Supercritical Fluid Extract of Putranjiva roxburghii Wall. Seeds Mitigates Fertility Impairment in a Zebrafish Model

Putrajeevak (Putranjiva roxburghii Wall.; synonym Drypetes roxburghii (Wall.) Hurus) seeds have been used since ancient times in the treatment of infertility in the Ayurvedic system of medicine in India. In this study, the oil component of Putrajeevak seeds (PJSO) was extracted using the supercritical fluid extraction (SCFE) method using liquid CO₂ and the constituents were analyzed using gas chromatography-flame ionized detectorand high-performance thin-layer chromatography. PJSO contained trace amounts of β-sitosterol with oleic and linoleic acids as the major fatty acid constituents. Male and female zebrafish were mutagenized with N-ethyl-N-nitrosourea (ENU) and fish that produced less than 20 viable embryos were selected for the study. SCFE oil extracts from the P. roxburghii seeds were used in this study to reverse fertility impairment. The mutant fish were fed with PJSO for a period of 14 days and the rates of fertility, conception, and fecundity were determined with wild-type healthy fish as a breeding partner. Treatment with PJSO increased the ovarian follicle count as well as the number of mature eggs, while reducing the number of ovarian cysts. Sperm count as well as sperm motility were greatly enhanced in the ENU-mutagenized male zebrafish when treated with PJSO. The results obtained in this study demonstrate the effectiveness of P. roxburghii seed oil in reversing impaired fertility in both male and female zebrafish models.     

Effect of Water and Ethanol Extracts from Hericium erinaceus Solid-State Fermented Wheat Product on the Protection and Repair of Brain Cells in Zebrafish Embryos

Extracts from Hericium erinaceus can cause neural cells to produce nerve growth factor (NGF) and protect against neuron death. The objective of this study was to evaluate the effects of ethanol and hot water extracts from H. erinaceus solid-state fermented wheat product on the brain cells of zebrafish embryos in both pre-dosing protection mode and post-dosing repair mode. The results showed that 1% ethanol could effectively promote zebrafish embryo brain cell death. Both 200 ppm of ethanol and water extracts from H. erinaceus solid-state fermented wheat product protected brain cells and significantly reduced the death of brain cells caused by 1% ethanol treatment in zebrafish. Moreover, the zebrafish embryos were immersed in 1% ethanol for 4 h to cause brain cell damage and were then transferred and soaked in the 200 ppm of ethanol and water extracts from H. erinaceus solid-state fermented wheat product to restore the brain cells damaged by the 1% ethanol. However, the 200 ppm extracts from the unfermented wheat medium had no protective and repairing effects. Moreover, 200 ppm of ethanol and water extracts from H. erinaceus fruiting body had less significant protective and restorative effects on the brain cells of zebrafish embryos. Both the ethanol and hot water extracts from H. erinaceus solid-state fermented wheat product could protect and repair the brain cells of zebrafish embryos damaged by 1% ethanol. Therefore, it has great potential as a raw material for neuroprotective health product.     

Study on the Joint Toxicity of BPZ,BPS, BPC and BPF to Zebrafish

Bisphenol Z (BPZ), bisphenol S (BPS), bisphenol C (BPC), and bisphenol F (BPF) had been widely used as alternatives to bisphenol A (BPA), but the toxicity data of these bisphenol analogues were very limited. In this study, the joint toxicity of BPZ, BPS, BPC, and BPF to zebrafish (Danio rerio) was investigated. The median half lethal concentrations (LC50) of BPZ, BPS, BPC, and BPF to zebrafish for 96 h were 6.9 × 10⁵ µM, 3.9 × 10⁷ µM, 7.1 × 10⁵ µM, and1.6 × 10⁶ µM, respectively. The joint toxicity effect of BPF–BPC (7.7 × 10⁵–3.4 × 10⁵µM) and BPZ–BPC (3.4 × 10⁵–3.5 × 10⁵µM) with the same toxic ratio showed a synergistic effect, which may be attributed to enzyme inhibition or induction theory. While the toxicity effect of the other two bisphenol analogue combined groups and multi-joint pairs showed an antagonistic effect due to the competition site, other causes need to be further explored. Meanwhile, the expression levels of the estrogen receptor genes (ERα, ERβ1) and antioxidant enzyme genes (SOD, CAT, GPX) were analyzed using a quantitative real-time polymerase chain reaction in zebrafish exposure to LC₅₀ of BPZ, BPS, BPC, and BPF collected at 24, 48, 72, and 96 h. Relative expression of CAT, GPX, and ERβ1 mRNA declined significantly compared to the blank control, which might be a major cause of oxidant injury of antioxidant systems and the disruption of the endocrine systems in zebrafish.     

