Hepatotoxicity and mechanism of Rhododendri Mollis Flos based on zebrafish model / 中国中药杂志

This study used the zebrafish model to explore the hepatotoxicity of Rhododendri Mollis Flos(RMF). The mortality was calculated according to the number of the survival of zebrafish larvae 4 days after fertilization under different concentration of RMF, and the dose-toxicity curve was fitted to preliminarily evaluate the toxicity of RMF. The liver phenotypes under the sublethal concentration of RMF in the treatment group and the blank control group were observed by hematoxylin-eosin(HE) staining and acridine orange(AO) staining. Meanwhile, the activities of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were determined to confirm the hepatotoxicity of RMF. Real-time quantitative polymerase chain reaction(real-time PCR) and Western blot were used to determine the expressions of genes and proteins in zebrafish larvae. Gas chromatography time-of-flight mass spectrometry(GC-TOF-MS) was used to conduct untargeted metabolomics testing to explore the mechanism. The results showed that the toxicity of RMF to zebrafish larvae was dose-dependent, with 1 100 μg·mL~(-1) of the absolute lethal concentration and 448 μg·mL~(-1) of sublethal concentration. The hepatocyte apoptosis and degeneration appeared in the zebrafish larvae under the sublethal concentration of RMF. The content of ALT and AST in zebrafish larvae at the end of the experiment was significantly increased in a dose-dependent manner. Under the sublethal concentration, the expressions of genes and proteins related to apoptosis in zebrafish larvae were significantly increased as compared with the blank control group. The results of untargeted metabolomics showed that the important metabolites related to the he-patotoxicity of RMF were mainly enriched in alanine, aspartic acid, glutamic acid, and other pathways. In conclusion, it is inferred that RMF has certain hepatotoxicity to zebrafish larvae, and its mechanism may be related to apoptosis.

Study on liver diseases with zebrafish as an important tool / 中国中药杂志

With the increasing incidence of hepatobiliary diseases, it is particularly important to understand the role of molecular, cellular and physiological factors in the clinical diagnosis and treatment with traditional Chinese medicine(TCM) in the development of liver disease. Appropriate animal models can help us identify the possible mechanisms of relevant diseases. Danio rerio(zebrafish) model was traditionally used to study embryonic development, and has been gradually used in screening and evaluation of liver diseases and relevant drug in recent years. Zebrafish embryos develop rapidly and the digestive organs of 5-day-old juvenile fish are all mature. At this stage, they may develop hepatobiliary diseases induced by developmental defects or compounds. Zebrafish liver is similar to human liver in cell composition, function, signal transduction, response to injury and cell process mediating liver disease. Furthermore, due to the high conservation of genes and proteins between humans and zebrafish, zebrafish becomes an alternative system for studying basic mechanisms of liver disease. Therefore, genetic screening could be performed to identify new genes involving specific disease processes, and chemical screening could be made for drugs in specific processes. This paper briefly introduced the experimental properties of zebrafish as model system, emphasized the study progress of zebrafish models for pathological mechanism of liver diseases, especially fatty liver, and drug screening and evaluation, so as to provide ideas and techniques for the future liver toxicity assessment of TCM.

The PTD domain of Tat protein enhance GFP protein delivering into myeloma cell SP2/0 / 生物工程学报

In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E. coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.