Zebrafish Larvae Model Research Articles

Therapeutic Potential of Deflamin against Colorectal Cancer Development and Progression.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that play a crucial role in tumor microenvironment remodeling, contributing to inflammatory and angiogenic processes, and ultimately promoting tumor maintenance and progression. Several studies on bioactive polypeptides isolated from legumes have shown anti-migratory, anti-MMPs, and anti-tumor effects, potentially constituting novel strategies for both the prevention and progression of cancer. In this work, we investigated the anti-tumor role of deflamin, a protein oligomer isolated from white lupine seeds (Lupinus albus) reported to inhibit MMP-9 and cell migration in colorectal cancer (CRC) cell lines. We found that deflamin exerts an inhibitory effect on tumor growth and metastasis formation, contributing to increased tumor apoptosis in the xenotransplanted zebrafish larvae model. Furthermore, deflamin resulted not only in a significant reduction in MMP-2 and MMP-9 activity but also in impaired cancer cell migration and invasion in vitro. Using the xenograft zebrafish model, we observed that deflamin inhibits collagen degradation and angiogenesis in the tumor microenvironment in vivo. Overall, our work reveals the potential of deflamin as an agent against CRC development and progression.

P003 The Effect of Centella Asiatica on Inactive Frequency and Movement Distance in Insomnia Zebrafish (Danio rerio) Larvae Model

Abstract Introduction Insomnia is a global health problem. Zebrafish (Danio rerio) is an ideal model for studying sleep disorder due to its diurnal cycle. Centella Asiatica (CA) is thought to have an anti-insomnia effect, but its mechanism is not clearly understood. The aim of this study is to evaluate the effect of CA on insomnia zebrafish larvae model by observing sleep parameters including inactive frequency and movement distance. Methods This experimental study is divided into five groups of zebrafish larvae consisting of control group, insomnia group with 24 hours of light exposure for 7 days, insomnia group with CA concentration of 2.5, 5, and 10 µg/mL. Observation of zebrafish larval movement was carried out on 5, 6, and 7 dpf (day post fertilization) during 30 minutes before and after turning off light exposure. Results There were significant differences on 5, 6, and 7 dpf between the normal group, insomnia group, and all treatment groups with CA addition on inactive frequency and movement distance in light and dark conditions (p<0.05). Discussion This study showed that CA has an anti-insomnia effect. CA can improve sleep quality of insomnia zebrafish larvae model by increasing inactive frequency and shortening movement distance. Keywords Centella Asiatica, zebrafish, inactive frequency, movement distance.

STUDYING OF POTENTIAL ANXIOLYTIC ACTIVITY OF ANGELICA ADZHARICA M. PIMEN. METHANOLIC EXTRACT

Anxiety disorders have become the number one mental health issue in developed countries. The number of people living with anxiety and depressive disorders rose significantly because of the COVID-19 pandemic. Although generally considered as safe and effective, the currently available anxiolytic therapy is responsible for numerous side effects and addiction. Therefore, there is an imperious demand for discovering more efficient and cheaper new groups of pharmaceuticals (“green anxiolytic”). There is a big number of research, studying plants containing coumarins as active compounds, especially those belonging to the Apiaceae family. Laboratory studies already suggest the anti-anxiety activity of various extracts of Angelica Archangelica. Therefore, the aim of our study was to obtain the methanolic extract of Angelica adzharica and evaluate the potential anxiolytic effect based on the zebrafish larvae model. According to our knowledge, this is the first report studying the anxiolytic effect of Angelica adzharica extract based on the zebrafish larvae.