Imaging mass spectrometry of gold nanoparticles in a tissue section as an immunohistochemical staining mass probe

For analysis of low abundance peptides in a tissue section, immunohistochemical staining through antibody‐antigen interaction is a usual technique. The antibody is conjugated with a probe moiety that aids in highly sensitive detection. Gold nanoparticles, which show excellent chemical stability and variation of surface modifications, are expected to act as a sensitive mass probe to desorb gold ions (Au⁺, Au₂⁺, Au₃⁺) that are distinguishable from fragment ions from organic molecules. Here, green fluorescent proteins (GFP) in a tissue section of a transgenic zebrafish were detected by the gold mass probe conjugated with antibodies. Due to the efficient ionization and desorption of gold ions, imaging mass spectrometry of Au₂⁺ ions indicated the distribution of gold nanoparticles stained in a tissue section, and the mass signal distribution was consistent with the area where the GFP‐expressing cells were distributed. Conventional immunofluorescence techniques showed intense autofluorescence that come from intrinsic fluorophores in the tissue section. In contrast, the gold nanoparticles acted as an immunostaining mass probe that displayed significantly lower background signals.     

超高效液相色谱-串联质谱法测定斑马鱼性腺中13种类固醇激素

建立了超高效液相色谱-串联质谱测定雌性斑马鱼性腺中13种类固醇激素的分析方法。雌鱼性腺分别经预冷的乙腈、乙酸乙酯浸提后,氨基(NH2)、亲水-亲脂平衡介质(HLB)固相萃取柱富集净化,ACQUITY UPLCBEH C18色谱柱进行梯度分离。采用多反应监测模式扫描,基质匹配外标法定量。研究了不同提取溶剂对13种类固醇激素的提取效果,考察了固相萃取柱的净化效果,并对斑马鱼性腺进行类固醇激素加标回收率实验,结果表明,13种分析物的平均加标回收率为74.8%~127.0%,相对标准偏差为3.9%~15.8%,方法检出限为0.006~0.300μg/kg。该方法简便、准确、灵敏,可应用于斑马鱼性腺类固醇激素生理水平的检测。     

斑马鱼胚胎在电离辐射生物学研究中的应用

斑马鱼胚胎最初为研究发育生物学与分子遗传学的重要模式动物,20 世纪90 年代关于斑马鱼胚胎研究的文章每年不足100 篇,到本世纪初期已上升到每年1 000 余篇,目前这个数字正呈指数增长,斑马鱼胚胎的研究范围与应用价值正也随之逐步提升。该文综述了斑马鱼胚胎在电离辐射研究领域中的应用,包括电离辐射诱导斑马鱼的毒性作用、电离辐射引起的DNA损伤及其信号转导机制,以及利用斑马鱼对辐射防护剂与增敏剂进行评价。在此基础上还特别介绍了中国科学院近代物理研究所利用斑马鱼胚胎在放射生物学研究上取得的进展,其中包括一种具有物理激发、化学刺激作用的线粒体内活性氧(ROS) 诱导剂,在非条件刺激下利用斑马鱼检测该物质的毒性,发现6 10 μL/mL 的该物质无胚胎发育毒性,然而,在一定波长的光刺激下可造成DNA双链断裂,在理论上是一种潜在的辐射增敏剂。Zebrafish embryo is a classic animal model to study developmental and molecular biology. The relatedworks using zebrafish embryo arise from less than 100 papers per year in 90 s to more than 1 000 papers per year at the beginning of 21st century. The research scope and application value of zebrafish embryo in biology studies are gradually extending, along with the exponential increase of related papers. Here, the applications of zebrafish embryo in the field of radiation biology are reviewed, including radiation-induced toxicity, signal transduction in response to radiation-induced DNA damage, and the assessment of radio-protection and radiosensitizers, respectively. The progress in radiation biology employing zebrafish embryo in Institute of Modern Physics has also been reported, includinga novel mitochondrial reactive oxygen species inducer, which can be triggered by exogenous physical signal and exert chemical stimulation. This inducer shows no obvious embryo toxicity to zebrafish under 10 μL/mL. It can induce DNA double strand breaks if activated by certain wavelength of lights. Itisa potential radiosensitizerin theory.     

