RESUMO
An effective method by high-speed countercurrent chromatography coordinated with silver nitrate for the preparative separation of sterones and triterpenoid saponins from Achyranthes bidentata Blume was developed. Methyl tert-butyl ether/n-butanol/acetonitrile/water (4:2:3:8, v/v/v/v) was selected for 20-hydroxyecdysone (compound 1), chikusetsusaponin IVa methyl ester (compound 4), 2'-glycan-11-keto-pigmented saponin V (compound 5), as well as a pair of isomers of 25S-inokosterone (compound 2) and 25R-inokosterone (compound 3), which were further purified by silver nitrate coordinated high-speed countercurrent chromatography. What is more, dichloromethane/methanol/isopropanol/water (6:6:1:4, v/v/v/v) was applied for calenduloside E (compound 6), 3ß-[(O-ß-d-glucuronopyranosyl)-oxy]-oleana-11,13-dien-28-oic acid (compound 7), zingibroside R1 (compound 8) and chikusetsusaponin IVa (compound 9). Adding Ag+ to the solvent system resulted in unique selectivity for 25R/25S isomers of inokosterone, which increased the complexing capability and stability of Ag+ coordinated 25S-inokosterone, as well as the α value between them. These results were further confirmed by the computational calculation of geometry optimization and frontier molecular orbitals assay. Comprehensive mass spectrometry and nuclear magnetic resonance analysis demonstrated the structures of the obtained compounds.
Assuntos
Achyranthes , Colestenos , Ácido Oleanólico/análogos & derivados , Saponinas , Distribuição Contracorrente , Achyranthes/química , Nitrato de Prata , Extratos Vegetais/química , Água/química , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesium parvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P. parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11 nM and 41.5 ± 0.6 nM after incubations times of 3 and 24 h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl-, Na+, or Ca2+ were performed. Cl- removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P. parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl- ionoregulation may be integral to prymnesin toxicity.
Assuntos
Colestenos , Haptófitas , Lipoproteínas , Animais , Humanos , Brânquias , Linhagem Celular , Células Epiteliais , ColoRESUMO
Cholestenoic acid (CA) has been reported as an important biomarker of many severe diseases, but its physiological and pathological roles remain unclear. This study aimed to investigate the potential role of CA in hepatic lipid homeostasis. Enzyme kinetic studies revealed that CA specifically activates DNA methyltransferases 1 (DNMT1) at low concentration with EC50 = 1.99 × 10-6 M and inhibits the activity at higher concentration with IC50 = 9.13 × 10-6 M, and specifically inhibits DNMT3a, and DNMT3b activities with IC50= 8.41 × 10-6 M and IC50= 4.89 × 10-6 M, respectively. In a human hepatocyte in vitro model of high glucose (HG)-induced lipid accumulation, CA significantly increased demethylation of 5mCpG in the promoter regions of over 7,000 genes, particularly those involved in master signaling pathways such as calcium-AMPK and 0.0027 at 6 h. RNA sequencing analysis showed that the downregulated genes are affected by CA encoding key enzymes, such as PCSK9, MVK, and HMGCR, which are involved in cholesterol metabolism and steroid biosynthesis pathways. In addition, untargeted lipidomic analysis showed that CA significantly reduced neutral lipid levels by 60% in the cells cultured in high-glucose media. Administration of CA in mouse metabolic dysfunction-associated steatotic liver disease (MASLD) models significantly decreases lipid accumulation, suppresses the gene expression involved in lipid biosynthesis in liver tissues, and alleviates liver function. This study shows that CA as an endogenous epigenetic regulator decreases lipid accumulation via epigenetic regulation. The results indicate that CA can be considered a potential therapeutic target for the treatment of metabolic disorders.NEW & NOTEWORTHY To our knowledge, this study is the first to identify the mitochondrial monohydroxy bile acid cholestenoic acid (CA) as an endogenous epigenetic regulator that regulates lipid metabolism through epigenome modification in human hepatocytes. The methods used in this study are all big data analysis, and the results of each part show the global regulation of CA on human hepatocytes rather than narrow point effects.
