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Quantitative real-time PCR analysis of hmox1 mRNA in untreated (wild-type (WT) n = 75; sapje CTRL, n = 75) and in treated (gingerol 4 µM, n = 75) homozygous sapje larvae. The error bars show the standard error of the mean (SEM). * p ≤ 0.05, ns p > 0.05.
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Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited neuromuscular disorder that causes loss of muscle mass and motor skills. In the era of genomic medicine, there is still no known cure for DMD. In clinical practice, there is a growing awareness of the possible importance of nutrition in neuromuscular dise...
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Background
This trial is to evaluate the effects of supplementation of Aureobasidium pullulans-N-163 strain produced 1,3-1,-6 beta glucan in young patients with Duchenne muscular dystrophy (DMD).
Methods
Twenty-seven male subjects aged 5-19 years with DMD were included, nine in the control arm and 18 in the treatment arm to receive N-163 beta gluc...
Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of si...
Abnormal dystrophin production due to mutations in the dystrophin gene causes Duchenne Muscular Dystrophy (DMD). Cases demonstrate considerable genetic and disease progression variability. It is unclear if specific gene mutations are prognostic of outcomes in this population. We conducted a retrospective cohort study of DMD patients followed at 17...
The nematode worm Caenorhabditis elegans has been used extensively to enhance our understanding of the human neuromuscular disorder Duchenne Muscular Dystrophy (DMD). With new arising clinically relevant models, technologies and treatments, there is a need to reconcile the literature and collate the key findings associated with this model.
Background:
Impaired muscle regeneration is a hallmark of Duchenne muscular dystrophy (DMD), a neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. The lack of heme oxygenase-1 (HO-1, Hmox1), a known anti-inflammatory and cytoprotective enzyme, was shown to aggravate DMD pathology.
Methods:
We evaluated the role of HO-...
Citations
... Moreover, patients with epilepsy and neurodegenerative disorders looking for treatments without the strong side effects of synthetic drugs are now becoming increasingly interested in naturally derived compounds [38][39][40]. Nutraceuticals have been recognized to possess pleiotropic biological activity, and have the dual advantage of low toxicity and high tolerability [40][41][42]. In this regard, cannabinoids, the bioactive compounds of the Cannabis sativa plant [39], appear promising. ...
Background: Valproic acid (VPA), an anticonvulsant used in epilepsy, has deleterious effects on embryonic development and is considered an environmental risk factor for autism spectrum disorders. There is a growing need for easy and rapid ways to study its effects on embryonic development. The zebrafish model is a cost and time-effective tool able to facilitate mechanistic studies and high-throughput drug screening. Epileptic patients are increasingly looking to natural compounds to avoid the strong side effects of synthetic drugs, and cannabinoids appear promising. We evaluated the potential of cannabidiol to mitigate the negative effects of VPA on developing zebrafish embryos. Methods: Wild-type AB embryos, untreated or exposed to VPA (5, 10 or 20 µM) and/or cannabidiol (1, 2 or 3 µM), were evaluated at up to 120 hours post-fertilization. Developmental endpoints: survival, hatching, heart rate, morphology, and locomotor behavior (tail coiling and visual motor response test). Three replicates were evaluated per group, for a total of 120 larvae per treatment. Results: Although VPA-treated groups showed significantly reduced survival rates compared to control group fish (p ≤ 0.01), zebrafish simultaneously treated with VPA (5 and 10 µM) and cannabidiol (3 µM) displayed survival rates similar to those of untreated controls. Hatching rate, body length and eye area were not influenced by any treatment, but the highest VPA dose and all cannabidiol doses caused a significant increase in burst activity (p ≤ 0.0001). Compared with controls, the pericardial area was larger only in larvae treated with VPA at the highest concentration (p ≤ 0.01). Each VPA treatment caused tachycardia (p ≤ 0.0001), while cannabidiol 3 µM induced bradycardia (p ≤ 0.01). Finally, simultaneous treatment with VPA 5 µM and cannabidiol 3 µM avoided almost all the adverse effects of the two compounds administered individually, stabilizing heart rate and locomotor behavior at control levels. Conclusions: This study adds further information on the embryotoxic effect of VPA in the zebrafish model and offers new insights into the use of cannabidiol as an alternative natural drug able to mitigate the deleterious effects of VPA. Multi-laboratory large-scale validation and new genomic and molecular analyses are required to clarify the mechanism of action of VPA on developing embryos and the role of cannabidiol as a potential natural protective agent against its toxic effects. Keywords: zebrafish; valproic acid; autism spectrum disorders; cannabidiol; valproate; neurodevelopment
... Several lead compounds able to ameliorate major disease symptoms, such as muscle fibrosis, inflammation, dysregulation of calcium homeostasis, oxidative stress, and mitochondrial dysfunction, have been discovered by using DMD sapje and sapje-like zebrafish models generated with ENU mutagenesis (see Table 2) [117,118,137,198,[204][205][206][207][208], LGMD2I zebrafish models harboring the fkrp mutation (see Table 3) [76], the zebrafish transgenic model of myotonic dystrophy harboring multiple CUG repetitions in its genome [209], zebrafish models of collagen VI deficiency congenital myopathies (Table 1) [119,210,211], and candyfloss zebrafish models [33]. ...
