Liwen Han

Oxidative stress‐mediated developmental toxicity induced by isoniazide in zebrafish embryos and larvae

Isoniazide (INH) is an important first‐line drug that is used to treat tuberculosis. However, the effect of INH on fetal growth has not yet been elucidated, and the mechanism of INH‐induced developmental toxicity is still unknown. In the present study, we employed zebrafish embryos and larvae to investigate the developmental toxicity of INH. The survival rates of the embryos and larvae as well as the hatching rates of embryos were significantly reduced. Morphological abnormalities, including spinal curvature, yolk retention, swimming bladder absence, tail bending and shorter body lengths were induced by INH. Histopathological analysis showed loose cell‐to‐cell contacts and large vacuoles in the larval hepatocytes. Thin intestinal walls, frayed gut villi and widespread cell lysis were observed in the intestines of the larvae in the higher concentration (8, 16 mm) exposure groups. In addition, exposure to high doses (≥ 6 mm) of INH significantly reduced the locomotor capacity of the zebrafish larvae. INH significantly increased the levels of reactive oxygen species and malondialdehyde and decreased the superoxide dismutase activity in zebrafish larvae, which suggested that oxidative stress was induced and that the antioxidant capacity was inhibited. Superoxide dismutase 1 and liver fatty acid‐binding protein mRNA levels were significantly downregulated, while the GSTP2 and cytochrome P450 3A mRNA levels were significantly upregulated in the INH‐exposed zebrafish larvae. The overall results indicated that INH caused a dose‐ and time‐dependent increase in developmental toxicity and that oxidative stress played an important role in the developmental toxicity induced by INH in zebrafish larvae. Copyright

L-FABP-deficiency provoked oxidative stress, inflammation and apoptosis-mediated hepatotoxicity induced by pyrazinamide on zebrafish larvae.

ABSTRACT Pyrazinamide (PZA) is an essential antitubercular drug, but little is still known about its hepatotoxicity potential. This study examined the effects of PZA exposure on zebrafish (Danio rerio) larvae and the mechanisms underlying its hepatotoxicity. A transgenic line of zebrafish larvae that expressed enhanced green fluorescent protein (EGFP) in the liver was incubated with 1, 2.5, and 5 mM PZA from 72 h postfertilization (hpf). Different endpoints such as mortality, morphology changes in the size and shape of the liver, histological changes, transaminase analysis and apoptosis, markers of oxidative and genetic damage, as well as the expression of certain genes were selected to evaluate PZA-induced hepatotoxicity. Our results confirm the manner of PZA dose-dependent hepatotoxicity. PZA was found to induce marked injury in zebrafish larvae, such as liver atrophy, elevations of transaminase levels, oxidative stress, and hepatocyte apoptosis. To further understand the mechanism behind PZA-induced hepatotoxicity, changes in gene expression levels in zebrafish larvae exposed to PZA for 72 h postexposure (hpe) were determined. The results of this study demonstrated that PZA decreased the expression levels of liver fatty acid binding protein (L-FABP) and its target gene, peroxisome proliferator-activated receptor α (PPAR-α), and provoked more severe oxidative stress and hepatitis via the upregulation of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and transforming growth factor β (TGF-β). These findings suggest that L-FABP-mediated PPAR-α downregulation appears to be a hepatotoxic response resulting from zebrafish larva liver cell apoptosis, and L-FABP can be used as a biomarker for the early detection of PZA-induced liver damage in zebrafish larvae.

Gastrodin Suppresses Pentylenetetrazole-Induced Seizures Progression by Modulating Oxidative Stress in Zebrafish

Pentylenetetrazole (PTZ)-induced seizures in Zebrafish models are now widely accepted for investigating human disease epilepsy. In epilepsy, the generation of oxidative stress contributes to the brain injury. Although Gastrodin (GAS) has been reported to have anticonvulsant activities, its effects on zebrafish seizure models and the underlying mechanism remain unclear. In this study, we evaluated the effects of GAS pretreatment on PTZ-induced seizures in zebrafish larvae and investigated the underlying mechanism related to its anti-oxidative defense. We found for the first time that GAS significantly decreased seizure-like behavior and extended the latency period to the onset of seizures. In addition, after exposure to GAS, anti-oxidative activity was observed in PTZ-induced seizures by measurement of antioxidant enzymes activities and oxidative stress-related genes expression. The overall results indicate that GAS attenuates PTZ-induced seizures in a concentration-dependent manner and modulates oxidative stress to potentially protect larval zebrafish from further seizures. Furthermore, our results have provided novel insights into GAS related therapy of seizures and associated neurological disorders.