Assuntos
Colestenos , Epigênese Genética , Pró-Proteína Convertase 9 , Humanos , Animais , Camundongos , Pró-Proteína Convertase 9/metabolismo , Cinética , Hepatócitos/metabolismo , Fígado/metabolismo , Lipídeos , Glucose/metabolismo , Metabolismo dos Lipídeos/genéticaRESUMO
Oxysterols are a family of over 25 cholesterol metabolites naturally produced by enzymatic or radical oxidation. They are involved in many physiological and pathological pathways. Although their activity has been mainly attributed to the modulation of the Liver X Receptors (LXR), it is currently accepted that oxysterols are quite promiscuous compounds, acting at several targets at the same time. The promiscuity of the oxysterols with the Estrogen Receptor α (ERα) is crucial in several pathologies such as ER+ breast cancer, inflammation and atherosclerosis. Regarding this matter, we have previously reported the synthesis, LXR activity and binding mode of a family of cholestenoic acid analogs with a modified side chain. Here we report the transcriptional activity on the ERα triggered by these compounds and details on the molecular determinants involved in their activities in order to establish structure-activity relationships to shed light over the molecular basis of the promiscuity of these compounds on ER/LXR responses. Our results show that 3ß-hydroxy-5-cholestenoic acid can interact with the ERα receptor in a way similar to 26-hydroxycholesterol and is an agonist of the receptor. Using molecular dynamics simulations, we were able to predict the ERα activity of a set of cholestenoic acid analogs with changes in the flexibility and/or steric requirements of the side chain, some of which exhibited selective activation of ERα or LXR.
Assuntos
Receptor alfa de Estrogênio , Oxisteróis , Colestenos/química , Receptor alfa de Estrogênio/genética , Receptores X do Fígado/agonistas , Oxisteróis/químicaRESUMO
BACKGROUND: Diabetes mellitus is a chronic carbohydrate metabolism disorder, which could develop a series of complications and thus lead to poor quality of life or even mortality. Antrodia camphorata is a rare parasitic fungus and has been proven to be effective for treating type II diabetes. PURPOSE: This study aims to evaluate the anti-diabetic activities of A. camphorata and its main compound antcin K, as well as to demonstrate the mechanisms. STUDY DESIGN AND METHODS: Network pharmacology was used to explore the potential targets of 12 major compounds of A. camphorata on diabetes. The core targets were analyzed by protein-protein interactions and the key pathways were enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG). The anti-diabetic effects of A. camphorata and antcin K were evaluated using a high-fat diet (HFD)-induced diabetic mice model and oral glucose tolerance test (OGTT). The mRNA expressions were assessed using qPCR. RESULTS: Network pharmacology revealed 17 core targets between the 12 compounds and diabetes. The insulin resistance and NF-κB signaling pathways were enriched using KEGG. Five insulin resistance-related targets were focused on and antcin K (1/2) was discovered in the compound-target-pathway network. In vivo studies exhibited that A. camphorata and antcin K could dose-dependently reduce blood levels of glucose and lipids, decrease serum levels of insulin and leptin, and increase serum levels of adiponectin in HFD mice (p < 0.05). The mechanism could be through modulating the expressions of Tnfα, Il6, and Pparγ. The OGTT test also showed the down-regulatory effects of A. camphorata and antcin K on blood glucose. CONCLUSION: This study demonstrates that A. camphorata and its major compound antcin K possess potent anti-diabetic effects. The mechanism may be through the insulin resistance pathway.