PHARMACO-TECHNOLOGICAL ASSESSMENT OF SAKHARE LAKE

Anxiety disorders have become the number one mental health issue in developed countries. The number of people living with anxiety and depressive disorders rose significantly because of the COVID-19 pandemic. Although generally considered as safe and effective, the currently available anxiolytic therapy is responsible for numerous side effects and addiction. Therefore, there is an imperious demand for discovering more efficient and cheaper new groups of pharmaceuticals (“green anxiolytic”). There is a big number of research, studying plants containing coumarins as active compounds, especially those belonging to the Apiaceae family. Laboratory studies already suggest the anti-anxiety activity of various extracts of Angelica Archangelica. Therefore, the aim of our study was to obtain the methanolic extract of Angelica adzharica and evaluate the potential anxiolytic effect based on the zebrafish larvae model. According to our knowledge, this is the first report studying the anxiolytic effect of Angelica adzharica extract based on the zebrafish larvae.

Withaferin A targets the membrane of Pseudomonas aeruginosa and mitigates the inflammation in zebrafish larvae; an in vitro and in vivo approach

Infections due to multidrug-resistant Pseudomonas aeruginosa are prevalent among patients with cystic fibrosis. The emergence of antibiotic-resistant pathogens necessitated the development of novel low-risk natural antibacterial compounds. Herbal medicines are used from dates of the origin of mankind and still serve their purpose as therapeutic agents. We demonstrated the antibacterial activity of Withaferin A extracted from the traditional herb, ashwagandha or winter cherry (Withania somnifera). Withaferin A exhibits strong antibacterial activity against P. aeruginosa with a minimum inhibitory concentration of 60 μM and minimum bactericidal concentration of 80 μM. Results obtained from membrane stabilization assay and electron microscopic analysis showed that Withaferin A acts by damaging the cell membrane of P. aeruginosa. Additionally, we investigated oxidative stress and inflammatory response after Withaferin A treatment in P. aeruginosa infected zebrafish larvae model. The results indicate that the level of ROS, and its related lipid peroxidation and apoptosis were significantly reduced after treated with Withaferin A. Consequently, an increment in antioxidant enzymes level such as superoxide dismutase (SOD) and catalase (CAT) was observed. Macrophage localization experiment showed a smaller number of localized macrophages in zebrafish, which indicates the reduction in inflammatory response. In conclusion, Withaferin A could serve as an alternative natural product in the treatment of infections caused by P. aeruginosa.

Epimedin C Alleviates Glucocorticoid-Induced Suppression of Osteogenic Differentiation by Modulating PI3K/AKT/RUNX2 Signaling Pathway.

Secondary osteoporosis is triggered mostly by glucocorticoid (GC) therapy. Dexamethasone (DEX) was reported to inhibit osteogenic differentiation in zebrafish larvae and MC3T3-E1 cells in prior research. In this research, we primarily examined the protective impacts of epimedin C on the osteogenic inhibition impact of MC3T3-E1 cells and zebrafish larvae mediated by DEX. The findings illustrated no apparent toxicity for MC3T3-E1 cells after administering epimedin C at increasing dosages from 1 to 60 μM and no remarkable proliferation in MC3T3-E1 cells treated using DEX. In MC3T3-E1 cells that had been treated using DEX, we discovered that epimedin C enhanced alkaline phosphatase activities and mineralization. Epimedin C could substantially enhance the protein expression of osterix (OSX), Runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALPL) in MC3T3-E1 cells subjected to DEX treatment. Additionally, epimedin C stimulated PI3K and AKT signaling pathways in MC3T3-E1 cells that had been treated using DEX. Furthermore, in a zebrafish larvae model, epimedin C was shown to enhance bone mineralization in DEX-mediated bone impairment. We also found that epimedin C enhanced ALPL activity and mineralization in MC3T3-E1 cells treated using DEX, which may be reversed by PI3K inhibitor (LY294002). LY294002 can also reverse the protective impact of epimedin C on DEX-mediated bone impairment in zebrafish larval. These findings suggested that epimedin C alleviated the suppressive impact of DEX on the osteogenesis of zebrafish larval and MC3T3-E1 cells via triggering the PI3K and AKT signaling pathways. Epimedin C has significant potential in the development of innovative drugs for the treatment of glucocorticoid-mediated osteoporosis.