Muscular dystrophies (MDs) are a heterogeneous group of myopathies characterized by progressive muscle weakness leading to death from heart or respiratory failure. MDs are caused by mutations in genes involved in both the development and organization of muscle fibers. Several animal models harboring mutations in MD-associated genes have been developed so far. Together with rodents, the zebrafish is one of the most popular animal models used to reproduce MDs because of the high level of sequence homology with the human genome and its genetic manipulability. This review describes the most important zebrafish mutant models of MD and the most advanced tools used to generate and characterize all these valuable transgenic lines. Zebrafish models of MDs have been generated by introducing mutations to muscle-specific genes with different genetic techniques, such as (i) N-ethyl-N-nitrosourea (ENU) treatment, (ii) the injection of specific morpholino, (iii) tol2-based transgenesis, (iv) TALEN, (v) and CRISPR/Cas9 technology. All these models are extensively used either to study muscle development and function or understand the pathogenetic mechanisms of MDs. Several tools have also been developed to characterize these zebrafish models by checking (i) motor behavior, (ii) muscle fiber structure, (iii) oxidative stress, and (iv) mitochondrial function and dynamics. Further, living biosensor models, based on the expression of fluorescent reporter proteins under the control of muscle-specific promoters or responsive elements, have been revealed to be powerful tools to follow molecular dynamics at the level of a single muscle fiber. Thus, zebrafish models of MDs can also be a powerful tool to search for new drugs or gene therapies able to block or slow down disease progression.
... Zebrafish eggs are transparent, allowing the observation of morphogenetic changes and organogenesis in real time (39). In particular, embryos have been shown to represent a valuable tool to study both pro-oxidant (40) and pro-inflammatory (41) phenomena, as well as to investigate the therapeutic potential of nutraceuticals (42) and natural products (43). Additionally, embryos have rapid development with embryogenesis being complete by 72 hours post fertilization (hpf) and most organs fully developed by 96 hpf. ...
Oxidative stress is due to an unbalance between pro-oxidants, such as reactive oxygen (ROS) and nitrogen (RNS) species, and antioxidants/antioxidant system. Under physiological conditions these species are involved in different cellular processes such as cellular homeostasis and immune response, while an excessive production of ROS/RNS has been linked to the development of various diseases such as cancer, diabetes, and Alzheimer's disease. In this context, the naturally occurring dipeptide carnosine has shown the ability to scavenge ROS, counteract lipid peroxidation, and inhibit proteins oxidation. Titanium dioxide nanoparticles (TiO2-NPs) have been widely used to produce cosmetics, in wastewater treatment, in food industry, and in healthcare product. As consequence, these NPs are often released into aquatic environments. The Danio rerio (commonly called zebrafish) embryos exposure to TiO2-NPs did not affect the hatching rate, but induced oxidative stress. According to this scenario, in the present study, we first investigated the effects of carnosine exposure and of a sub-toxic administration of TiO2-NPs on the development and survival of zebrafish embryos/larvae measured through the acute embryo toxicity test (FET-Test). Zebrafish larvae represent a useful model to study oxidative stress-linked disorders and to test antioxidant molecules, while carnosine was selected based on its well-known multimodal mechanism of action that includes a strong antioxidant activity. Once the basal effects of carnosine were assessed, we then evaluated its effects on TiO2-NPs-induced oxidative stress in zebrafish larvae, measured in terms of total ROS production (measured with 2,7-dichlorodihydrofluorescein diacetate probe) and protein expression by immunohistochemistry of two cellular stress markers, 70 kDa-heat shock protein (Hsp70) and metallothioneins (MTs). We demonstrated that carnosine did not alter the phenotypes of both embryos and larvae of zebrafish at different hours post fertilization. Carnosine was instead able to significantly decrease the enhancement of ROS levels in zebrafish larvae exposed to TiO2-NPs and its antioxidant effect was paralleled by the rescue of the protein expression levels of Hsp70 and MTs. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of pathologies characterized by oxidative stress such as neurodegenerative disorders.
