RESUMO
Doxorubicin (DOX) is a potent anti-neoplastic agent with cumulative cardiotoxicity. DOX-induced cardiotoxicity has been shown to depend on the different dosing times. However, the basis for determining the dosing time to minimize DOX-induced cardiotoxicity and the underlying mechanisms remain incompletely understood. Here we first showed that SIRT3, the major mitochondrial deacetylase, is negatively correlated to DOX-induced cardiotoxicity through the regulation of ATP production, mitochondrial membrane potential (MMP) level and ROS level in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Then, we used in vivo experiments to demonstrate that DOX significantly reduced the SIRT3 expression and the SIRT3 activity as reflected by the increased AcK68MnSOD/MnSOD ratio in rats after six weeks of treatment. Notably, the activity of SIRT3 had an obvious diurnal rhythm pattern in the myocardium of healthy rats. More importantly, an obvious lower AcK68MnSOD/MnSOD ratio was observed in rat hearts with DOX administrated at Zeitgeber time (ZT) 9 (ZT 0 was the time lights were turned on) than ZT1, which represent the peak and trough of SIRT3 activity. Moreover, DOX ZT9 reduced the body weight loss, extended the survival period, improved the heart function and alleviated the myocardial lesions compared to DOX ZT1. Mechanistic investigations demonstrated that DOX ZT1 significantly reduced ATP production, oxygen consumption rate (OCR) at various respiration states, MMP level and MnSOD activity and enhanced the H2O2 level compared with CON ZT1, whereas there was no significant effect for DOX ZT9 compared with CON ZT9. Taken together, dosing at the peak time of SIRT3 activity reduced DOX-induced cardiotoxicity, which may be related to the increased endogenous tolerance against the mitochondrial dysfunction and oxidative stress caused by DOX.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Cardiotoxicidade/prevenção & controle , Doxorrubicina/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 3/metabolismo , Animais , Antibióticos Antineoplásicos/administração & dosagem , Cardiotoxicidade/etiologia , Cardiotoxicidade/metabolismo , Cardiotoxicidade/patologia , Linhagem Celular , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos Sprague-DawleyRESUMO
The tripartite motif (TRIM)-containing proteins exhibit various activities and play important roles in the immune system through regulating signaling pathways. Bloodthirsty gene is a multigene subset of TRIM genes. In this study we identified and characterized a new member of the bloodthirsty subset of TRIM genes, btr20, in zebrafish (Danio rerio). The gene is located on chromosome 19 and forms a cluster with btr18, btr21, btr22 and an E3 ubiquitin ligase TRIM39-like gene. Deduced btr20 represents a RBCC-B30.2 TRIM protein containing 544 amino acids. The mRNA expression level of btr20 was highest in intestine and gill, followed by in spleen and kidney. Challenge experiment with Aeromonas hydrophila strain NJ-1 showed that the levels of btr20 and NF-κB mRNA were remarkably upregulated in the four tissues mentioned above. btr20 was localized in the cytoplasm and formed aggregate in human embryonic kidney cell line 293T. In vitro self-ubiquitylation experiment demonstrated that btr20 has E3 ubiquitin ligase activity that can be self-ubiquitylated with most E2 enzymes, especially UbcH6. The results suggested that btr20 may involve in the anti-microbial activity in the immune system as an E3 ubiquitin ligase.
Assuntos
Aeromonas/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Transporte Proteico , Análise de Sequência de Proteína , Frações Subcelulares/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish-whitespotted bamboo shark (Chiloscyllium plagiosum Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1-3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Splicing de RNA , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Clonagem Molecular , DNA Complementar , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Filogenia , Tubarões , UbiquitinaçãoRESUMO
Akirin is a nuclear factor involved in innate immune responses of arthropods and mammals. In this study we have cloned an Akirin2 gene, pdakirin2, from freshwater Chinese loach (Paramisgurnus dabryanus) and characterized its biological functions. Phylogenetic analysis revealed deduced PdAkirin2 had high sequence identities to Akirin2 homologs from fish and mammals (70-91%), it contained two conserved nuclear localization signals (NLSs) with verified sub-cellular localization. Quantitative real-time (qRT)-PCR analysis indicated that PdAkirin2 was present in a wide range of loach tissues and showed up-regulation with challenges of Aeromonas hydrophila NJ-1, LPS and poly I:C. PdAkirin2 as an immune factor had significant effects on the expression of cytokines (TNFα, IFN-α, IFN-γ, IL-4 and IL-1ß) and transcription factor NF-κB. This study provides insights into the potential role of PdAkirin2 in the innate immune system.
