Alcohol-associated liver disease (ALD) encompasses a range of hepatic abnormalities, including isolated alcoholic steatosis, steatohepatitis, and cirrhosis. The flavanone-7-O-glycoside narirutin (NRT), the primary flavonoid in citrus peel, has antioxidant, anti-inflammatory, and lipid-lowering activity. We investigated the effects of NRT on liver injury induced by alcohol and explored the underlying mechanisms. Zebrafish larvae were used to investigate the effects of NRT on acute exposure to ethanol (EtOH). Liver phenotypic, morphological, and biochemical assessments were performed to evaluate the hepatoprotective effects of NRT. Network pharmacology and molecular docking analyses were conducted to identify candidate targets of NRT in EtOH-induced liver injury. A drug affinity responsive target stability (DARTS) assay was conducted to evaluate the binding of NRT to mitogen-activated protein kinase 14 (MAPK14). The mechanism of action of NRT was validated by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis. The liver phenotypic, morphological, and biochemical assessments revealed that NRT has potential therapeutic effects against acute EtOH-induced liver injury. RT-qPCR confirmed that NRT reversed the change in the expression of genes related to oxidative stress, lipogenesis, and the endoplasmic reticulum (ER)/unfolded protein response pathway. Network pharmacology and molecular docking analyses identified potential targets of NRT's protective effects and confirmed that NRT regulates the p38 MAPK signaling pathway by targeting mitogen-activated protein kinase 14 (MAPK14). NRT mitigates alcohol-induced liver injury by preventing lipid formation, protecting the antioxidant system, and suppressing ER stress-induced apoptosis through MAPK14 modulation.

Erythropoietic protoporphyria (EPP1) results in painful photosensitivity and severe liver damage in humans due to the accumulation of fluorescent protoporphyrin IX (PPIX). While zebrafish (Danio rerio) models for porphyria exist, the utility of ferrochelatase (fech) knockout zebrafish, which exhibit EPP, for therapeutic screening and biological studies remains unexplored. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated fech-knockout zebrafish larvae as a model of EPP1 for drug screening. CRISPR/Cas9 was employed to generate fech-knockout zebrafish larvae exhibiting morphological defects without lethality prior to 9 days post-fertilization (dpf). To assess the suitability of this model for drug screening, ursodeoxycholic acid (UDCA), a common treatment for cholestatic liver disease, was employed. This treatment significantly reduced PPIX fluorescence and enhanced bile-secretion-related gene expression (abcb11a and abcc2), indicating the release of PPIX. Acridine orange staining and quantitative reverse transcription polymerase chain reaction analysis of the bax/bcl2 ratio revealed apoptosis in fech^−/− larvae, and this was reduced by UDCA treatment, indicating suppression of the intrinsic apoptosis pathway. Neutral red and Sudan black staining revealed increased macrophage and neutrophil production, potentially in response to PPIX-induced cell damage. UDCA treatment effectively reduced macrophage and neutrophil production, suggesting its potential to alleviate cell damage and liver injury in EPP1. In conclusion, CRISPR/Cas9-mediated fech^−/− zebrafish larvae represent a promising model for screening drugs against EPP1.

