of several key genes, involved in blood glucose regulation, was detected by real-time PCR in the small intestine, skeletal muscle and liver. The expression of glucose transporter type 2 (GLUT2), sodium-glucose transporter 1 (SGLT1) and sodium-potassium ATPase (Na-K/ATPase) in the small intestine were generally increased by Dex, and GLUT2 mRNA expression was significantly up-regulated (P < 0.05). In liver, the expression of genes involved in gluconeogenesis including glucose-6-phosphatase catalytic subunit (G6PC), cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) and pyruvate carboxylase (PC), were significantly down-regulated by Dex. However, the protein expression levels of PCK1 & PCK2 were significantly increased by Dex, suggesting a post-transcriptional regulation. In dorsal longissimus, the mRNA expression of genes associated with gluconeogenesis and the insulin signaling pathway were generally up-regulated by Dex, but the mRNA expression of two markers of muscle atrophy, namely F-box protein 32 (FBXO32/Atrogin1) and muscle RING-finger protein 1 (MuRF1), was not altered by Dex. CONCLUSIONS: Taken together, these results indicate that chronic administration of a low dosage of Dex induces hyperglycemia mainly through gluconeogenesis activation in the goat liver.
RESUMO
Chronic stress seriously threatens welfare and health in animals and humans. Consecutive dexamethasone (Dex) injection was used to mimic chronic stress previously. In order to investigate the effect of chronic stress on hepatic lipids metabolism, in this study, 10 healthy male goats were randomly allocated into two groups, one received a consecutive injection of Dex via intramuscularly for 3â¯weeks (Dex group), the other received the same volume of saline as the control group (Con group). Hepatic health and triglyceride (TG) metabolism were analyzed and compared between two groups. The data showed that a significant decrease of TG in plasma and the liver was significantly decreased by Dex (Pâ¯<â¯.05), while the hepatic nonesterified fatty acid (NEFA) concentration was increased compared to the Con group (Pâ¯<â¯.05). Consistent with the decrease of TG level, the activity of hepatic lipoprotein lipase (LPL) and hepatic lipase (HL) enzymes activities were significantly enhanced by Dex. Real-time PCR results showed that the mRNA expression of sterol regulatory element binding transcription factor 1 (SREBP-1), acyl-CoA dehydrogenase long chain (ACADL) and acyl-CoA synthetase bubblegum family member 1 (ACSBG1) genes in liver was significantly up-regulated by chronic Dex injection (Pâ¯<â¯.05), whereas perilipin 2 (PLIN2) and adipose triglyceride lipase (ATGL) mRNA expression was significantly decreased by Dex (Pâ¯<â¯.05). In addition, no obvious damages were observed in the liver in both Con and Dex groups demonstrating by the sirius red staining, HE staining as well as several biochemical parameters related to the functional status of hepatocytes. Our data indicate that chronic Dex exposure decreases TG levels in the circulation and the liver through activating lipolysis and inhibiting lipogenesis without causing hepatic damages in the growing goats.
Assuntos
Dexametasona/uso terapêutico , Cabras , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Triglicerídeos/sangue , Animais , Dexametasona/farmacologia , Humanos , MasculinoRESUMO
BACKGROUND: It is well known that feeding a high concentrate (HC) diet to lactating ruminants likely induces subacute ruminal acidosis (SARA) and leads to a decrease in milk fat production. However, the effects of feeding a HC diet for long periods on milk fatty acids composition and the mechanism behind the decline of milk fat still remains poorly understood. The aim of this study was to investigate the impact of feeding a HC diet to lactating dairy goats on milk fat yield and fatty acids composition with an emphasis on the mechanisms underlying the milk fat depression. Seventeen mid-lactating dairy goats were randomly allocated to three groups. The control treatment was fed a low-concentrate diet (35% concentrate, n = 5, LC) and there were two high-concentrate treatments (65% concentrate, HC), one fed a high concentrate diet for a long period (19 wks, n = 7, HL); one fed a high concentrate diet for a short period of time (4 wk, n = 5, HS). Milk fat production and fatty acids profiles were measured. In order to investigate the mechanisms underlying the changes in milk fat production and composition, the gene expression involved in lipid metabolism and DNA methylation in the mammary gland were also analyzed. RESULTS: Milk production was increased by feeding the HC diet in the HS and HL groups compared with the LC diet (P < 0.01), while the percentage of milk fat was lower in the HL (P < 0.05) but not in the HS group. The total amount of saturated fatty acids (SFA) in the