Aqueous Extract of Psiloxylon mauritianum, Rich in Gallic Acid, Prevents Obesity and Associated Deleterious Effects in Zebrafish.

Obesity has reached epidemic proportions, and its prevalence tripled worldwide between 1975 and 2016, especially in Reunion Island, a French overseas region. Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island registered in the French pharmacopeia, has recently gained interest in combating metabolic disorders because of its traditional lipid-lowering and “anti-diabetic” use. However, scientific data are lacking regarding its toxicity and its real benefits on metabolic diseases. In this study, we aim to determine the toxicity of an aqueous extract of P. mauritianum on zebrafish eleutheroembryos following the OECD toxicity assay (Organization for Economic Cooperation and Development, guidelines 36). After defining a non-toxic dose, we determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) that this extract is rich in gallic acid but contains also caffeoylquinic acid, kaempferol and quercetin, as well as their respective derivatives. We also showed that the non-toxic dose exhibits lipid-lowering effects in a high-fat-diet zebrafish larvae model. In a next step, we demonstrated its preventive effects on body weight gain, hyperglycemia and liver steatosis in a diet-induced obesity model (DIO) performed in adults. It also limited the deleterious effects of overfeeding on the central nervous system (i.e., cerebral oxidative stress, blood-brain barrier breakdown, neuro-inflammation and blunted neurogenesis). Interestingly, adult DIO fish treated with P. mauritianum display normal feeding behavior but higher feces production. This indicates that the “anti-weight-gain” effect is probably due to the action of P. mauritianum on the intestinal lipid absorption and/or on the microbiota, leading to the increase in feces production. Therefore, in our experimental conditions, the aqueous extract of P. mauritianum exhibited “anti-weight-gain” properties, which prevented the development of obesity and its deleterious effects at the peripheral and central levels. These effects should be further investigated in preclinical models of obese/diabetic mice, as well as the impact of P. mauritianum on the gut microbiota.

The effect of Centella asiatica ethanolic extract on expression of glucose transporter 1 and osteocalcin on stunting larvae of Zebrafish (Danio rerio)

Stunting is known as length for age below minus two standard deviations (<-2 SD) of the WHO Child Growth Standard nutritional status. Several causes are identified, including exposure to pesticides (rotenone), which works by inhibiting mitochondrial complex I as an ATP-producing site and generate oxidative stress that affects glucose delivery and bone elongation involving GLUT 1 and osteocalcin. Centella asiatica is a high antioxidant herbal plant with anti-inflammatory properties. This study aimed to investigate the effect of Centella asiatica ethanolic extract on the rotenone-induced stunting zebrafish (Danio rerio) larvae model through GLUT 1 and osteocalcin expressions. This study found that 12.5 ppb of rotenone can induce about 6 dpf (day post-fertilization) zebrafish larvae into stunting. The effect of Centella asiatica ethanolic extract on increasing body length in stunting zebrafish larvae works by increasing the expression of GLUT 1 and osteocalcin expressions. The correlation test showed a positive and strong relationship between this ethanolic extract level with GLUT 1 and Osteocalcin expression. Thus, the Centella asiatica ethanolic extract effectively promotes the body length by elevating the expression of GLUT 1 and osteocalcin in stunting zebrafish larvae.

Pro-inflammatory cytokine molecules from Boswellia serrate suppresses lipopolysaccharides induced inflammation demonstrated in an in-vivo zebrafish larval model.