Assuntos
Cipriniformes/genética , Cipriniformes/imunologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Imunidade Inata , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cipriniformes/classificação , Citocinas/genética , Citocinas/metabolismo , Doenças dos Peixes/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência/veterináriaRESUMO
Cold bending forming is a complex forming process, and its product quality is closely related to the forming process parameters. To mitigate issues such as bulging and waviness arising from the extension of the material at the edges during the forming process of thin-walled circular tubes, a comprehensive comparative analysis was conducted on four forming methods. This analysis determined that the combined bending method is the optimal forming technique for the equipment. For the impact of different parameters on the equivalent plastic strain distribution of the product and the force on the rollers, numerical simulations were carried out using the software COPRA (COPRA FEA RF 2023.1) after designing the pattern diagram based on the integrated bending method. The results showed that different processing speeds on the equivalent plastic strain distribution and work hardening of the plate have little effect. As the spacing between the upper and lower rollers increases, the equivalent plastic strain of the plate to a certain extent and the value of the moment of the rollers is significantly reduced. Analyzing the performance characteristics of high-strength steel materials from the aspects of the thickness strain and cross-sectional forming of the plate, this verifies the advantages of forming high-strength steel plates. The numerical simulation results of this study are in good agreement with actual production experimental results.
RESUMO
Xylanase is a crucial hydrolytic enzyme that degrades plant polysaccharides in the rumen. To date, there is no information on the genetic composition and expression characteristics of ruminal xylanase during feeding cycles of ruminants. Here, the major xylanase of the glycoside hydrolase family 10 (GH 10) from the rumen of small-tail Han sheep was investigated during a feeding cycle. We identified 44 distinct GH 10 xylanase gene fragments at both the genomic and transcriptional levels. Comparison of their relative abundance showed that results from the evaluation of functional genes at the transcriptional level are more reliable indicators for understanding fluctuations in xylanase levels. The expression patterns of six xylanase genes, detected at all time points of the feeding cycle, were investigated; we observed a complex trend of gene expression over 24 h, revealing the dynamic expression of xylanases in the rumen. Further correlation analysis indicated that the rumen is a dynamic ecosystem where the transcript profiles of xylanase genes are closely related to ruminal conditions, especially rumen pH and bacterial population. Given the huge diversity and changing composition of enzymes over the entire rumen, this research provides valuable information for understanding the role of functional genes in the digestion of plant material.
Assuntos
Dieta/métodos , Glicosídeo Hidrolases/biossíntese , Metagenoma , Polissacarídeos/metabolismo , Rúmen/microbiologia , Transcriptoma , Animais , Glicosídeo Hidrolases/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , OvinosRESUMO
ß-defensins are a large family of multi-disulfide-bonded peptides with broad-spectrum antimicrobial activities that contribute to innate host defense in many organisms, but little information is available about ß-defensins produced by freshwater fish lacking scales. We therefore cloned and identified a ß-defensin gene from Chinese loach (Paramisgurnus dabryanus) by designing degenerate primers and using thermal asymmetric interlaced PCR. This gene is the first defensin gene ever identified in a non-scaled freshwater fish. Annotation of the protein domain architecture showed that the putative Chinese loach ß-defensin contains the signature motif of six conserved cysteines within the mature peptide, an aspect similar to ß-defensins of other marine fish. We also used quantitative real-time PCR to investigate the expression pattern of the Chinese loach ß-defensin gene, mRNA of which could be observed in various tissues. After challenge with the pathogenic bacterium Aeromonas hydrophila, ß-defensin expression was induced in the eye, gill, skin, and spleen of the adult loach. The bioactivity of the recombinant P. dabryanus ß-defensin was examined against pathogenic bacteria, and the results suggest that this class 2 ß-defensin has potential applications for treatment of bacterial infections.
