Phenolic constituents with anti-inflammatory and cytotoxic activities from the rhizomes of Iris domestica.

Four undescribed flavonoid glucosides (iridins B-C, tectoridin A and ampelopsinin A); one undescribed phenolic glucoside (diplostephioside B); one undescribed phenolic compound (phenanthrenetriol A); and seventeen known compounds were isolated from the rhizomes of Iris domestica. The chemical structures of the undescribed compounds were established by spectroscopic/spectrometric data interpretation using HRESIMS, NMR, and ECD. Tectoridin A, nigricin A and naringenin exhibited anti-inflammatory activities with inhibition rates of 53.71%, 57.68% and 88.71%, respectively, against the NF-κB signaling pathway at a concentration of 10 µM. 4'-O-methylnyasol (10 µM) exhibited 84.91% antiproliferative activity against the K562 human leukemia cell line with an IC50 value of 4.20 µM.

New stilbenes and phenolic constituents from the rhizomes of Belamcanda chinensis.

A pair of stilbenes with γ-lactam unit [(+)-1 and (-)-1], a new phenolic glucoside (2), and a new isoflavone glucoside (3), together with two known compounds (4-5) were isolated from the rhizomes of Belamcanda chinensis. The chemical structures of the undescribed compounds were elucidated on the basis of detailed spectroscopic analyses. Compounds 1, 4, and 5 (10 µM) exhibited anti-inflammatory activities with inhibition rates of 30.46%, 60.34%, and 37.91%, respectively, against the NF-κB signaling pathway.

Validation, Optimization, and Application of the Zebrafish Developmental Toxicity Assay for Pharmaceuticals Under the ICH S5(R3) Guideline.

The zebrafish as an alternative animal model for developmental toxicity testing has been extensively investigated, but its assay protocol was not harmonized yet. This study has validated and optimized the zebrafish developmental toxicity assay previously reported by multiple inter-laboratory studies in the United States and Europe. In this study, using this classical protocol, of 31 ICH-positive compounds, 23 compounds (74.2%) were teratogenic in zebrafish, five had false-negative results, and three were neither teratogenic nor non-teratogenic according to the protocol standard; of 14 ICH-negative compounds, 12 compounds (85.7%) were non-teratogenic in zebrafish and two had false-positive results. After we added an additional TI value in the zebrafish treated with testing compounds at 2 dpf along with the original 5 dpf, proposed a new category as the uncategorized compounds for those TI values smaller than the cutoff both at 2 dpf and 5 dpf but inducing toxic phenotypes, refined the testing concentration ranges, and optimized the TI cut-off value from ≥ 10 to ≥ 3 for compounds with refined testing concentrations, this optimized zebrafish developmental assay reached 90.3% sensitivity (28/31 positive compounds were teratogenic in zebrafish) and 88.9% (40/45) overall predictability. Our results from this study strongly support the use of zebrafish as an alternative in vivo method for screening and assessing the teratogenicity of candidate drugs for regulatory acceptance.

Probiotics Modulate Intestinal Motility and Inflammation in Zebrafish Models.

This study was aimed to assess effects of three strains of probiotics Lactobacillus acidophilus NCFM, Lactobacillus rhamnosus HN001, and Bifidobacterium animalis subsp. lactis Bi-07 on the intestinal motility and inflammation in the zebrafish models. The intestinal motility model was established using 5 days postfertilization (dpf) zebrafish administered with a fluorescent dye Nile red at 10 ng/mL for 16 h, followed by probiotics treatment for 24 h and the intestinal motility was inversely proportional to the intestinal fluorescence intensity that was quantitatively measured by image analysis. The intestinal inflammation was induced by treating 3 dpf neutrophil fluorescent zebrafish with 0.0125% of trinitrobenzenesulfonic acid for 48 h. Probiotics were administered at low, moderate, and high concentrations determined based on maximum tolerable concentration through soaking. All three strains of probiotics promoted intestinal movement, of which B. animalis subsp. lactis Bi-07 was most potent at lower concentrations. L. rhamnosus HN001 and B. animalis subsp. lactis Bi-07 had the therapeutic effects on the intestinal inflammation and the inflammation-associated mucosal damage recovery. The anti-inflammatory mechanisms of L. rhamnosus HN001 was related to both reduce inflammatory factor interleukin-6 (IL-6) and restored tissue repair factor transforming growth factor-ß-1 (TGFß-1); whereas B. animalis subsp. lactis Bi-07 was probably only associated with TGFß-1 elevation. Using larval zebrafish models for probiotics screening and assessment would speed up product research and development and improve products' efficacy and quality.