Boswellia serrate is an ancient and highly valued ayurvedic herb. Its extracts have been used in medicine for centuries to treat a wide variety of chronic inflammatory diseases. However, the mechanism by which B. serrata hydro alcoholic extract inhibited pro-inflammatory cytokines in zebrafish (Danio rerio) larvae with LPS-induced inflammation remained unknown. LC-MS analysis was used to investigate the extract's phytochemical components. To determine the toxicity of B. serrata extract, cytotoxicity and embryo toxicity tests were performed. The in-vivo zebrafish larvae model was used to evaluate the antioxidant and anti-inflammatory activity of B. serrata extract. According to an in silico study using molecular docking and ADMET, the compounds acetyl-11-keto-boswellic and 11-keto-beta-boswellic acid present in the extract had higher binding affinity for the inflammatory specific receptor, and it is predicted to be an orally active molecule. In both in-vitro L6 cells and in-vivo zebrafish larvae, 160µg/mL concentration of extract caused a high rate of lethality. The extract was found to have a protective effect against LPS-induced inflammation at concentrations ranged between 10 and 80µg/mL. In zebrafish larvae, 80µg/mL of treatment significantly lowered the level of intracellular ROS, apoptosis, lipid peroxidation, and nitric oxide. Similarly, zebrafish larvae treated with B. serrata extract (80µg/mL) showed an increased anti-inflammatory activity by lowering inflammatory specific gene expression (iNOS, TNF-α, COX-2, and IL-1). Overall, our findings suggest that B. serrata can act as a potent redox scavenger against LPS-induced inflammation in zebrafish larvae and an inhibitor of specific inflammatory genes.

Effect of Fixation Methods on Biochemical Characteristics of Green Teas and Their Lipid-Lowering Effects in a Zebrafish Larvae Model.

Fixation is a key process contributing to different flavors of green tea and pan-fire and steam were the common fixation methods applied conventionally. In this study, pan-fired green tea (PGT) and steamed green tea (SGT) produced by different fixation methods were compared in characteristic biochemicals including volatile compounds, amino acids, catechins and alkaloids, together with evaluating their tastes and lipid-lowering effects. PGT and SGT could be distinguished clearly by orthogonal partial least squares discriminant analysis (OPLS-DA) and heatmap. SGT had higher contents of volatile alcohols (44.75%) with green and floral attributes, while PGT had higher contents of volatile esters (22.63%) with fruity and sweet attributes. Results of electronic tongue analysis showed that PGT and SGT had similar taste of strong umami and sweetness, but little astringency and bitterness. In addition, amino acids were more abundant in PGT (41.47 mg/g in PGT, 33.79 mg/g in SGT), and catechins were more abundant in SGT (111.36 mg/g in PGT, 139.68 mg/mg in SGT). Zebrafish larvae high-fat model was applied to study the lipid-lowering effects of PGT and SGT. Results showed that both SGT and PGT had lipid-lowering effects, and the lipid level was decreased to 61.11 and 54.47% at concentration of 300 mg/L compared to high-fat group, respectively. Generally, different fixation methods of pan-fire and steam showed significant effects on aroma and contents of characteristic chemical compounds (amino acids and catechins) of green tea, but no effects on the taste and lipid-lowering activity.

Anti-osteoporosis effects of Anemarrhenae Rhizoma / Phellodendri Chinensis Cortex herb pair and its major active components in diabetic rats and zebrafish

