[Systematic comparison of two kinds of Bufonis Venenum derived from different Bufo gargarizans subspecies based on metabolomics and antitumor activity].

This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.

Developmental neurotoxicity and toxic mechanisms induced by olaquindox in zebrafish.

Olaquindox (OLA) has been widely used as an animal feed additive in China for decades; however, its toxicity and toxic mechanisms have not been well investigated. In this study, the developmental neurotoxicity and toxic mechanisms of OLA were evaluated in zebrafish. Zebrafish embryos were exposed to different concentrations of OLA (25-1,000 mg/L) from 6 to 120 hours post fertilization (hpf). OLA exposure resulted in many abnormal phenotypes in zebrafish, including shortened body length, notochord degeneration, spinal curvature, brain apoptosis, damage of axon and peripheral motor neuron, and hepatotoxicity. Interestingly, OLA increased zebrafish spontaneous tail coiling, while reduced locomotor capacity. Quantitative polymerase chain reaction (Q-PCR) showed that the expression levels of nine marker genes for nervous system functions or development, namely, α1-tubulin, glial fibrillary acidic protein (gfap), myelin basic protein (mbp), synapsinII a (syn2a), sonic hedgehog a (shha), encoding HuC (elavl3), mesencephalic astrocyte-derived neurotrophic factor (manf) growth associated protein 43 (gap43), and acetylcholinesterase (ache) were all down-regulated significantly in zebrafish after treated with OLA. Besides, the anti-apoptotic and pro-apoptotic genes bcl-2/bax ratio was reduced. These results show that OLA exposure could cause severe developmental neurotoxicity in the early stages of zebrafish life and OLA might induce neurotoxicity by inhibiting the expression of neuro-developmental genes and promoting apoptosis.

A natural complex product Yaocha reduces uric acid level in a live zebrafish model.

INTRODUCTION: This study was aimed to assess uric acid (UA)-lowering effect and its possible mechanisms of a natural complex product Yaocha in a live zebrafish model. METHODS: The zebrafish high UA model was established by feeding 5 dpf zebrafish with both an uricase inhibitor potassium oxonate at 10 mM and an UA synthesis precursor xanthine sodium at 0.5 mM for 24 h. Yaocha was administered to the high UA zebrafish through soaking at 3 various concentrations, with allopurinol as a positive control. UA level, xanthine oxidase (XOD) activity, and mRNA expression of hypoxanthine guanine-phosphoribosyltransferases transferase (HPRT1) and organic anion transporter 1 (OAT1) were measured. RESULTS: Yaocha effectively reduced UA level and inhibited xanthine oxidase (XO) activity in the high UA zebrafish. Yaocha could be a potential therapeutics for hyperuricemia through up-regulating HPRT1 and OAT1 gene expression and suppressing XO activity. DISCUSSION: These results suggested that Yaocha hold a potential for high UA prevention and therapy, possibly through inhibiting UA production and promoting urate secretion and purine conversion.

Development of zebrafish demyelination model for evaluation of remyelination compounds and RORγt inhibitors.

RAR-related orphan receptor-γt (RORγt) directs differentiation of proinflammatory T helper 17 cells and is a potential therapeutic target for chronic autoimmune and inflammatory diseases including multiple sclerosis. In this study, zebrafish at days post fertilization treated with ethidium bromide (EB) at a concentration of 75 µM for 72 h were determined as the optimum conditions for the demyelination model development. Zebrafish motility was recorded automatically using a video-track motion detector and quantitative myelin assay was measured by FluoroMyelin staining. A well-known remyelination agent thyroxine (T4) was tested to confirm whether EB-induced motility and myelin damage could be rescued. Two RORγt lead inhibitors GSK805 and SR1001 were assessed for their therapeutic effects on remyelination, axon regeneration, motor neuron promotion and anti-inflammation. T4 significantly improved EB-induced motility dysfunction and myelin damage and promoted myelin basic protein (MBP) regeneration in the demyelinated zebrafish. GSK805 and SR1001 enhanced remyelination in a dose-dependent manner and promoted MBP regeneration. Both GSK805 and SR1001 markedly recovered EB-induced axon and motor neuron damage, and exhibited significantly inhibitory effects of neutrophil infiltration and macrophage recruitment. These results indicate that EB treatment can induce zebrafish demyelination; and the zebrafish demyelination model in combination with quantitative motility and myelin assays is a predictive, reproducible and relatively high throughput screening for rapidly in vivo identification of remyelination compounds and RORγt inhibitors.

A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

Heart failure is a leading cause of death and the development of effective and safe therapeutic agents for heart failure has been proven challenging. In this study, taking advantage of larval zebrafish, we developed a zebrafish heart failure model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days postfertilization) were treated with verapamil at a concentration of 200 µM for 30 min, which were determined as optimum conditions for model development. Tested drugs were administered into zebrafish either by direct soaking or circulation microinjection. After treatment, zebrafish were randomly selected and subjected to either visual observation and image acquisition or record videos under a Zebralab Blood Flow System. The therapeutic effects of drugs on zebrafish heart failure were quantified by calculating the efficiency of heart dilatation, venous congestion, cardiac output, and blood flow dynamics. All 8 human heart failure therapeutic drugs (LCZ696, digoxin, irbesartan, metoprolol, qiliqiangxin capsule, enalapril, shenmai injection, and hydrochlorothiazide) showed significant preventive and therapeutic effects on zebrafish heart failure (p < 0.05, p < 0.01, and p < 0.001) in the zebrafish model. The larval zebrafish heart failure model developed and validated in this study could be used for in vivo heart failure studies and for rapid screening and efficacy assessment of preventive and therapeutic drugs.