... In the current study, using a microinjection technique, we attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves at early stages of embryogenesis, with the aim of evaluating the short-term effects of the treatment on mortality, locomotor behavior, and gene expression. The zebrafish is already considered an ideal organism for neurobiology and genetics research and for exploring possible new drug treatments [31][32][33][34][35]. The use of zebrafish, rather than mammalian models, for genetic and drug studies offers numerous advantages, such as high fecundity, egg transparency, rapid ex utero embryogenesis (which allows noninvasive drug treatments at early embryonic stages) and affordability [36][37][38]. ...
Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.
... The cyprinidae zebrafish (Danio rerio) is a well-established biomedicine and aquaculture research animal model [7][8][9][10]. As a model for aquaculture studies, zebrafish enables lower research costs and possesses a well-known digestive system from both the morphofunctional and microbiome points of view [11,12], making it a useful model to assess the relationship between nutrition and health [13,14]. ...
The current study evaluated the effects of hydrolyzable and condensed tannins from chestnut and quebracho wood, respectively (TSP, Silvafeed®), on zebrafish with intestinal inflammation induced by a plant-based diet (basal diet). Four experimental diets were prepared as follows: the basal diet + 0 TSP, the basal diet + TSP at 0.9 g/Kg of feed, the basal diet + TSP at 1.7 g/Kg of feed, and the basal diet + TSP at 3.4 g/Kg of feed. Eighty-four zebrafish (Danio rerio) were fed for 12 days with the experimental diets. In zebrafish fed the basal diet, intestine integrity appeared to be altered, with damaged intestinal villi, high immunoexpression of tumor necrosis factor-α (TNFα) and cyclooxygenase 2 (COX2), and high expression of the cox2, interleukin 1 (il-1b), interleukin 8 (cxcl8-l1), and tnfα genes. The tannin treatment partially restored intestinal morphology and downregulated the expression of cytokines. The best activity was detected with 1.7 and 3.4 g/kg of feed. In the guts of all groups, Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes were the most represented phyla. The most represented genera were Plesiomonas and Sphingomonas, belonging to the Proteobacteria phylum; Cetobacterium, belonging to the Fusobacteria phylum; and Lactobacillus, belonging to the Firmicutes phylum. No significant differences were detected among groups, except for a slight decrease in the Fusobacteria phylum and slight increases in the Shewanella and Bacteroides genera with TSP. In conclusion, these results suggest that tannins can improve the zebrafish intestinal inflammation caused by a terrestrial-plant-based diet in a dose-dependent manner.
... erefore, they can be used to construct many pathological models. For example, sleep deprivation model was induced by light stimulation [49], depression model was induced by reserpine [50], thrombus model was induced by phenylhydrazine (PHZ) [51], epilepsy model was induced by pentylenetetrazol (PTZ) [52], vascular insufficiency model was induced by tyrosine kinase inhibitor [53], vascular defect model was induced by axitinib [54], and sapje strain model was always used for muscle atrophy [55]. GCs are only recognized to induce osteoporosis and cannot be used to induce other diseases in zebrafish. ...
