Figure 4

Ethanol treatment of zebrafish larvae increased the extrahepatic ATP and Ado levels. (a) Oil Red O staining of the liver (black line) of 6 days postfertilization (dpf) wild-type larvae with or without 1% or 2% ethanol (EtOH) exposure. Lower-magnification sagittal images (top panel) and higher-magnification images (bottom panel) are shown. Scale bar 200 µm. (b) Representative hematoxylin and eosin (HE)-stained images of the liver sections (red dotted line) from zebrafish larvae. Lipid droplets are seen in 1% EtOH-exposed larvae (white arrowheads). Scale bars 40 µm (top panel) and 20 µm (bottom panel). (c) Hepatic mRNA levels of inflammatory genes tnfa and il1b, inflammasome-related gene nlrp3, and fibrosis marker mmp9 were determined in zebrafish larvae with or without 1% or 2% EtOH exposure using qRT-PCR. (d,e) Representative images of the liver of GRAB_ATP zebrafish larvae at 6 dpf. The GFP fluorescence intensity in the liver was increased in response to EtOH treatment (bottom panel). Liver: white dotted line. Scale bar 100 μm (d). Quantification of the change in the GFP fluorescence intensity in GRAB_ATP zebrafish larvae with or without EtOH treatment (n = 10, 10, 11, each group) (e). (f,g) Representative images of the liver of GRAB_Ado zebrafish larvae at 6 dpf. The GFP fluorescence intensity in the liver was increased in response to EtOH treatment (bottom panel). Liver: white dotted line. Scale bar 100 μm (f). Quantification of the change in the GFP fluorescence intensity in GRAB_Ado zebrafish larvae with or without EtOH treatment (n = 11, 12, 9, each group) (g). Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.