Ethnopharmacological relevanceAnemarrhenae Rhizoma/Phellodendri Chinensis Cortex (AR/PCC) herb pair has been widely used in traditional Chinese medicines for the treatment of diabetic osteoporosis. However, the anti-diabetic osteoporotic active components of AR/PCC remain unclear. This study aimed to explore the major active ingredients in AR/PCC for its protective effects against bone deterioration induced by diabetes. Materials and methodsThe aqueous extracts of AR/PCC with different proportions (AR:PCC = 1:3, 1:2, 1:1, 2:1 and 3:1, w/w) were prepared. Streptozotocin-induced diabetic rats were orally administrated with the AR/PCC extracts. The absorbed phytochemical compounds in serum of diabetic rats were identified by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry method and their contents in the AR/PCC extracts were determined by high performance liquid chromatography-ultraviolet detector-evaporative light scattering detector method. The absorbed compounds in the extracts were considered as the major potential active components in AR/PCC, and their combination was defined as M-AR/PCC. A component-knockout approach was applied to evaluate the contribution of each compound in M-AR/PCC. The larvae and adults of diabetic zebrafish models were then used to evaluated the anti-diabetic osteoporotic performance of the M-AR/PCC. The real-time reverse transcription polymerase chain reaction technique was applied to study the regulation effects of M-AR/PCC on osteogenesis and osteoclastgensis in diabetic zebrafish models. ResultsThe phenotypes of diabetic osteoporosis rats induced by streptozotocin were reversed by the oral administration of AR/PCC extracts with different ratios, as evidenced by the increased bone mineral density, bone volume density, trabecular thickness, trabecular number, and decreased trabecular separation of femoral metaphysis. Seven phytochemical compounds were detected in the serum and their contents in AR/PCC varied dramatically with different proportions, including 1 xanthone glycoside and 6 alkaloids. By using diabetic zebrafish larvae model and compound-knockout strategy, each compound in M-AR/PCC were proved to play an indispensable role in the positive regulatory actions in the bone mass of diabetic zebrafish. Furthermore, the herb pair with a ratio of 1:1 and the related M-AR/PCC showed the best therapeutic effects on diabetic osteoporosis. They showed similar performances on the inhibition of the tartrate-resistant acid phosphatase activity and the promotion of the alkaline phosphatase activity in diabetic adult zebrafish model. The M-AR/PCC treatment could decrease the blood glucose, upregulate the mRNA expression levels of osteoblast-related genes (alp, runx2b and opg) and downregulate the expression of osteoclast-related genes (acp5α, rankl and sost) in streptozotocin-induced zebrafish. ConclusionAR/PCC herb pair and its major active components possess potent anti-diabetic osteoporotic effect on streptozotocin-induced in vivo models. The combination of the seven active compounds derived from AR/PCC herbal pair could be a potential agent for protection against osteoporosis associated with diabetes.

Alleviative effects of a novel strain Bacillus coagulans XY2 on copper-induced toxicity in zebrafish larvae

Copper (Cu) is a kind of micronutrient element that is essential for human metabolism. However, it is also considered as an environmental pollutant which is toxic to organisms at a high concentration level. Probiotics, regarded as beneficial microorganisms for promoting human health, have functions of antioxidant capacity, immune-enhancing properties, intestinal barrier protection and regulation. Several studies have reported that probiotics show positive effects on alleviating and intervening heavy metals toxicity. However, evidence for relieving copper-induced toxicity by probiotics is still limited. In this study, we firstly conducted a zebrafish larvae model to screen out microorganisms which are helpful for CuSO4 toxicity resistance and one novel strain named as Bacillus coagulans XY2 was discovered with the best protective activity. B. coagulans XY2 significantly reduced the mortality of zebrafish larvae exposed to 10 µmol/L CuSO4 for 96 hr, as well as alleviated the neutrophils infiltration in the larvae lateral line under a 2 hr exposure. B. coagulans XY2 exhibited a high in vitro antioxidant activity and against CuSO4-induced oxidative stress in zebrafish larvae by up-regulating sod1, gstp1 and cat gene transcriptional levels and relevant enzymatic activities. CuSO4 stimulated the inflammation process resulting in obvious increases of gene il-1β and il-10 transcription, which were suppressed by B. coagulans XY2 intervention. Overall, our results underline the bio-function of B. coagulans XY2 on protecting zebrafish larvae from copper toxicity, suggesting the potential application values of probiotics in copper toxicity alleviation on human and the environment.

