Application of Mycobacterium marinum infected zebrafish for evaluation of anti-tuberculosis drugs / 药学学报

Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium. tuberculosis. In recent years, with the emergence of drug-resistant forms, the development of new anti-tuberculosis drugs is urgently needed. In this study, we used Mycobacterium marinum (M. marinum), which is highly similar to M. tuberculosis, to establish a M. marinum infected-zebrafish model and quantitative PCR (qPCR) method for bacterial count analysis. The results showed that injecting M. marinum into the yolk sac is an efficient and convenient way to infect zebrafish embryos. By counting the survival rate of infected zebrafish and the number of bacteria in zebrafish by Ziehl-Neelsen staining, we analyzed the efficacy of isoniazid and rifampicin as anti-tuberculosis drugs and the synergistic effect of drugs. The results suggested that three evaluation methods exhibit good consistency. This study demonstrated that zebrafish-M. marinum infection model combined with qPCR analysis is a simple and efficient method for in vivo screening and evaluation of anti-tuberculosis drugs. Animal experiments were carried out in accordance with the provisions for animal ethics in the Regulations on Laboratory Animals of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

Analysis of the key networks, metabolic pathways, and regulation substances of hypoxia based on the omics and zebrafish model / 中国药理学与毒理学杂志

OBJECTIVE Hypoxia is associated with many complicated pathophysiological and biochemical processes that integrated and regulated via the key gene, protein and endogenous metabolite levels. Up to date, the exact molecular mechanism of hypoxia still remains unclear. In this work, we further explore the molecular mechanism of hypoxia and adaption to attenuate the damage in zebrafish model that have potential to resist hypoxic environment. METHODS The hypoxic zebrafish model was established in different concentration of oxygen with 3%,5%,10%,21% in water. The brain tissue was separated and the RNA-seq was used to identify the differentially expressed genes. The related endogenous metabolites profiles were obtained by LC-HDMS, and the multivariate statistics was applied to discover the important metabolites candidates in hypoxic zebrafish. The candidates were searched in HMDB, KEGG and Lipid Maps databases. RESULTS The zebrafish hypoxic model was successfully constructed via the different concentration of oxygen, temperature and hypoxic time. The activities of the related hypoxic metabolic enzymes and factors including HIF-1a, actate dehydrogenase (LDH) and citrate synthase (CS) were evaluated. Significant differences (P<0.05 and fold change >2) in the expression of 422 genes were observed between the normal and 3% hypoxic model. Among them, 201 genes increased depended on the lower concentration of oxygen. 53 metabolites were identified that had significant difference between the hypoxia and control groups (P<0.05, fold change>1.5 and VIP>1.5). The ten key metabolites were increased gradually while six compounds were decreased. The endogenous hypoxic metabolites of phenylalanine, D-glucosamine-6P and several important lipids with the relevant hub genes had similar change in hypoxic model. In addition, the metabolic pathways of phenylalanine, glutamine and glycolipid were influenced in both the levels of genes and metabolites. CONCLUSION The up- regulation of phenylalanine, D- glucosamine- 6P and lipid may have further understanding of protective effect in hypoxia. Our data provided an insight to further reveal the hypoxia and adaptation mechanism.

Development of drug toxicity and novel strategy for toxicity of Chinese materia medica based on zebrafish model / 中草药

With the advantages of high throughput activity screening and toxicity evaluation, zebrafish has become popular model organism in pharmaceutical research in recent years. Being integrated advantages both in vivo and in vitro, with high efficiency and low cost, evaluation using zebrafish has become an effective method in quick screening of early safety of compounds on abroad. In past years, studies on toxicity of traditional Chinese medicine (TCM) with zebrafish model have attracted more attention in China. The development in drug toxicity studies based on zebrafish model was reviewed, and novel strategy for toxicity of TCM using zebrafish was suggested in the paper.

Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model

BACKGROUND/OBJECTIVES: In this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model. MATERIALS/METHODS: We investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model. RESULTS: Among the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate. CONCLUSION: The results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.

Production of a compound against methicillin resistant Staphylococcus aureus (MRSA) from Streptomyces rubrolavendulae ICN3 & its evaluation in zebrafish embryos.

Background & objectives: Antibiotic resistance in pathogens has become a serious problem worldwide. Therefore, the search for new antibiotics for drug resistanct pathogens is an important endeavor. The present study deals with the production of anti-methicillin resistant Staphylococcus aureus (MRSA) potential of Streptomyces rubrolavendulae ICN3 and evaluation of anti-MRSA compound in zebrafish embryos. Methods: The antibiotic production from S. rubrolavendulae ICN3 was optimized in solid state fermentation and extracted. The antagonistic activity was confirmed against MRSA and purified in silica gel column and reverse phase - HPLC with an absorption maximum at 215 nm. Minimal inhibitory concentration of the compound was determined by broth microdilution method. zebrafish embryos were used to evaluate the extract/compound for its minimal inhibition studies, influences on heart beat rates, haematopoietic blood cell count and lethal dose values. Results: Streptomyces rubrolavendulae ICN3 showed potent antagonistic activity against MRSA with a zone of 42 mm. The minimum inhibitory concentration was calculated as 500 μg/ml of the crude extract and the purified C23 exhibited 2.5 μg/ml in in vitro assay. The LC50 value of the anti MRSA compound C23 was calculated as 60.49 μg/ml and the MRSA treated embryos survived in the presence of purified compound C23 at a dose of 10 μg/ml. Interpretation & conclusions: our results suggested that the compound was potent with less toxic effects in zebrafish embryonic model system for MRSA infection. Further structural evaluation and analysis in higher mammalian model system may lead to a novel drug candidate for drug resistant Staphylococcus aureus.

The Zebrafish Model for the Study on Hair Cell Development / 听力学及言语疾病杂志

Objective This study aims to examine the development of the posterior lateral line of the zebrafish and establish ant model to study the process of hair cell differentiation and regeneration .Methods We observed the posterior lateral line system formation by DAPI immunohistochemistry and whole mount in situ hybridization .We further evaluated hair cells differentiation within neuromast by using Transgenic Tg (Brn3c:mGFP) zebrafish and stained the functional hair cells by the mechanotransduction marker FM 1 -43FX .We labelled proliferating cells in primordium and neuromast by addition of BrdU to the system water .Results The posterior lateral line primordium originated from a sensory placode and started its journey at around 20 hours post fertilization to migrate along the horizontal myoseptum to the tail -tip with a constant speed (1 .7somite/hour) .The primordium depositd five or six neuromasts spaced along the body ,and two or three terminal neuromasts at the tail -tip at 48 hours post fertiliza-tion .At 3 ,5 and 7 days post fertilization ,zebrafish contained 5 .68 ± 1 .46 ,10 .1 ± 0 .99 ,and 12 .45 ± 1 .32 hair cells per neuromast ,respectively .Furthermore ,the average number of FM1-43FX stained hair cells within each neuro-mast were 3 .68 ± 1 .11 ,8 .18 ± 1 .86 ,and 10 .22 ± 1 .24 ,respectively .Conclusion We establish the development model of hair cells in zebrafish lateral line neuromast and suggest that 3 to 7 days post fertilization is an important period for lateral line neuromast differentiation .This study would be useful for underlying the mechanisms of hair cell differentiation and regeneration .