Theoretical Exploring of a Molecular Mechanism for Melanin Inhibitory Activity of Calycosin in Zebrafish

Tyrosinase is an oxidase that is the rate-limiting enzyme for controlling the production of melanin in the human body. Overproduction of melanin can lead to a variety of skin disorders. Calycosin is an isoflavone from Astragali Radix, which is a traditional Chinese medicine that exhibits several pharmacological activities including skin whitening. In our study, the inhibitory effect of calycosin on melanin production is confirmed in a zebrafish in vivo model by comparing with hydroquinone, kojic acid, and arbutin, known as tyrosinase inhibitors. Moreover, the inhibitory kinetics of calycosin on tyrosinase and their binding mechanisms are determined using molecular docking techniques, molecular dynamic simulations, and free energy analysis. The results indicate that calycosin has an obvious inhibitory effect on zebrafish pigmentation at the concentration of 7.5 μM, 15 μM, and 30 μM. The IC₅₀ of calycosin is 30.35 μM, which is lower than hydroquinone (37.35 μM), kojic acid (6.51 × 10³ μM), and arbutin (3.67 × 10⁴ μM). Furthermore, all the results of molecular docking, molecular dynamics simulations, and free energy analysis suggest that calycosin can directly bind to the active site of tyrosinase with very good binding affinity. The study indicates that the combination of computer molecular modeling and zebrafish in vivo assay would be feasible in confirming the result of the in vitro test and illustrating the target-binding information.     

Three New Phthalide Glycosides from the Rhizomes of Cnidium officinale and Their Recovery Effect on Damaged Otic Hair Cells in Zebrafish

The extract from Cnidium officinale rhizomes was shown in a prior experiment to markedly recover otic hair cells in zebrafish damaged by neomycin. The current study was brought about to identify the principal metabolite. Column chromatography using octadecyl SiO₂ and SiO₂ was performed to isolate the major metabolites from the active fraction. The chemical structures were resolved on the basis of spectroscopic data, including NMR, IR, MS, and circular dichroism (CD) data. The isolated phthalide glycosides were assessed for their recovery effect on damaged otic hair cells in neomycin-treated zebrafish. Three new phthalide glycosides were isolated, and their chemical structures, including stereochemical characteristics, were determined. Two glycosides (0.1 μM) showed a recovery effect (p < 0.01) on otic hair cells in zebrafish affected by neomycin ototoxicity. Repeated column chromatography led to the isolation of three new phthalide glycosides, named ligusticosides C (1), D (2), and E (3). Ligusticoside C and ligusticoside E recovered damaged otic hair cells in zebrafish.     

Screening of Potential Anti-Thrombotic Ingredients from Salvia miltiorrhiza in Zebrafish and by Molecular Docking

Background: Danshen (DS), the dry root of Salvia miltiorrhiza Bge., has been used in traditional Chinese medicine (TCM) for many years to promote blood circulation and to inhibit thrombosis. However, the active ingredients responsible for the anti-thrombotic effect and the underlying mechanisms are yet to be fully elucidated. Methods: Molecular docking was used to predict the active ingredients in DS and their potential targets by calculating the scores of docking between DS ingredients and thrombosis-related proteins. Then, a chemical-induced zebrafish thrombosis model was applied to confirm their anti-thrombotic effects. Result: The molecular docking results indicated that compared to the control ligand, higher docking scores were observed for several compounds in DS, among which salvianolic acid B (SAB), lithospermic acid (LA), rosmarinic acid (MA), and luteolin-7-O-β-d-glucoside (LG) could attenuate zebrafish caudal vein thrombosis and recover the decrease in heart red blood cells (RBCs) in a dose-dependent manner. Conclusions: Our study showed that it is possible to screen the potential active components in natural products by combining the molecular docking method and zebrafish in vivo model.