Assuntos
Antrodia , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Animais , Colestenos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Camundongos , Farmacologia em Rede , Polyporales , Qualidade de VidaRESUMO
Antrodia cinnamomea is a well-known medicinal mushroom in Taiwan that exhibits anti-inflammatory biological activities. In rheumatoid arthritis (RA), chronic inflammation and angiogenesis driven by proinflammatory cytokines reflect the severity of the disease. Although biological treatments have improved the outlook for RA, no healing exists. Moreover, the available pharmacotherapies do not work for all patients and drug safety is a major consideration. Investigations into plant-based medicines hope to reveal better, more tolerable agents. We examined whether Antcin K, a phytosterol isolated from A. cinnamomea, has anti-angiogenic activity in RA. The GSE12021 gene dataset from the Gene Expression Omnibus (GEO) database was examined for levels of vascular endothelial growth factor (VEGF) expression in 10 RA and 10 osteoarthritis (OA) synovial tissue samples. In clinical samples, VEGF expression was analyzed by immunohistochemical staining and ELISA in normal and RA synovial tissue, as well as OA and RA synovial fluid. Collagen-induced arthritis (CIA) and control tissue was stained with hematoxylin and eosin (H&E) for histological changes; Safranin O/Fast Green staining examined histopathological changes and evidence of bone erosion. Human RA synovial fibroblasts (RASFs) were incubated with Antcin K and cell viability was examined by the MTT assay. VEGF mRNA expression was detected in RASFs using qPCR. Antcin K significantly inhibited VEGF expression and ameliorates endothelial progenitor cell (EPC) migration and tube formation in RASFs by downregulating the phospholipase C-γ/protein kinase C-α pathway. Antcin K also induced anti-angiogenic effects in human RASFs without cytotoxicity. PRACTICAL APPLICATIONS: Analysis of GEO dataset samples and human synovial fluids or synovial tissues revealed higher VEGF levels in rheumatoid arthritis (RA) samples compared with osteoarthritis (OA) and healthy control samples. VEGF levels were also higher in mice with collagen-induced arthritis (CIA) than in healthy controls. Antcin K markedly suppressed VEGF expression in human RA synovial fibroblasts and inhibited the migration and tube formation of epithelial progenitor cells (EPCs) by downregulating the phospholipase C-γ/protein kinase C-α pathway. Further investigations are warranted to examine the effects of Antcin K in other angiogenesis-associated disorders.
Assuntos
Artrite Reumatoide , Fator A de Crescimento do Endotélio Vascular , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Colestenos , Fibroblastos , Humanos , Camundongos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Nematodes are presumably the most abundant Metazoa on Earth, and can even be found in some of the most hostile environments of our planet. Various types of hypobiosis evolved to adapt their life cycles to such harsh environmental conditions. The five most distal major clades of the phylum Nematoda (Clades 8-12), formerly referred to as the Secernentea, contain many economically relevant parasitic nematodes. In this group, a special type of hypobiosis, dauer, has evolved. The dauer signalling pathway, which culminates in the biosynthesis of dafachronic acid (DA), is intensively studied in the free-living nematode Caenorhabditis elegans, and it has been hypothesized that the dauer stage may have been a prerequisite for the evolution of a wide range of parasitic lifestyles among other nematode species. Biosynthesis of DA is not specific for hypobiosis, but if it results in exit of the hypobiotic state, it is one of the main criteria to define certain behaviour as dauer. Within Clades 9 and 10, the involvement of DA has been validated experimentally, and dauer is therefore generally accepted to occur in those clades. However, for other clades, such as Clade 12, this has hardly been explored. In this review, we provide clarity on the nomenclature associated with hypobiosis and dauer across different nematological subfields. We discuss evidence for dauer-like stages in Clades 8 to 12 and support this with a meta-analysis of available genomic data. Furthermore, we discuss indications for a simplified dauer signalling pathway in parasitic nematodes. Finally, we zoom in on the host cues that induce exit from the hypobiotic stage and introduce two hypotheses on how these signals might feed into the dauer signalling pathway for plant-parasitic nematodes. With this work, we contribute to the deeper understanding of the molecular mechanisms underlying hypobiosis in parasitic nematodes. Based on this, novel strategies for the control of parasitic nematodes can be developed.
Assuntos
Adaptação Fisiológica , Colestenos/metabolismo , Estágios do Ciclo de Vida , Nematoides/crescimento & desenvolvimento , Nematoides/fisiologia , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Nematoides/classificação , Nematoides/genéticaRESUMO
OBJECTIVE: To elucidate the active compounds and the molecular mechanism of Cyathula Officinalis as a drug treatment for rheumatoid arthritis (RA). METHODS: The target genes of active ingredients from Cyathula Officinalis were obtained from bioinformatics analysis tool for the molecular mechanism of traditional Chinese medicine. The protein-protein interaction between the target genes were analyzed using STRING and Genemania. The transcriptome of RA patients compared to healthy people (GSE121894) were analyzed using R program package Limma. The relative expression of the target genes was obtained from the RNA-seq datasets. The molecular docking analyses were processed based on the molecular model of estrogen receptor 1 (ESR1) binding with estradiol (PDB ID:1A52). The binding details were analyzed by SYBYL. RESULTS: Inokosterone, ecdysterone, and cyaterone were the 3 active ingredients from Cyathula Officinalis that bind to target genes. Of all the significantly changed genes from RA patients, ESR1, ADORA1, and ANXA1 were significantly increased in mRNA samples of RA patients. CONCLUSION: ESR1, the transcription factor that binds inokosterone in the molecular binding analysis, is the target protein of Cyathula Officinalis.