A Zebrafish Thrombosis Model for Assessing Antithrombotic Drugs.

Thrombosis is a leading cause of death and the development of effective and safe therapeutic agents for thrombotic diseases has been proven challenging. In this study, taking advantage of the transparency of larval zebrafish, we developed a larval zebrafish thrombosis model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days post fertilization) were treated with phenylhydrazine (PHZ) and a testing drug for 24 h. Tested drugs were administered into the zebrafish either by direct soaking or circulation microinjection. Antithrombotic efficacy was quantitatively evaluated based on our previously patented technology characterized as an image analysis of the heart red blood cells stained with O-dianisidine staining. Zebrafish at 2 dpf treated with PHZ at a concentration of 1.5 µM for a time period of 24 h were determined as the optimum conditions for the zebrafish thrombosis model development. Induced thrombosis in zebrafish was visually confirmed under a dissecting stereomicroscope and quantified by the image assay. All 6 human antithrombotic drugs (aspirin, clopidogrel, diltiazem hydrochloride injection, xuanshuantong injection, salvianolate injection, and astragalus injection) showed significant preventive and therapeutic effects on zebrafish thrombosis (p < 0.05, p < 0.01, & p < 0.001) in this zebrafish thrombosis model. The larval zebrafish thrombosis model developed and validated in this study could be used for in vivo thrombosis studies and for rapid screening and efficacy assessment of antithrombotic drugs.

Bioactive C21 Steroidal Glycosides from the Roots of Cynanchum otophyllum That Suppress the Seizure-like Locomotor Activity of Zebrafish Caused by Pentylenetetrazole.

Six new C21 steroidal glycosides, cynotophyllosides A-F (1-6), together with 16 known compounds, were isolated from the roots of Cynanchum otophyllum. The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. The three major components, otophylloside F (15), otophylloside B (17), and rostratamine 3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside (18), suppressed the seizure-like locomotor activity caused by pentylenetetrazole in zebrafish. Preliminary structure-activity relation studies revealed that a pregnene skeleton with a C-12 ester group (ikemaoyl > cinnamoyl > hydroxy > p-hydroxybenzoyl) and a C-3 sugar chain consisting of three 2,6-dideoxysaccharide units is essential for this suppressive activity.

Rapid analysis of hypolipidemic drugs in a live zebrafish assay.

INTRODUCTION: Hyperlipidemia is the most common form of dyslipidemia, which is the key risk factor for cardiovascular disease and stroke. The development of effective and safe drug treatments for hyperlipidemia has been proven challenging. METHODS: In this study, taking advantage of the transparency of larval zebrafish, we developed a zebrafish hyperlipidemia model for drug screening and efficacy assessment. Zebrafish at 5 d.p.f (days post fertilization) were fed with 0.1% egg yolk for 48 h (hours), followed by drug treatment for 24h or 48 h. Tested drugs were administered into the zebrafish by direct soaking. Drug effect was evaluated based on quantitative analysis of Oil Red O (ORO) in zebrafish vena caudalis. RESULTS: All 5 human hypolipidemic drugs (simvastatin, lovastatin, ezetimibe, bezafibrate and hyodesoxycholic acid) showed significant hypolipidemic effects (p<0.01) in a dose-dependent manner in the zebrafish hyperlipidemia model. 'We also found a well-known Chinese tea Pu-erh tea significantly reduced lipids in this model (p<0.001 and p<0.01). DISCUSSION: Our results demonstrate that the zebrafish hyperlipidemia model developed and validated in this study could be used for in vivo hyperlipidemia studies and drug screening and for assessing hypolipidemic drugs with different mechanisms.

A zebrafish phenotypic assay for assessing drug-induced hepatotoxicity.

INTRODUCTION: Numerous studies have confirmed that zebrafish and mammalian toxicity profiles are strikingly similar and the transparency of larval zebrafish permits direct in vivo assessment of drug toxicity including hepatotoxicity in zebrafish. METHODS: Hepatotoxicity of 6 known mammalian hepatotoxic drugs (acetaminophen [APAP], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. Zebrafish liver degeneration was originally screened visually, quantified using an image-based morphometric analysis and confirmed by histopathology. RESULTS: All the tested mammalian hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish, whereas the non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% (8/8) as compared with mammalian results, suggesting that hepatotoxic drugs in mammals also caused similar hepatotoxicity in zebrafish. DISCUSSION: Larval zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity. This convenient, reproducible animal model saves time and money for drug discovery and can serve as an intermediate step between cell-based evaluation and conventional animal testing of hepatotoxicity.