Activation of BDNF-TrkB signaling pathway-regulated brain inflammation in pentylenetetrazole-induced seizures in zebrafish

Abstract Seizures are sustained neuronal hyperexcitability in brain that result in loss of consciousness and injury. Understanding how the brain responds to seizures is critical to help developing new therapeutic strategies for epilepsy, a neurological disorder characterized by recurrent and unprovoked seizures. However, the mechanisms underlying seizure‐dependent alterations of biological properties are poorly understood. In this study, we analyzed gene expression profiles of the zebrafish heads that were undergoing seizures and identified 1776 differentially expressed genes. Gene‐regulatory network analysis revealed that BDNF‐TrkB signaling pathway positively regulated brain inflammation in zebrafish during seizures. Using K252a, a TrkB inhibitor to block BDNF‐TrkB signaling pathway, attenuated pentylenetetrazole (PTZ)‐induced seizures, which also confirmed BDNF‐TrkB mediated inflammatory responses including regulation of il1&bgr; and nf&kgr;b, and neutrophil and macrophage infiltration of brain. Our results have provided novel insights into seizure‐induced brain inflammation in zebrafish and anti‐inflammatory related therapy for epilepsy. HighlightsGene expression profiling of seizures identifies 1776 differentially expressed genes.Regulatory analysis suggests BDNF‐TrkB regulated brain inflammation during seizures.Pretreatment of K252a to block BDNF‐TrkB inhibits the PTZ‐induced seizure responses.Blocking BDNF‐TrkB inhibits seizure‐induced increase of inflammatory‐related genes.Activation of BDNF‐TrkB during seizures triggers leukocyte infiltration into the head.

Targeted lipidomics profiling of marine phospholipids from different resources by UPLC-Q-Exactive Orbitrap/MS approach

Marine phospholipids (MPLs) are valuable components that can be applied within diverse areas like nutrition, pharmacy, and medicine as well as basic scientific research, particularly due to their high concentration of long chain polyunsaturated fatty acids. In this work, lipidomics approach by UPLC-Q-Exactive Orbitrap/MS was used for the identification, quantification, comparison, and characterization of phospholipids (PLs) from three different marine resources (shrimp head, codfish roe, and squid gonad). In total, 310 PL molecular species containing 34 different structures of fatty acid chains were identified simultaneously by Lipidsearch software. Significant differences between three groups in the PL classes, compositions and contents were revealed. A list of characteristic PL species that represent significant differences among groups was determined by lipidomics analysis. Until now, the information about the deep comprehensive description of PL from marine sources is limited. Thus, this study will give a very potential starting point to develop MPL products and establish the quality standards for different marine raw materials.

The role of hepatic antioxidant capacity and hepatobiliary transporter in liver injury induced by isopsoralen in zebrafish larvae

Isopsoralen is the main component of the Chinese medicine psoralen, which has antitumour activity and can be used for the treatment of osteoporosis. However, the mechanism behind its hepatotoxicity has not yet been elucidated. In this study, the hepatotoxicity of isopsoralen was investigated using zebrafish. Isopsoralen treatment groups of 25, 50 and 100 μM were established. The mortality, liver morphology changes, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver histopathology and mRNA levels of liver injury–related genes in zebrafish larvae were measured. The results showed that isopsoralen resulted in the development of malformed zebrafish, dose-dependent increases in ALT and AST, decreased liver fluorescence and weakened fluorescence intensity. Histopathological examination showed that high-dose isopsoralen caused a large number of vacuolated structures in the larvae liver. The polymerase chain reaction results showed a significant decrease in the mRNA levels of genes related to antioxidant capacity (lfabp, gstp2 and sod1) and drug transport (mdr1, mrp1 and mrp2), indicating that isopsoralen significantly inhibited liver antioxidant capacity and drug efflux capacity in zebrafish larvae. Isopsoralen is hepatotoxic to zebrafish larvae via inhibition of drug transporter expression resulting in the accumulation of isopsoralen in the body and decreased antioxidant capacity, leading to liver injury.

Tenacissoside H exerts an anti-inflammatory effect by regulating the nf-κb and p38 pathways in zebrafish