Reviewing toxicokinetics with a focus on metabolism of new psychoactive substances in the zebrafish (larvae) model

AbstractZebrafish (Danio rerio) share around 70% of their genome with humans and many important enzymes for drug metabolism such as some cytochrome p450s have direct orthologs in zebrafish. To date, several studies showed a similar metabolism to humans in general. Furthermore, although using adult fish as model organism requires approval by an Ethics Committee, using larvae until 120 h postfertilization does not necessarily need any approval at least in the European Union. All these aspects seem to be beneficial for using zebrafish (larvae) in toxicokinetic studies for the so‐called new psychoactive substances. These compounds are expected to have similar effects as traditional drugs of abuse but are often not listed as controlled drugs when they first appear on the market. However, no information about their toxicokinetics is available when they appear, which is particularly critical concerning their biotransformation. This knowledge is important for example, for developing urine‐based screening procedures or for predicting drug–drug interactions. This focus article aims to briefly introduce into the topic of using zebrafish (larvae) in the context of toxicokinetic studies, particularly metabolism studies, and will highlight some aspects such as the route of administration, which are important to consider when using this model.This article is categorized under: Toxicology > New Psychoactive Substances Toxicology > Analytical

GR15 peptide of S-adenosylmethionine synthase (SAMe) from Arthrospira platensis demonstrated antioxidant mechanism against H2O2 induced oxidative stress in in-vitro MDCK cells and in-vivo zebrafish larvae model

GR15 is a short molecule or peptide composed of aliphatic amino acids and possesses to have antioxidant properties. The GR15, 1GGGAFSGKDPTKVDR15 was identified from the protein S-adenosylmethionine synthase (SAMe) expressed during the sulfur departed state of Arthrospira platensis (spirulina or cyanobacteria). The in-silico assessment and the structural features of GR15 showed its antioxidant potency. Real-time PCR analysis found the up-regulation of ApSAMe expression on day 15 against oxidative stress due to 10 mM H2O2 treatment in A. platensis (Ap). The antioxidant activity of GR15 was accessed by the cell-free antioxidant assays such as ABTS, SARS, HRAS and NO; the results showed dose-dependent antioxidant activity. The toxicity assay was performed in both in vitro and in vivo models, in which peptide does not exhibit any toxicity in MDCK cell and zebrafish embryos. The intercellular ROS reduction potential of GR15 peptide was also investigated in both in vitro and in vivo models including LDH assay, antioxidant enzymes (SOD and CAT), and fluorescent staining assay (DCFDA, Hochest and Acridine orange sting) was performed; the results showed that the GR15 peptide was effectively reduced the ROS level. Further, RT-PCR demonstrated that GR15 enhanced the antioxidant property and also up-regulated the antioxidant gene, thus reduced the ROS level in both in vitro and in vivo models. Based on the results obtained from this study, we propose that GR15 has the potential antioxidant ability; hence further research can be directed towards the therapeutic product or drug development against disease caused by oxidative stress.

Kaempferol Ameliorates the Inhibitory Activity of Dexamethasone in the Osteogenesis of MC3T3-E1 Cells by JNK and p38-MAPK Pathways

Kaempferol has been reported to exhibit beneficial effect on the osteogenic differentiation in mesenchymal stem cells (MSC) and osteoblasts. In our previous study, dexamethasone (DEX) demonstrated inhibitory effect on MC3T3-E1 cells differentiation. In this study, we mainly explored the protective effect of kaempferol on the inhibitory activity of DEX in the osteogenesis of MC3T3-E1 cells. We found that kaempferol ameliorated the proliferation inhibition, cell cycle arrest, and cell apoptosis and increased the activity of alkaline phosphatase (ALP) and the mineralization in DEX-treated MC3T3-E1 cells. Kaempferol also significantly enhanced the expression of osterix (Osx) and runt-related transcription factor 2 (Runx2) in MC3T3-E1 cells treated with DEX. In addition, kaempferol attenuated DEX-induced reduction of cyclin D1 and Bcl-2 expression and elevation of p53 and Bax expression. Kaempferol also activated JNK and p38-MAPK pathways in DEX-treated MC3T3-E1 cells. Furthermore, kaempferol improved bone mineralization in DEX-induced bone damage in a zebrafish larvae model. These data suggested that kaempferol ameliorated the inhibitory activity of DEX in the osteogenesis of MC3T3-E1 cells by activating JNK and p38-MAPK signaling pathways. Kaempferol exhibited great potentials in developing new drugs for treating glucocorticoid-induced osteoporosis.