Osteoporosis is a degenerative disease that endangers human health. At present, chemical drugs used for osteoporosis have serious side effects. Therefore, it is valuable to search herbs with high safety and good curative effect in antiosteoporosis. Erzhi formula (EZF), an ancient classic compound, has been reported to have a beneficial effect in antiosteoporosis, but its mechanism is unclear. In this paper, the active compounds of EZF were found in Systems Pharmacology Database, and gene targets related to osteoporosis were obtained in GeneCards. The GO functional and KEGG pathway enrichment analysis were performed by Metascape. The network of “components-targets-signal pathway” was constructed by Cytoscape. Next, molecular docking between the active components and hub genes related to the PI3K-Akt signaling pathway was conducted by Autodock. In the verification experiment, the zebrafish induced by prednisolone (PNSL) was used to reproduce glucocorticoid-induced osteoporosis (GIOP) model, and then the reversal effects of EZF were systematically evaluated according to the behavior, skull staining area, bone mineralization area (BMA), average optical density (AOD), and cumulative optical density (COD). Finally, it was shown that 24 components in EZF could regulate 39 common gene targets to exert antiosteoporosis effect. Besides, the main regulatory mechanisms of EZF were 4 signaling pathways: PI3K-Akt, JAK-STAT, AGE-RAGE, and cancer pathway. In PI3K-Akt signaling pathway, wedelolactone, dimethyl wedelolactone, specnuezhenide, ursolic acid, acacetin, beta-sitosterol, apigenin, and kaempferol can bind tightly with EGF, IL-2, and IL-4 genes. Compared with the model group, the moving distance, swimming speed, and cumulative swimming time of zebrafish in EZF group were significantly increased (P
... So far, the majority of scientific research on the effect of cannabinoids was based on in vitro approaches or on rodent models, but recently, the zebrafish (Danio rerio) has gained attractiveness as a powerful in vivo model for complementing and expanding the findings of these existing studies. The zebrafish is, nowadays, widely used as an animal model for pharmacological screening [35][36][37][38], owing to its excellent adaptation to laboratory conditions and its advantageous features already reviewed elsewhere [39][40][41][42][43][44]. In drug screenings, zebrafish are able to absorb small molecules across the skin from the surrounding water, at all stages of development [31]. ...
... The gold standard for predictive analysis of chemical risks to humans remains vertebrate toxicity studies [35]. In this context, zebrafish larvae have become a tool widely used to assess the toxic effects of chemicals, drugs, and natural compounds [36,38]. The present study evaluated, for the first time, the effects of a whole cannabis extract diluted in DMSO on zebrafish development, locomotion and mRNA expression of some ECS-related genes (cnr1 and cnr2). ...
... Distance moved, velocity and movement cumulative time were measured as zebrafish locomotor behavior indicators on untreated and treated larvae at 5 dpf. To this purpose, the DanioVision system and EthoVision XT12 software (Noldus Information Technology, Wageningen, The Netherlands) were used as previously described by Licitra et al. [36]. Together with 300 µL of the solution treatment for a single larva, 32 individuals of each group were transferred from the rearing dishes to a 96 multiwell plate and placed into the DanioVision system for 1 h of acclimatization. ...
Historically, humans have used Cannabis sativa for both recreational and medical purposes. Nowadays, cannabis-based products have gained scientific interest due to their beneficial effects on several syndromes and illnesses. The biological activity of cannabinoids is essentially due to the interaction with the endocannabinoid system and zebrafish (Danio rerio) is a very well-known and powerful in vivo model for studying such specific interactions. The aim of the study was to investigate the effects of different doses of a Cannabis sativa whole extract (dissolved in DMSO) on zebrafish eggs hatchability, embryo post-hatching survival, larvae locomotion behavior and mRNA gene expression. The results showed the absence of toxicity and no significant differences were observed between treatments for both embryos hatching and survival rate. In addition, larvae exposed to the cannabis extract at the highest dose (containing 1.73 nM and 22.3 nM of THC and CBD, respectively) showed an increased locomotion compared to control and DMSO treated groups. Moreover, qRT-PCR analysis showed that the highest dosage of can-nabis induced an over-expression of cnr1 and cnr2 cannabinoid receptors. In conclusion, the ex-position of zebrafish larvae to the whole extract of Cannabis sativa showed no negative effects on embryo development and survival and enhanced the larvae locomotor performances. These findings may open to possible Cannabis sativa applications in human pharmacology, as well as other animal sector.
... Total RNA was extracted from larvae at 120 hpf using Quick RNA miniprep (Zymo Research, Irvine, USA) according to the manufacturer's instruction. cDNA and qRT-PCR were performed as described in [21]. Relative expression levels of the gene were calculated using the 2 −∆∆Ct method [59]. ...
Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular childhood disorder that causes progressive muscle weakness and degeneration. A lack of dystrophin in DMD leads to inflammatory response, autophagic dysregulation, and oxidative stress in skeletal muscle fibers that play a key role in the progression of the pathology. ß-glucans can modulate immune function by modifying the phagocytic activity of immunocompetent cells, notably macrophages. Mitochondrial function is also involved in an important mechanism of the innate and adaptive immune responses, owing to high need for energy of immune cells. In the present study, the effects of 1,3-1,6 ß-glucans on five-day-old non-dystrophic and dystrophic (sapje) zebrafish larvae were investigated. The effects of the sonication of β-glucans and the dechorionation of embryos were also evaluated. The results showed that the incidence of dystrophic phenotypes was reduced when dystrophic embryos were exposed to 2 and 4 mg L−1 of 1,3-1,6 ß-glucans. Moreover, when the dystrophic larvae underwent 8 mg L−1 treatment, an improvement of the locomotor performances and mitochondrial respiration were observed. In conclusion, the observed results demonstrated that 1,3-1,6 ß-glucans improve locomotor performances and mitochondrial function in dystrophic zebrafish. Therefore, for ameliorating their life quality, 1,3-1,6 ß-glucans look like a promising diet supplement for DMD patients, even though further investigations are required.
Background
This exploratory case-control study is to evaluate the effects of supplementation of Aureobasidium pullulans-N-163 strain produced 1,3–1,− 6 beta glucan in young patients with Duchenne muscular dystrophy (DMD).
Methods
Twenty-seven male subjects aged 5–19 years with DMD were included, nine in the control arm and 18 in the treatment arm to receive N-163 beta glucan along with conventional therapies for 45 days. While performing the analysis, steroid usage was also taken into consideration, those not administered steroids (Steroid -ve) (Control, n = 5; treatment, n = 9), those administered steroids (Steroid +ve) (Control, n = 4; treatment, n = 9).
Results
IL-6 showed a significant decrease in the treatment groups, especially the N-163 Steroid -ve group. IL-13 decreased in both treatment groups and TGF-β levels showed a significant decrease in the treatment groups, especially the N-163 Steroid –ve group, (p < 0.05). Dystrophin levels increased by up to 32% in the treatment groups compared to the control. Medical research council (MRC) grading showed slight improvement in muscle strength improvement in 12 out of 18 patients (67%) in the treatment group and four out of nine (44%) subjects in the control group.
Conclusion
Supplementation with the N-163 beta glucan food supplement produced beneficial effects: a significant decrease in inflammation and fibrosis markers, increase in serum dystrophin and slight improvement in muscle strength in DMD subjects over 45 days, thus making this a potential adjunct treatment for DMD after validation.
Trial registration
The study was registered in Clinical trials registry of India, CTRI/2021/05/033346. Registered on 5th May, 2021.
Sepsis is a life-threatening condition caused by an abnormal immune response induced by infection with no approved or specific therapeutic options. We present our perspectives for the therapeutic management of sepsis through a four-way approach: (1) infection control through immune enhancement; (2) immune suppression during the initial hyper-inflammatory phase; (3) balanced immune-modulation to counter the later immune-paralysis phase; and (4) advantageous effects on metabolic and coagulation parameters throughout. COVID-19 is a virus-triggered, accelerated sepsis-like reaction that is associated with the rapid progress of an inflammatory cascade involving a cytokine storm and multiorgan failure. Here, we discuss the potential of the biological response modifiers, β-glucans (BRMGs), in the management of sepsis based on their beneficial effects on inflammatory-immune events in COVID-19 clinical studies. In COVID-19 patients, apart from metabolic regulation, BRMGs, derived from a black yeast, Aureobasidium pullulans strain AFO-202, have been reported to stimulate immune responses. BRMGs, produced by another strain (N-163) of A. pullulans, have been implicated in the beneficial regulation of inflammatory markers and immunity, namely IL-6, C-reactive protein (CRP), D-Dimer, ferritin, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-C-reactive protein ratio (LCR), leucocyte-to-C-reactive protein ratio (LeCR), and leukocyte-to-IL-6 ratio (LeIR). Agents such as these β-glucans, which are safe as they have been widely consumed by humans for decades, have potential as adjuncts for the prevention and management of sepsis as they exert their beneficial effects across the spectrum of processes and factors involved in sepsis pathology, including, but not limited to, metabolism, infection, inflammation, immune modulation, immune enhancement, and gut microbiota.