Ponatinib-induced ischemic stroke in larval zebrafish for drug screening.

Conventional mammalian ischemic stroke models for drug screening are technically challenging, laborious and time-consuming. In this study, using Ponatinib as an inducer, we developed and characterized a zebrafish ischemic stroke model. This zebrafish ischemic stroke had the cerebral vascular endothelial injury, thrombosis, reduced blood flow, inflammation and apoptosis as well as the reduced motility. The zebrafish ischemic stroke model was validated with 6 known human therapeutic drugs of ischemic stroke (Aspirin, Clopidogrel, Naoxintong capsules, Edaravone, Xingnaojing injection, Shuxuening injection). The mRNA levels of the neovascularization-related gene (vegfaa) and vascular endothelial growth factor receptor gene (VEGFR), neurodevelopment related genes (mbp and α1-tubulin), brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) were significantly downregulated; whereas apoptosis-related genes (caspase-3, caspase-7, caspase-9 and bax/bcl-2), and inflammatory factor genes (IL-1ß, IL-6, IL-10, TNF-α and NF-κB) were remarkably upregulated in the model. These results suggest that the pathophysiology of Ponatinib-induced zebrafish ischemic stroke is similar to that of human ischemic stroke patients and this whole animal model could be used to study the complex cellular and molecular pathogenesis of ischemic stroke and to rapidly identify therapeutic agents.

Fenobucarb-induced developmental neurotoxicity and mechanisms in zebrafish.

Fenobucarb (2-sec-butylphenyl methylcarbamate, BPMC) is an extensively used carbamate insecticide. Its developmental neurotoxicity and the underlying mechanisms have not been well investigated. In this study, zebrafish embryos were exposed to various concentrations of BPMC from 6 hpf (hours post fertilization, hpf) to 120 hpf. BPMC induced developmental toxicity with reduced motility in larval zebrafish. The spinal cord neutrophil infiltration, increased ROS production, caspase 3 and 9 activation, central nerve and peripheral motor neuron damage, axon and myelin degeneration were observed in zebrafish treated with BPMC generally in a dose-dependent manner. The expression of eight marker genes for nervous system function or development, namely, a1-tubulin, shha, elavl3, gap43, syn2a, gfap, mbp and manf, was significantly downregulated following BPMC exposure. AChE activity reduction and ache gene expression suppression was also found significantly in BPMC-treated zebrafish. These results indicate that BPMC is highly toxic to zebrafish and that BPMC induces zebrafish developmental neurotoxicity through pathways involved in inflammation, oxidative stress, degeneration and 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.

[The prevent miscarriage effects of Yun Kang oral liquid on threatened abortion rat induced by kidney deficiency and luteum inhibition].

