Survival rate of zebrafish larvae exposed to FRA for 48 h. Data are presented as the mean ± SD. Each experiment was performed 3 times with 30 randomly selected zebrafish larvae. ** p < 0.01 vs. control.

Hepatotoxic effects of FRA on zebrafish larvae. Treatment with different concentrations of FRA for 48 h blackened the liver area of zebrafish larvae (A) and increased the gray value (B). AO staining showed that the proportion of apoptotic hepatocytes had increased (C). Changes to levels of ALT (D), AST (E), TBIL (F), and DBIL (G) in zebrafish larvae treated with FRA for 48 h. Data are presented as the mean ± SD. Each experiment was performed 3 times with 40 randomly selected zebrafish larvae. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control.

Effects of FRA on liver morphology. The liver is indicated with red arrows. Low-magnification images were captured at 200× and high-magnification images at 630×.

At 48 h after FRA treatment, flow cytometry was performed to determine ROS levels (A) and average fluorescence intensity (B). Data are presented as the mean ± SD. Each experiment was performed 3 times with 50 randomly selected zebrafish larvae. ** p < 0.01 vs. control.

Effects of FRA on the expression levels of bile acid transporters and components of the JNK/p53 pathway. FRA inhibited mRNA expression of the bile acid transporters P-gp (A), Bsep (B), and NTCP (C), and increased those of the apoptotic genes JNK1 (D), caspase-3 (E), and p53 (F). The protein levels of JNK, p-JNK (H), p53, p-p53 (I), Bax (J), and cleaved caspase-3 (K) were determined by western blot analysis. GAPDH was used as an internal reference to normalize the results (G). Data are presented as the mean ± SD. Each experiment was performed 3 times with 40 randomly selected zebrafish larvae. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control.

Inhibitory effects of KCZ against FRA-induced hepatotoxicity. (A) KCZ reduced FRA-induced mortality of zebrafish larvae. The biochemical indices of ALT (B), AST (C), TBIL (D), and DBIL (E) decreased significantly after KCZ treatment. (F–H) Morphological observation and AO staining to determine the degree of liver injury and apoptosis. Data are presented as the mean ± SD. Each experiment was performed 3 times with 40 randomly selected zebrafish larvae. ** p < 0.01 vs. control; *** p < 0.001 vs. control; ^#p < 0.05 vs. FRA + KCZ; ^##p < 0.01 vs. FRA+KCZ.

KCZ altered the expression patterns of transporter and apoptotic proteins inhibited by FRA. The mRNA expression levels of P-gp (A), Bsep (B), Ntcp (C), Cyp3a65 (D), p53 (E), and caspase-3 (F) were detected by qRT-PCR analysis. (G) The protein expression level of cleaved caspase-3 was detected by western blot analysis. Data are presented as the mean ± SD. Each experiment was performed 3 times with 40 randomly selected zebrafish larvae. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control; ^## p < 0.01 vs. FRA + KCZ; ^### p < 0.001 vs. FRA + KCZ.

KCZ restored the amino acid metabolism spectrum altered by FRA. (A) PCA scores of zebrafish larvae samples in the control, FRA, KCZ, and KCZ and FRA groups. The 95% confidence intervals are shown. (B) A heatmap showing changes in the contents of 19 amino acids related to zebrafish liver injury obtained by GC-MS/MS. Each experiment was performed 3 times with 40 randomly selected zebrafish larvae.

Chemical structures of fraxinellone (FRA) and its metabolites.