Assuntos
Artrite Reumatoide , Preparações Farmacêuticas , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Colestenos , Receptor alfa de Estrogênio , Humanos , Simulação de Acoplamento MolecularRESUMO
Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate the development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole hydroxysteroid dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal-associated neurons (CANs) and excretory cells whose homologous cells in Caenorhabditis elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.
Assuntos
Diapausa/genética , Regulação da Expressão Gênica no Desenvolvimento , Comportamento de Busca por Hospedeiro , Rabditídios/crescimento & desenvolvimento , Animais , Colestenos/metabolismo , Besouros/metabolismo , Besouros/parasitologia , Loci Gênicos , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Larva , Redes e Vias Metabólicas/genética , Mutação , Neurônios/metabolismo , Odorantes , Feromônios/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Rabditídios/genética , Olfato/genéticaRESUMO
Every cell in vertebrates possesses the machinery to synthesise cholesterol and to metabolise it. The major route of cholesterol metabolism is conversion to bile acids. Bile acids themselves are interesting molecules being ligands to nuclear and G protein-coupled receptors, but perhaps the intermediates in the bile acid biosynthesis pathways are even more interesting and equally important. Here, we discuss the biological activity of the different intermediates generated in the various bile acid biosynthesis pathways. We put forward the hypothesis that the acidic pathway of bile acid biosynthesis has primary evolved to generate signalling molecules and its utilisation by hepatocytes provides an added bonus of producing bile acids to aid absorption of lipids in the intestine.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Hepatócitos/metabolismo , Redes e Vias Metabólicas , Animais , Ácidos e Sais Biliares/química , Colestenos/química , Colestenos/metabolismo , Colesterol/química , Hepatócitos/citologia , Humanos , Modelos Químicos , Estrutura Molecular , Oxisteróis/química , Oxisteróis/metabolismoRESUMO
Extracts from Taiwan's traditional medicinal mushroom, Antrodia cinnamomea, exhibit anti-inflammatory activities in cellular and preclinical studies. However, this paper is the first to report that Antcin K, a triterpenoid isolated from A. cinnamomea, inhibits proinflammatory cytokine production in human rheumatoid synovial fibroblasts (RASFs), which are major players in rheumatoid arthritis (RA) disease. In our analysis of the mechanism of action, Antcin K inhibited the expression of three cytokines (tumor necrosis factor alpha [TNF-α], interleukin 1 beta [IL-1ß] and IL-8) in human RASFs; cytokines that are crucial to RA synovial inflammation. Notably, incubation of RASFs with Antcin K reduced the phosphorylation of the focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and nuclear factor-κB (NF-κB) signaling cascades, all of which promote cytokine production in RA. Intraperitoneal injections of Antcin K (10 mg/kg or 30 mg/kg) attenuated paw swelling, cartilage degradation and bone erosion, and decreased serum levels of TNF-α, IL-1ß, IL-8 in collagen-induced arthritis (CIA) mice; in further experiments, IL-6 levels were similarly reduced. The inhibitory effects of Antcin K upon TNF-α, IL-1ß and IL-8 expression in human RASFs was achieved through the downregulation of the FAK, PI3K, AKT and NF-κB signaling cascades. Our data support clinical investigations using Antcin K in RA disease.