Abstract Marsdenia tenacissima exhibits biological activity with heat‐clearing and detoxifying properties, relieving coughs and asthma and exerting anticancer and anti‐HIV effects. Tenacissioside H (TH) is a Chinese medicine monomer extracted from the dried stem of Marsdenia tenacissima. We investigated the in vivo anti‐inflammatory activity of TH using three different zebrafish inflammation models: local inflammation induced by tail cutting, acute inflammation induced by CuSO4, and systemic inflammation induced by lipopolysaccharide (LPS). Real time‐polymerase chain reaction (RT‐PCR) was used to elucidate the mechanism of TH action against LPS‐induced inflammatory responses. Our results showed TH significantly reduced the number of macrophages in the injured zebrafish tail, inhibited CuSO4‐induced migration of macrophages toward the neural mound, and decreased the distribution of macrophages in tail fin compared to LPS‐treated group. Furthermore, TH inhibits LPS‐induced inflammation responses in zebrafish by modulating the nuclear factor &kgr;B (nf‐&kgr;b) and p38 pathways to regulate inflammatory cytokines, such as tumor necrosis factor‐&agr; (tnf‐&agr;), cyclooxygenase (cox‐2), interleukin‐1b (il‐1b), interleukin‐8 (il‐8), interleukin‐10 (il‐10), nitric oxide synthase (nos2b) and prostaglandin E synthase (ptges). In conclusion, TH possesses anti‐inflammation activity via the regulation of the nf‐&kgr;b and p38 pathways. This finding provides a reference for the clinical application of Xiaoaiping (the trade name of Marsdenia tenacissima extract). Graphical abstract Figure. No Caption available. HighlightsTH significantly inhibits inflammatory responses in zebrafish in three different inflammatory models.TH could down‐regulate inflammatory cytokines against LPS‐induced inflammatory responses, such as tnf‐&agr; and nos2b.TH inhibits LPS‐induced inflammation responses in zebrafish by modulating the nuclear factor &kgr;B and p38 pathways.

Developmental toxicity induced by PM2.5 through endoplasmic reticulum stress and autophagy pathway in zebrafish embryos

The aims of this study were to investigate the mechanism underlying the developmental toxicity of fine particulate matter (PM2.5) and provide a more thorough understanding of the toxicity of PM2.5 in an ecological environment. Zebrafish embryos at 4 h post-fertilization were exposed to PM2.5 at doses of 200, 300, 400, 500, 600 and 800 μg/mL for 120 h. The mortality, hatching rate, morphology score, body length, locomotor capacity, histological changes, antioxidant defense system, leukocyte migration, inflammation-related gene mRNA expression, endoplasmic reticulum stress (ERS) and autophagy were evaluated to study PM2.5-induced developmental toxicity and its underlying mechanisms. PM2.5 exposure significantly increased the mortality and malformations and reduced the hatching rate and body length of the zebrafish. PM2.5 significantly reduced the locomotor capacity of zebrafish larvae, increased the levels of ROS and disturbed the antioxidant defense system in zebrafish larvae. In addition, a histological examination showed that the heart, liver, intestines and muscle of the PM2.5-treated zebrafish exhibited abnormal changes and a significant increase in cellular autophagic accumulation. RT-PCR showed that the expression of genes related to inflammation (tgfβ and cox2), ERS (hspa5, chop, ire1, xbp1s, and atf6) and autophagy (lc3, beclin1 and atg3) pathways was significantly increased in the PM2.5-treated zebrafish, indicating that PM2.5 induced inflammation and promoted ERS and autophagy responses via the activation of the IRE1-XBP1 and ATF6 pathways. Together, our data indicate that PM2.5 induced a dose- and time-dependent increase in developmental toxicity to zebrafish embryos. Additionally, ERS and autophagy may play important roles in PM2.5-induced developmental toxicity.

LC-MS/MS Identification of Novel Saponins from the Viscera of Sea Cucumber Apostichopus japonicus

Sea cucumbers, soft-bodied wormlike echinoderms, are traditionally consumed in China. Sea cucumber viscera are often discarded during production. In this study, we characterized saponins present in the viscera of the Chinese sea cucumber Apostichopus japonicus. Sea cucumber saponins were obtained by a two-stage process consisting of foam fractionation and macroporous resin separation. Furthermore, the saponins were characterized by HPLC-Q-TOF-MS. Three novel saponins, i.e., apostichoposide A–C, were detected and characterized based on their unique fragmentation patterns in the negative ion mode tandem MS spectra as well as isolation and NMR studies. The abundance of saponins in sea cucumber viscera holds promise for industrial applications.

A rapid assessment for predicting drug-induced hepatotoxicity using zebrafish.

INTRODUCTION Zebrafish have been used as a model to access drug-induced hepatotoxicity. However, individual differences occur in the liver development of zebrafish. METHODS We used a transgenic line of zebrafish that expressed enhanced green fluorescent protein (EGFP) in the liver and then used a calculation of the liver index area, a potentially new endpoint of hepatotoxicity, to evaluate drug-induced liver injury. To further validate the reliability of the liver area index as a quick evaluation of zebrafish liver function damage, the liver area index level was correlated with hepatic transaminase activities using the Pearson correlation coefficient and confirmed by histopathology. RESULTS Zebrafish larvae treated with high doses of the known mammalian hepatotoxic drugs carbaryl, isoniazide, and pyrazinamide showed significantly decreased liver area index levels, which are suggestive of liver injury and correspond with the higher alanine transaminase (ALT) and aspartate transaminase (AST) activities and histological liver alterations. The results showed a significant negative correlation between the degree of liver injury and the liver area index level. DISCUSSION Our data support the use of the liver area index as a reliable and comparable indicator to screen hepatotoxic agents using the zebrafish model.