milk was not changed by feeding the HC diet, whereas the levels of unsaturated fatty acids (UFA) and monounsaturated fatty acids (MUFA) were markedly decreased in the HL group compared with the LC group (P < 0.05). Among these fatty acids, the concentrations of C15:0 (P < 0.01), C17:0 (P < 0.01), C17:1 (P < 0.01), C18:1n-9c (P < 0.05), C18:3n-3r (P < 0.01) and C20:0 (P < 0.01) were markedly lower in the HL group, and the concentrations of C20:0 (P < 0.05) and C18:3n-3r (P < 0.01) were lower in the HS group compared with the LC group. However, the concentrations of C18:2n-6c (P < 0.05) and C20:4n-6 (P < 0.05) in the milk fat were higher in the HS group. Real-time PCR results showed that the mRNA expression of the genes involved in milk fat production in the mammary gland was generally decreased in the HL and HS groups compared with the LC group. Among these genes, ACSL1, ACSS1 & 2, ACACA, FAS, SCD, FADS2, and SREBP1 were down-regulated in the mammary gland of the HL group (P < 0.05), and the expressions of ACSS2, ACACA, and FADS2 mRNA were markedly decreased in the HS goats compared with the LC group (P < 0.05). In contrast to the gene expression, the level of DNA methylation in the promoter regions of the ACACA and SCD genes was increased in the HL group compared with the LC group (P < 0.05). The levels of ACSL1 protein expression and FAS enzyme activity were also decreased in the mammary gland of the HL compared with the LC group (P < 0.05). CONCLUSIONS: Long-term feeding of a HC diet to lactating goats induced milk fat depression and FAs profile shift with lower MUFAs but higher SFAs. A general down-regulation of the gene expression involved in the milk fat production and a higher DNA methylation in the mammary gland may contribute to the decrease in milk fat production in goats fed a HC diet for long time periods.
RESUMO
Studies on the effect of a high-concentrate (HC) diet on the hindgut microbiota and metabolome of ruminants are rarely reported. We used 454 pyrosequencing of 16S rDNA genes and gas chromatography-mass spectrometry to evaluate the effects of long-term feeding (HL) or short-term (HS) feeding of an HC diet on changes in bacterial microbiota and their metabolites in the hindgut, with Guanzhong goat as a ruminant model. Results indicated that an HC diet decreased bacterial diversity and induced metabolic disorder in the hindgut. The levels of lactate, endotoxin (lipopolysaccharide, LPS), and volatile fatty acid concentrations were higher in the intestinal digesta of the HC goats than in those of the LC goats (P < 0.05). The level of beta-alanine decreased, whereas the levels of stigmasterol and quinic acid decreased in the cecal and colonic digesta of the HC goats. At the genus level, the abundance of Clostridium and Turicibacter was significantly increased in both the colonic and cecal digesta of the HC goats. Several potential relationships between metabolites and several microbial species were revealed in this study. The mRNA expression of the genes functionally associated with nutrients transport, including NHE2, NHE3, MCT1, and MCT4 were significantly downregulated in the colonic mucosa by the HC diet (P < 0.05). The expression levels of the genes related to the inflammatory response, including TLR4, MYD88, TNF-α, and IL-1ß were markedly upregulated in the cecal mucosa by the HC diet (P < 0.05). Our results indicate that an HC diet induces microbiota dysbiosis, metabolic disorders, and mucosal damage in the hindgut of goats.
RESUMO
There is limited knowledge about the impact of long-term feeding a high-concentrate (HC) diet on rumen microbiota, metabolome, and host cell functions. In this study, a combination of mass spectrometry-based metabolomics techniques, 454 pyrosequencing of 16S rDNA genes, and RT-PCR was applied to evaluate the changes of ruminal microbiota composition, ruminal metabolites, and related genes expression in rumen epithelial cells of lactating goats received either a 35% concentrate diet or a 65% concentrate diet for 4 or 19 weeks, respectively. Results show that feeding a HC diet reduced the microbiota diversity and led to the disorders of metabolism in the rumen. The concentrations of lactate, phosphorus, NH3-N and endotoxin Lipopolysaccharide in ruminal fluids, and plasma histamine, lactate and urine N (UN) were increased significantly in goats fed with a HC diet. A significant increase of genes expression related to volatile fatty acids transport, cell apoptosis, and inflammatory responses were also observed in goats fed with a HC diet. Correlation analysis revealed some potential relationships between bacteria abundance and metabolites concentrations. Our findings indicate that a HC diet can induce ruminal microbiota dysbiosis and metabolic disorders, thus increasing risks to host health and potential harm to the environment.