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Cartilagem/metabolismo , Colestenos/farmacologia , Citocinas/antagonistas & inibidores , Membrana Sinovial/efeitos dos fármacos , Animais , Células Cultivadas , Colestenos/uso terapêutico , Citocinas/biossíntese , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Humanos , Interleucina-1beta/antagonistas & inibidores , Interleucina-8/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Membrana Sinovial/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Environmental signals often control central life history decisions, including the choice between reproduction and somatic maintenance. Such adaptive developmental plasticity occurs in the nematode Caenorhabditis elegans, where environmental cues govern whether larvae will develop directly into reproducing adults or arrest their development to become stress-resistant dauer larvae. Here, we identified a natural variant underlying enhanced sensitivity to dauer-inducing cues in C. elegans: a 92-bp deletion in the cis-regulatory region of the gene eak-3. This deletion reduces synthesis or activity of the steroid hormone dafachronic acid (DA), thereby increasing environmental sensitivity for dauer induction. Consistent with known pleiotropic roles of DA, this eak-3 variant significantly slows down reproductive growth. We experimentally show that, although the eak-3 deletion can provide a fitness advantage through facilitated dauer production in stressful environments, this allele becomes rapidly outcompeted in favorable environments. The identified eak-3 variant therefore reveals a trade-off in how hormonal responses influence both the pace of developmental timing and the way in which environmental sensitivity controls adaptive plasticity. Together, our results show how a single mutational event altering hormonal signaling can lead to the emergence of a complex life history trade-off.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Colestenos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/crescimento & desenvolvimento , Adaptação Fisiológica/genética , Alelos , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Pleiotropia Genética , Larva/genética , Mutação , Sequências Reguladoras de Ácido Nucleico/genética , Deleção de Sequência , Transdução de SinaisRESUMO
Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.
Assuntos
Colestenos/farmacologia , Dirofilaria immitis/crescimento & desenvolvimento , Dirofilariose/prevenção & controle , Doenças do Cão/prevenção & controle , Proteínas de Helminto/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Colestenos/uso terapêutico , Colesterol/metabolismo , Dirofilaria immitis/efeitos dos fármacos , Dirofilaria immitis/metabolismo , Dirofilariose/parasitologia , Doenças do Cão/parasitologia , Cães , Proteínas de Helminto/agonistas , Interações Hospedeiro-Parasita , Larva/efeitos dos fármacos , Larva/metabolismo , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Muda/efeitos dos fármacos , Células NIH 3T3 , Domínios Proteicos , Receptores Citoplasmáticos e Nucleares/agonistasRESUMO
Cholesterol is one of the hallmarks of animals. In vertebrates, the cholesterol synthesis pathway (CSP) is the primary source of cholesterol that has numerous structural and regulative roles [1]. Nevertheless, the few invertebrates tested for cholesterol synthesis show complete sterol auxotrophy [2-6], raising questions about how animals thrive without cholesterol synthesis and about the prevalence of sterol auxotrophy in animals. In the nematode Caenorhabditis elegans (C. elegans), sterols are the precursors of the steroid hormone dafachronic acid that coordinates development to adulthood [7, 8]; thus, sterol-deprived C. elegans arrest at the diapause "dauer" larval stage [9]. Using this system, we have identified a pathway that converts plant and fungal sterols into cholesterol through the activity of enzymes with sequence similarity to specific human CSP enzymes. Based on this finding, we propose that two critical steps shaped the evolution of animal sterol auxotrophy: (1) the loss of the orthologs of the first three enzymes of the CSP and (2) the co-opting of other downstream enzymes of the CSP for the utilization of dietary sterols. Using this mechanistic signature, we studied the evolution of cholesterol auxotrophy across the animal kingdom. Complete sets of CSP enzymes in basal animals suggest that the loss of cholesterol synthesis occurred during animal evolution. A sterol auxothropy signature in the genomes of many invertebrates, including nematodes and most arthropods, suggests widespread cholesterol auxotrophy in animals. Thus, we propose that this co-opted pathway supports widespread cholesterol auxotrophy by interkingdom interactions between cholesterol-auxotrophic animals and sterol-producing fungi and plants.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Caenorhabditis elegans/metabolismo , Colesterol/biossíntese , Esteróis/metabolismo , Animais , Colestenos/metabolismo , Larva/metabolismoRESUMO