Lateral inhibition is known to be mediated by Delta-associated Notch signaling pathway (Kunisch et al., 1994). Within equivalent cells in proneural domain, signal-sending cells expressed Delta induce Notch receptor in neighboring cells, which become neuroblasts or sensory organ precursors (Bray, 1998). It is well known that Hairy Enhancer-of-split, a transcriptional repressor, is expressed by the transcriptional complex consist of notch intracellular domain (NICD), CBF1/Su(H)/Lag1 (CSL) and Mastermind (Mam) in neighboring cells (Bray, 1998). In neighboring cells of neural ectoderm, Ngn1 as a proneural gene is repressed by a Hairy Enhancer-of-split-related protein (Kim et al., 1997; Takke et al., 1999). In contrast to neighboring cells, Mind bomb (Mib) promotes ubiquitination and endocytosis of Delta with extracellular domain of Notch (NECD) in signal-sending cells (Itoh et al., 2003). Jagged have been identified as a ligand of Notch receptors (Lindsell et al., 1995). Zebrafish Jagged2 expressed in the domain located progenitor cells of motor neuron (pMN domain) activate Notch and maintain a population of proliferating progenitors in the p3 domain (Yeo & Chitnis, 2007). Jagged-mediated Notch signaling regulate the temporal generation of late-born neurons, such as secondary motor neurons (sMN) and GABAergic KA” neurons in the ventral spinal cord (Yeo & Chitnis, 2007). The gain-of-function study using Tg[hsp70-Mib:EGFP] showed that the majority of cells in the p3 domain differentiated into KA” neurons after heat-shock during late neurogenesis (Ryu et al., 2015). These observations imply that Jagged may also be a substrate for Mib. Our preliminary data showed that Jagged2 was ubiquitinated by Mib in P19 cells, but not cytosolic form of intracellular domain of zebrafish Jagged2 (J2ICD) (unpublished data). This observation implicate that an adaptor might be required for ubiquitinating membrane-bounded Jagged ICD by cytosolic Mib. By performing yeast-two hybrid screen using zebrafish Jagged2 ICD as a bait, zebrafish PEX1 was isolated and identified a potential adaptor between Mib and Jagged2 ICD.

Global warming and extreme weather events pose a significant threat to global biodiversity, with rising water temperatures exerting a profound influence on fish conservation and fishery development. In this study, we used zebrafish as a model organism to explore the impact of a heat acclimation period on their survival rates. The results demonstrated that a 2-month heat acclimation period almost completely mitigated heat stress-induced mortality in zebrafish. Subsequent analysis of the surviving zebrafish revealed a predominance of hepatic mitochondria in a fission state. Remarkably, a short-term fasting regimen, which induced hepatic mitochondrial fission, mirrored the outcomes of the protective effect of heat acclimation and augmented animal survival under heat stress. Conversely, treatment with a mitochondrial fission inhibitor within the fasting group attenuated the elevated survival rate. Furthermore, zebrafish embryos subjected to brief heat acclimation also exhibited increased heat resistance, a trait diminished by a chemical intervention inhibiting mitochondrial fission. This suggests a shared mechanism for heat resistance between embryos and adult zebrafish. These findings underscore the potential use of inducing mitochondrial fission to enhance heat resistance in zebrafish, offering promise for fish biodiversity conservation in the face of global warming.

A comprehensive review has concluded that progenitor cells, which are distributed along the dorsal-ventral axis of the spinal cord in vertebrates, generate distinct types of neuron in each compartment (Briscoe & Ericson, 2001). Our previous study suggested that zebrafish Jagged2 interacts with progenitor cells in the adjacent p3 domain and plays a crucial role in maintaining proliferating neural progenitor cells in this region (Yeo & Chitnis, 2007). It was proposed that Jagged2 might bind to Notch expressed in the neural progenitor cells of the p3 domain, thereby facilitating signal transduction in a contact-dependent manner (Yeo & Chitnis, 2007). While the p3 domain in zebrafish is composed of a single cell layer, the pMN domain is composed of multiple cell layers (Yeo & Chitnis, 2007). Jagged2 is expressed in the pMN domain of the zebrafish, which encompasses neural precursors as well as various types of neurons (Yeo & Chitnis, 2007). Therefore, it can be concluded that Jagged2-mediated Notch signaling does not only occur between neural progenitors in the p3 domain and those in the pMN domain. This study investigates the activation and transmission mechanisms of Jagged2, which has previously been shown to activate the Notch signaling pathway.

Optimized histological techniques are crucial for visualizing cellular morphology across zebrafish tissues. Here, we report a rapid and reliable hematoxylin and Oil Red O (H-ORO) staining protocol for frozen sections that can be completed in less than three minutes. Mayer's hematoxylin is used for nuclear staining, followed by Oil Red O (ORO) to visualize lipid-rich structures such as the endomysium surrounding myofibers, white matter of the brain, and myelin layers of major axonal tracts. Importantly, our optimized H-ORO protocol preserves tissue integrity and minimizes artifacts such as myofiber shrinkage commonly observed with ethanol-based hematoxylin and eosin (H&E) staining in both frozen and paraffin sections.