Amberlyst-15 catalysed synthesis of novel indole derivatives under ultrasound irradiation: Their evaluation as serotonin 5-HT2C receptor agonists

A series of indole based novel Schiff bases was designed as potential agonists of 5-HT2C receptor that was supported by docking studies in silico. These compounds were synthesized via Amberlyst-15 catalysed condensation of an appropriate pyrazole based primary amine with the corresponding indole-3-aldehyde under ultrasound irradiation at ambient temperature. A number of target Schiff bases were obtained in good yields (77–87%) under mild conditions within 1 h. Notably, the methodology afforded the corresponding pyrazolo[4,3-d]pyrimidin-7(4H)-one derivatives when the primary amine was replaced by a secondary amine. Several Schiff bases showed agonist activity when tested against human 5-HT2C using luciferase assay in HEK293T cells in vitro. The SAR (Structure-Activity-Relationship) studies suggested that the imine moiety was more favorable over its cyclic form i.e. the corresponding pyrazolopyrimidinone ring. The Schiff bases 3b (EC50 1.8 nM) and 3i (EC50 5.7 nM) were identified as the most active compounds and were comparable with Lorcaserin (EC50 8.5 nM). Also like Lorcaserin, none of these compounds were found to be PAM of 5-HT2C. With ∼24 and ∼150 fold selectivity towards 5-HT2C over 5-HT2A and 5-HT2B respectively the compound 3i that reduced locomotor activity in zebrafish (Danio rerio) larvae model emerged as a promising hit molecule for further study.

Anti-seizure activity of African medicinal plants: The identification of bioactive alkaloids from the stem bark of Rauvolfia caffra using an in vivo zebrafish model

Ethnopharmacological relevanceEpilepsy is one of the major chronic diseases that does not have a cure to date. Adverse drug reactions have been reported from the use of available anti-epileptic drugs (AEDs) which are also effective in only two-thirds of the patients. Accordingly, the identification of scaffolds with promising anti-seizure activity remains an important first step towards the development of new anti-epileptic therapies, with improved efficacy and reduced adverse effects. Herbal medicines are widely used in developing countries, including in the treatment of epilepsy but with little scientific evidence to validate this use. In the search for new epilepsy treatment options, the zebrafish has emerged as a chemoconvulsant-based model for epilepsy, mainly because of the many advantages that zebrafish larvae offer making them highly suitable for high-throughput drug screening. Aim of the studyIn this study, 20 medicinal plants traditionally used in South Africa to treat epilepsy were screened for anti-epileptic activity using a zebrafish larvae model. Materials and methodsToxicity triaging was conducted on 120 crude extracts, 44 fractions and three isolated compounds to determine the maximum tolerated concentration (MTC) of each extract, fraction or compound. MTC values were used to guide the concentration range selection in bioactivity studies. The effectiveness of crude extracts, fractions and isolated compounds from Rauvolfia caffra Sond. in suppression of pentylenetetrazole (PTZ) induced seizure-like behaviour in a 6-dpf zebrafish larvae model was measured using the PTZ assay. ResultsFollowing a preliminary toxicity triage and bioactivity screen of crude extracts from 20 African plants used traditionally for the treatment and management of epilepsy, the methanolic extract of Rauvolfia caffra Sond. was identified as the most promising at suppressing PTZ induced seizure-like behaviour in a zebrafish larvae model. Subsequent bioactivity-guided fractionation and spectroscopic structural elucidation resulted in the isolation and identification of two tryptoline derivatives; a previously unreported alkaloid to which we assigned the trivial name rauverine H (1) and the known alkaloid pleiocarpamine (2). Pleiocarpamine was found to reduce PTZ-induced seizures in a dose-dependent manner. ConclusionsAccordingly, pleiocarpamine represents a promising scaffold for the development of new anti-seizure therapeutic compounds. Furthermore, the results of this study provide preliminary evidence to support the traditional use of Rauvolfia caffra Sond. in the treatment and management of epilepsy. These findings warrant further studies on the anti-epileptic potential of Rauvolfia caffra Sond. using other models.