BACKGROUND: While immune responses to the murine hookworm Nippostrongylus brasiliensis have been investigated, signaling pathways regulating development of infectious larvae (iL3) are not well understood. We hypothesized that N. brasiliensis would use pathways similar to those controlling dauer development in the free-living nematode Caenorhabditis elegans, which is formally known as the "dauer hypothesis." METHODS: To investigate whether dafachronic acid activates the N. brasiliensis DAF-12 homolog, we utilized an in vitro reporter assay. We then utilized RNA-Seq and subsequent bioinformatic analyses to identify N. brasiliensis dauer pathway homologs and examine regulation of these genes during iL3 activation. RESULTS: In this study, we demonstrated that dafachronic acid activates the N. brasiliensis DAF-12 homolog. We then identified N. brasiliensis homologs for members in each of the four canonical dauer pathways and examined their regulation during iL3 activation by either temperature or dafachronic acid. Similar to C. elegans, we found that transcripts encoding antagonistic insulin-like peptides were significantly downregulated during iL3 activation, and that a transcript encoding a phylogenetic homolog of DAF-9 increased during iL3 activation, suggesting that both increased insulin-like and DAF-12 nuclear hormone receptor signaling accompanies iL3 activation. In contrast to C. elegans, we observed a significant decrease in transcripts encoding the dauer transforming growth factor beta ligand DAF-7 during iL3 activation, suggesting a different role for this pathway in parasitic nematode development. CONCLUSIONS: Our data suggest that canonical dauer pathways indeed regulate iL3 activation in the hookworm N. brasiliensis and that DAF-12 may be a therapeutic target in hookworm infections.
Assuntos
Colestenos/farmacologia , Nippostrongylus/efeitos dos fármacos , Nippostrongylus/genética , Transdução de Sinais/efeitos dos fármacos , Temperatura , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Filogenia , RNA-SeqRESUMO
Under stressful conditions, the early larvae of C. elegans enter dauer diapause, a non-aging period, driven by the seemingly opposite influence of ascaroside pheromones (ASCRs) and steroid hormone dafachronic acids (DAs). However, the molecular basis of how these small molecules engage in competitive crosstalk in coordination with insulin/IGF-1 signaling (IIS) remains elusive. Here we report a novel transcriptional regulatory pathway that seems to operate between the ASCR and DA biosynthesis under ad libitum (AL) feeding conditions or bacterial deprivation (BD). Although expression of the ASCR and DA biosynthetic genes reciprocally inhibit each other, ironically and interestingly, such dietary cue-mediated modulation requires the presence of the competitors. Under BD, induction of ASCR biosynthetic gene expression required DA, while ASCR suppresses the expression of the DA biosynthetic gene daf-36. The negative regulation of DA by ASCR was IIS-dependent, whereas daf-36 regulation appeared to be independent of IIS. These observations suggest that the presence of ASCR determines the IIS-dependency of DA gene expression regardless of dietary conditions. Thus, our work defines a molecular basis for a novel reciprocal gene regulation of pheromones and hormones to cope with stressful conditions during development and aging.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Caenorhabditis elegans/fisiologia , Sinais (Psicologia) , Hormônios/genética , Hormônios/metabolismo , Feromônios/genética , Feromônios/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colestenos/metabolismo , Regulação da Expressão Gênica , Modelos Biológicos , Transdução de SinaisRESUMO
Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.