OBJECTIVE: Yun Kang oral liquid is a listed proprietary Chinese Medicine. To further evaluate its efficacy, this experiment established a kidney deficiency and luteum inhibition threatened abortion rat model to observe the effects of Yun Kang oral liquid. METHODS: Sixty pregnant rats were randomly divided into normal control group (NC), model group (MG), dydrogesterone group (DT, 3.02 mg/kg), and Yun Kang oral liquid low-dose group (YK-L, 4 ml/kg), medium dose group (YK-M, 6 ml/kg), high dose group (YK-H, 9 ml/kg), 10 in each group. On the first day of pregnancy, each administration group was treated with the test drug at the prescribed dose every morning, and the NC group and the MG group were given an equal volume of purified water for 10 days; the rats were intragastrically administrated every afternoon, except for the NC group. In addition, the other groups were intragastrically administered with hydroxyurea at a dose of 450 mg/kg for 9 days, and mifepristone was administered at a dose of 4.0 mg/kg on the 10th day. On the 9th day of pregnancy, behavioral signs such as back temperature, grasping force, pain threshold, and autonomic activity were measured in each group. On the 11th day of pregnancy, blood was collected from the abdominal aorta in each group to determine serum levels of estradiol (E2) , progesterone (P) and thromboxane B2 (TXB2) . Ovary and fetal uterus were removed, the number and diameter of embryos were observed, and the ovary and uterus indexes were calculated. RESULTS: Compared with the NC group, the back temperature, grip, pain threshold, number of spontaneous activities, number of embryos, embryo diameter, uterus index and serum E2, P, TXB2 levels in the MG group were decreased significantly (P＜0.05, 0.01). Compared with the MG group, the back temperature, grasping force, number of embryos, embryo diameter and serum E2 and P levels were increased significantly in each dose group (P＜0.05, 0.01); the pain threshold, autonomic activity, and uterus index of YK-M and YK-H group were increased significantly (P＜0.05); serum level of TXB2 in YK-H group were increased significantly (P＜0.05). CONCLUSION: Yun Kang oral liquid has a clear kidney-filling effect on rats with threatened abortion caused by kidney deficiency-luteal suppression. The mechanism may be related to raising serum E2, P, TXB2 levels, improving kidney deficiency and improving embryo quality.

[Effects of Yun Kang oral liquid on rats model of embryo implantation disorder].

OBJECTIVE: Through the establishment of abortion model caused by embryo implantation difficulties, exploring the role of Yun Kang oral liquid in protecting embryos. METHODS: The pregnant rats were divided into 6 groups:normal control group (NC), model group (MG), dydrogesterone group (DT), and three dose groups of low, medium and high levels of Yun Kang oral liquid (YK-L, YK-M, YK-H), 11 in each group.From the first day of pregnancy, daily intragastric administration, the dose of DT group was 3.02 mg/kg, and the doses of Yun Kang oral liquid were 4, 6, and 9 ml/kg, respectively.The rats in NC and MG were treated with an equal volume of purified water for 10 days.On the third day of pregnancy, except for the NC group, the other groups were injected with mifepristone subcutaneously at the back of the neck at a dose of 5 mg/kg to cause an embryo implantation barrier model.On the 10th day of pregnancy, blood was collected from the abdominal aorta in each group.Serum follicle stimulating hormone (FSH), interferon-γ (IFN-γ) and interleukin (IL-4) were measured by enzyme-linked immunosorbent assay.The number of embryo implantation was observed in the uterus, and the pathological changes of the uterus were observed by HE staining. RESULTS: Compared with the NC group, the number of embryo implantation and the serum levels of FSH and IL-4 in the MG group were decreased significantly (P< 0.05, 0.01), and pathological changes such as uterine glandular epithelial hyperplasia and inflammatory cell infiltration in the glandular cavity were observed.Compared with MG group, the number of embryo implantation and serum FSH and IL-4 levels of rats in YK-M and YK-H groups were increased significantly (P<0.05, 0.01).The pathological changes such as uterine glandular epithelial hyperplasia and inflammatory cell infiltration in the gland were also improved.There was no significant difference in serum IFN-γ levels between the groups. CONCLUSIONS: Yun Kang oral liquid may improve the endometrial pathological changes and increase the number of embryo implantation by increasing the levels of serum sex hormone FSH and immune cytokine IL-4 in embryo implantation impediment rats.