Synthetic resin acid derivatives selectively open the hKV 7.2/7.3 channel and prevent epileptic seizures.

About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hKV ) channel hKV 7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hKV 7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hKV 7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect. Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model. Fourteen resin acid derivatives were tested on hKV 7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hKV 7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hKV 7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle-relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hKV 11.1, hNaV 1.5, or hCaV 1.2, or on the amplitude of hKV 11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model. The described resin acid derivatives hold promise for new antiseizure medications, with reduced risk for adverse effects compared with retigabine.

Aggressiveness and Metastatic Potential of Breast Cancer Cells Co-Cultured with Preadipocytes and Exposed to an Environmental Pollutant Dioxin: An in Vitro and in Vivo Zebrafish Study.

Background:Breast cancer (BC) is a major public health concern, and its prognosis is very poor once metastasis occurs. The tumor microenvironment and chemical pollution have been suggested recently to contribute, independently, to the development of metastatic cells. The BC microenvironment consists, in part, of adipocytes and preadipocytes in which persistent organic pollutants (POPs) can be stored.Objectives:We aimed to test the hypothesis that these two factors (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extensively studied, toxic POP and the microenvironment) may interact to increase tumor aggressiveness.Methods:We used a co-culture model using BC MCF-7 cells or MDA-MB-231 cells together with hMADS preadipocytes to investigate the contribution of the microenvironment and 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD on BC cells. Global differences were characterized using a high-throughput proteomic assay. Subsequently we measured the BC stem cell–like activity, analyzed the cell morphology, and used a zebrafish larvae model to study the metastatic potential of the BC cells.Results:We found that coexposure to TCDD and preadipocytes modified BC cell properties; moreover, it induced the expression of ALDH1A3, a cancer stem cell marker, and the appearance of giant cancer cells with cell-in-cell structures (CICs), which are associated with malignant metastatic progression, that we demonstrated in vivo.Discussion:The results of our study using BC cell lines co-cultured with preadipocytes and a POP and an in vivo zebrafish model of metastasis suggest that the interactions between BC cells and their microenvironment could affect their invasive or metastatic potential. https://doi.org/10.1289/EHP7102

Coumarins from Seseli devenyense Simonk.: Isolation by Liquid-Liquid Chromatography and Potential Anxiolytic Activity Using an In Vivo Zebrafish Larvae Model.

Different types of anxiety disorders have become the number one mental health issue in developed countries. The search for new, safer and effective drug-like molecules among naturally derived substances faces two difficulties: an efficient method of isolation compounds with a high-purity and high-throughput animal model for activity assay. Thus, the aim of the present study was to isolate by liquid–liquid chromatography high-purity rare coumarins from the fruits of Seseli devenyense Simonk. and evaluate their anxiolytic effect (defined as reversed thimotaxis) using a 5-days post-fertilization (dpf) Danio rerio larvae model. Liquid–liquid chromatography enabled the isolation of one simple hydroxycoumarin (devenyol) and four pyranocoumarins (cis-khellactone, d-laserpitin, isolaserpitin and octanoyllomatin). The anxiolytic effect was defined as a decrease in the time spent in the boundaries of the living space (also described as reversed thigmotaxis). Our results show that all isolated courmarins exerted a significant influence on the anxiety behavior (anxiolytic activity) in the zebrafish larvae model. According to our knowledge, this is the first report of anxiolytic activity of pyranocoumarins and devenyol.