Assuntos
Colestenos/farmacologia , Colesterol/metabolismo , Receptores X do Fígado/química , Oxisteróis/metabolismo , Colestenos/química , Colesterol/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Metabolismo dos Lipídeos , Receptores X do Fígado/genética , Receptores X do Fígado/ultraestrutura , Análise em Microsséries , Conformação Molecular , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Oxisteróis/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Drug induced liver damage remains a prevalent concern in healthcare and may reduce the effectiveness of therapy by compromising therapeutic regimens. Many Commiphora species are known for their medicinal properties, and some of them are used traditionally for hepatoprotective effect. In the course of our drugs discovery from natural sources, phytosterols (lophenol (Lop) and lathosterol (Lat)), isolated from Commiphora kua were studied to evaluate their hepatoprotective effects in acetaminophen (APAP) induced hepatotoxicity in mice. AIMS AND OBJECTIVE: To evaluate the hepatoprotective effects of phytosterols isolated from C. kua using in vivo experimental model. MATERIALS AND METHODS: Mice of either sex were divided into 7 groups: Vehicle, silymarin (SLY), acetaminophen (APAP), Lop 25, Lop 50, Lat 25, Lat 50 (nâ¯=â¯5). Vehicle group received only vehicle (0.1% DMSO solution) for 7 days, APAP group received single dose of acetaminophen on day 7 and SLY group received silymarin for 7 days. Lop 25 and Lop 50 received low and high doses of Lop (25⯵g/kg BW and 50⯵g/kg BW), respectively, for 7 days, while Lat 25 and Lat 50 received low and high doses of Lat (25⯵g/kg BW and 50⯵g/kg BW) for 7 days. On day 7, all animals except Vehicle group kept fasted for 18â¯h and received APAP i. p. 400â¯mg/kg BW. After 20â¯h of APAP administration, the animals anesthetized with light chloroform and scarified by cervical decapitation. The blood serum and liver tissue samples were collected for biochemical and histopathological analysis. Liver function tests (LFTs) including lactate deydrogenase (LDH), alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and direct bilirubin) were used as biochemical parameters. While catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) were taken as anti-oxidant enzymes. RESULTS: Significant increase in levels of ALT, AST, ALP, LDH and direct bilirubin, and significant decrease in concentration of anti-oxidant enzymes (SOD, CAT and GSH) was observed in APAP-treated group. Similarly, histological slides showed obvious signs of damage to liver cells, reflecting acetaminophen induced hepatotoxicity. Treatment of test animals with phytosterols resulted in significant recovery of LFTs profile and concentration of anti-oxidant enzymes. Similarly, significant improvement of liver tissues was noted in histological analysis. CONCLUSIONS: Both phytosterols possessed hepatoprotective potential and should be further evaluated for acute toxicity studies and pharmacokinetics/pharmacodynamics profile.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colesterol/uso terapêutico , Commiphora , Extratos Vegetais/uso terapêutico , Substâncias Protetoras/uso terapêutico , Acetaminofen , Animais , Catalase/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestenos/farmacologia , Colestenos/uso terapêutico , Colesterol/farmacologia , Feminino , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Resinas Vegetais/química , Superóxido Dismutase/metabolismoRESUMO
Follicular fluid meiosis-activating sterol (FF-MAS) exerts beneficial effects on the meiotic resumption of mammalian oocytes and their subsequent early embryonic development, but the signaling pathway underlying these effects has not been elucidated. Therefore, the objective of the present study was to investigate whether the mitogen-activated protein kinase (MAPK) pathway is involved in the FF-MAS-induced in vitro resumption of meiosis in porcine oocytes. Porcine cumulus oocyte complexes (COCs) were allocated in several groups cultured in TCM-199 medium with different concentration of AY 9944-A-7 (20, 30, 40⯵mol/L) or ketoconazole (20⯵mol/L) to increase or decrease endogenous accumulation of FF-MAS. Each experimental condition was repeated at least six times. After maturation for 44â¯h, the resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) and the first polar body (PBI) extrusion, The relative expressions of related genes in MAPK pathway [c-mos, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1/2 (ERK1/2)] at both transcriptional and translational levels were detected to investigate the kinetic trend of expression throughout oocyte maturation in vitro in response to the addition of AY 9944-A-7 or ketoconazole to the maturation medium. Results indicated that AY 9944-A-7 promoted, while ketoconazole inhibited, the in vitro maturation (IVM) of porcine oocytes. Relative expression of meiosis related genes was upregulated by AY 9944-A-7 and downregulated by ketoconazole. With extended culturing time, c-mos mRNA expression levels reached their peak at 12â¯h of maturation and decreased gradually thereafter, while MEK, ERK1 and ERK2 expression increased after an initial decrease peaking at 44â¯h of culture in the AY 9944-A-7-group. And the trend of the protein expression of c-mos, MEK, ERK1/2 was basically consistent with the mRNA expression of these genes. These results imply that the endogenous accumulation of FF-MAS is beneficial to resumption of meiosis in porcine oocytes and that MAPK signaling is involved in FF-MAS-induced meiotic resumption.
Assuntos
Colestenos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Meiose/efeitos dos fármacos , Animais , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Cetoconazol/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/farmacologiaRESUMO
Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.