Transcriptome analysis reveals the early resistance of zebrafish larvae to oxidative stress.

Oxidative stress is one of most common environmental stresses encountered by fish, especially during their fragile larval stage. More and more studies are aimed at understanding the antioxidant defense mechanism of fish larvae. Herein we characterized the early resistance of zebrafish larvae to oxidative stress and investigated the underlying transcriptional regulations using RNA-seq. We found that pre-exposure of zebrafish larvae to 2 mM H2O2 for 1 or 3 h significantly improved their survival under higher doses of H2O2 (3 mM), suggesting the antioxidant defenses of zebrafish larvae were rapidly built under pre-exposure of H2O2. Comparative transcriptome analysis showed that 310 (185 up and 125 down) and 512 (331 up and 181 down) differentially expressed genes were generated after 1 and 3 h of pre-exposure, respectively. KEGG enrichment analysis revealed that protein processing in endoplasmic reticulum is a highly enriched pathway; multiple genes (e.g., hsp70.1, hsp70.2, and hsp90aa1.2) encoding heat shock proteins in this pathway were sharply upregulated presumably to correct protein misfolding and maintaining the cellular normal functions during oxidative stress. More importantly, the Keap1/Nrf2 system-mediated detoxification enzyme system was significantly activated, which regulates the upregulation of target genes (e.g., gstp1, gsr, and prdx1) to scavenger reactive oxygen species, thereby defending against apoptosis. In addition, the MAPK, as a transmitter of stress signals, was activated, which may play an important role in activating antioxidant system in the early stages of oxidative stress. Altogether, these findings demonstrate that zebrafish larvae rapidly establish resistance to oxidative stress, and this involves changes in protein processing, stress signal transmission, and the activation of detoxification pathways.

Flavokawain B alleviates LPS-induced acute lung injury via targeting myeloid differentiation factor 2.

Acute lung injury (ALI) is a sudden onset systemic inflammatory response. ALI causes severe morbidity and death and currently no effective pharmacological therapies exist. Natural products represent an excellent resource for discovering new drugs. Screening anti-inflammatory compounds from the natural product bank may offer viable candidates for molecular-based therapies for ALI. In this study, 165 natural compounds were screened for anti-inflammatory activity in lipopolysaccharide (LPS)-challenged macrophages. Among the screened compounds, flavokawain B (FKB) significantly reduced LPS-induced pro-inflammatory IL-6 secretion in macrophages. FKB also reduced the formation of LPS/TLR4/MD2 complex by competitively binding to MD2, suppressing downstream MAPK and NF-κB signaling activation. Finally, FKB treatment of mice reduced LPS-induced lung injury, systemic and local inflammatory cytokine production, and macrophage infiltration in lungs. These protective activities manifested as increased survival in the ALI model, and reduced mortality upon bacterial infection. In summary, we demonstrate that the natural product FKB protects against LPS-induced lung injury and sepsis by interacting with MD2 and inhibiting inflammatory responses. FKB may potentially serve as a therapeutic option for the treatment of ALI.

Transcriptome analysis reveals the importance of exogenous nutrition in regulating antioxidant defenses during the mouth-opening stage in oviparous fish.

Antioxidant system is crucial for protecting against environmental oxidative stress in fish life cycle. Although the effects of starvation on the antioxidant defenses in several adult fish have been defined, no relevant researches have been reported in the larval stage, particularly during the transition from endogenous to exogenous feeding. To clarify the molecular response of antioxidant system that occurs during the mouth-opening stage under starvation stress and explore its association with energy metabolism, we employed RNA-seq to analyze the gene expression profiles in zebrafish larvae that received a delayed first feeding for 3 days. Our data showed that delayed feeding resulted in downregulation of 7078 genes and upregulation of 497 genes. These differentially expressed genes are mainly involved in growth regulation (i.e., DNA replication and cell cycle), energy metabolism (i.e., glycolysis/gluconeogenesis and fatty acid metabolism), and antioxidant defenses. We demonstrated that the starved larvae are in an extremely malnourished state in the absence of exogenous nutrition, and the consequence is that numerous antioxidant genes are downregulated. Meanwhile, the antioxidant defenses also respond negatively to oxidative stress. After nutritional supply, the expression of these inhibited antioxidant genes was restored. These results suggest that the establishment of antioxidant defenses during the mouth-opening stage depends highly on exogenous nutrition. Our findings would contribute to comprehending the nutritional stress and metabolic switches during the mouth-opening stage and are essential for reducing high mortality in commercial fish farming.

A Model Construction of Starvation Induces Hepatic Steatosis and Transcriptome Analysis in Zebrafish Larvae.

Hepatic steatosis caused by starvation, resulting in non-alcoholic fatty liver disease (NAFLD), has been a research topic of human clinical and animal experiments. To understand the molecular mechanisms underlying the triggering of abnormal liver metabolism by starvation, thus inducing hepatic lipid accumulation, we used zebrafish larvae to establish a starvation-induced hepatic steatosis model and conducted comparative transcriptome analysis by RNA-seq. We demonstrated that the incidence of larvae steatosis is positively correlated with starvation time. Under starvation conditions, the fatty acid transporter (slc27a2a and slc27a6-like) and fatty acid translocase (cd36) were up-regulated significantly to promote extrahepatic fatty acid uptake. Meanwhile, starvation inhibits the hepatic fatty acid metabolism pathway but activates the de novo lipogenesis pathway to a certain extent. More importantly, we detected that the expression of numerous apolipoprotein genes was downregulated and the secretion of very low density lipoprotein (VLDL) was inhibited significantly. These data suggest that starvation induces hepatic steatosis by promoting extrahepatic fatty acid uptake and lipogenesis, and inhibits hepatic fatty acid metabolism and lipid transport. Furthermore, we found that starvation-induced hepatic steatosis in zebrafish larvae can be rescued by targeting the knockout cd36 gene. In summary, these findings will help us understand the pathogenesis of starvation-induced NAFLD and provide important theoretical evidence that cd36 could serve as a potential target for the treatment of NAFLD.