Assuntos
Cipriniformes/genética , Proteínas de Peixes/genética , Imunidade Inata , beta-Defensinas/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cipriniformes/imunologia , Cipriniformes/microbiologia , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária , beta-Defensinas/química , beta-Defensinas/metabolismoRESUMO
The biological significance of tripartite motif (TRIM) proteins is increasingly being appreciated due to their roles in a broad range of biological processes that associated with innate immunity. In this study, we have described the structural and functional analysis of TRIM3a from zebrafish. Annotation of domain architectures found that the TRIM3a fulfills the TRIM-NHL rule of domain composition with a Filamin/ABP280 domain and NHL repeats at its C-terminal region. In addition, the mRNA expression level of TRIM3a was the highest in brain, and with a relatively higher level in spleen, liver, and gill. A strong expression starting at 36 h post fertilization (hpf) was observed by real-time PCR and could be detected in brain by in situ hybridization, suggesting that TRIM3a protein might play an important role in brain development in zebrafish. Considering that TRIM3a has a RING finger domain, we expressed and purified the TRIM3a protein and performed ubiquitylation assays, our results showed that TRIM3a underwent self-polyubiquitylation in combination with E1, UbcH5c, biotin-ubiquitin in vitro. Meanwhile, TRIM3a-R without the RING domain was expressed and purified as well, in vitro ubiquitylation assays showed that the self-ubiquitylation of TRIM3a was dependent on its RING domain, suggesting that TRIM3a might function as a RING finger E3 ubiquitin ligase.
Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Especificidade de Órgãos , Filogenia , Domínios RING Finger , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/análise , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas com Motivo Tripartido , Ubiquitina/análise , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
Sweet tea (Lithocarpus litseifolius [Hance] Chun) is a new resource for food raw materials, with plenty of health functions. This study aimed to investigate the preventive effect and potential mechanism of sweet tea extract (STE) against ulcerative colitis (UC). Briefly, BABL/c mice were treated with STE (100 and 400 mg/kg) for 2 weeks to prevent 3% dextran sulfate sodium (DSS)-induced UC. It was found that STE supplementation significantly prevented DSS-induced UC symptoms; suppressed the levels of pro-inflammatory mediators, such as myeloperoxidase and tumor necrosis factor-α; increased the levels of anti-inflammatory cytokines; and up-regulated the expression of tight junction proteins (Zonula occludens-1 and Occludin). STE also altered the gut microbiota profile of UC mice by increasing Bacteroidetes, Lactobacillus, Akkermansia, Lachnospiraceae_NK4A136_group, and Alistipes and inhibiting Firmicutes, Proteobacteria, and Helicobacter, accompanied by a significant increase in the content of butyric acid. Moreover, STE increased the expression of G-protein-coupled receptor (GPR) 43 and GPR109A and inhibited the expression of histone deacetylase 3 (HDAC3) and nuclear factor-κB p65 (NF-κB p65) in the colon. In conclusion, this study indicated that STE has a good preventive effect on UC by regulating gut microbiota to activate butyrate-GPR-mediated anti-inflammatory signaling and simultaneously inhibit HDAC3/NF-κB inflammatory signaling.
Assuntos
Colite Ulcerativa , Colite , Microbioma Gastrointestinal , Animais , Anti-Inflamatórios/uso terapêutico , Ácido Butírico/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colite/prevenção & controle , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/prevenção & controle , Colo/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Chá/efeitos adversosRESUMO
Changes in gene expression occur as animals, including primates, age. Macaques have long been used as a model species for primate evolution and biomedical studies. Here, to study gene expression in Tibetan macaques (Macaca thibetana, TMs) and its differences to humans, we applied RNA-Seq to obtain the blood transcriptomes of 24 TMs. In total, 2 523 age-associated differentially expressed genes (DEGs) were identified. Several pathways and processes that regulate aging, including the FoxO signaling pathway, autophagy, and platelet activation, were significantly enriched in the up-regulated DEGs. Two significantly age-related modules were identified by weighted gene co-expression network analysis (WGCNA). The TMs and humans shared 279 common DEGs, including 111 up-regulated and 141 down-regulated genes with advancing age in the same expression direction. However, 27 age-related DEGs presented the opposite expression direction in TMs as that in humans. For example, INPPL1, with inhibitory effects on the B cell receptor signaling pathway, was up-regulated in humans but down-regulated in TMs. In general, our study suggests that aging is a critical factor affecting gene expression in the captive TM population. The similarities and differences in gene expression patterns between TMs and humans could provide new insights into primate evolution and benefit TM model development.
Assuntos
Envelhecimento/fisiologia , Regulação da Expressão Gênica/fisiologia , Macaca/sangue , Animais , Regulação para Baixo , Feminino , Humanos , Masculino , RNA-Seq , Análise de Sequência de RNA , Especificidade da Espécie , TranscriptomaRESUMO
Fifteen Math-DMA and 16 Math-DMB novel alleles of Tibetan macaques were identified.